Chimeric proteins comprising the constant region of immunoglobulins for treating hemophilia B (WO2005001025)

The application (WO2005001025) details recombinant fusion proteins attached to the constant region of heavy chains of immunoglobulins. They are found to be particularly useful for the treatment of hemostatic disorders, such as hemophilia B. It aims at engineering chimeric proteins comprising of a single molecule of human factor IX (FIX) and the constant region (Fc domain) of one or two heavy chain(s) of human IgG (rFIXFc). cDNA for rFIXFc was generated by a PCR. rFIXFc protein was isolated and purified from stably transfected mammalian cells. The concentration and clotting activity of rFIXFc were assessed in mice, rats, monkeys, and FIX-deficient mice and dogs, after intravenous administration. The half-life of rFIXFc activity is prolonged by three to fourfold, compared with rFIX, when administered intravenously in all animals. The generation of chimeric proteins, comprised of FIX fused to the Fc domain of IgG, extends the clotting activity of the recombinant molecule. rFIXFc represents a promising candidate for the treatment of patients with hemophilia B. The application claims the methods of making recombinant chimeric proteins comprising of one biologically active molecule fused to the Fc region of the heavy chain(s) of immunoglobulins and their use for therapy.     

Chimeric proteins comprising the constant region of immunoglobulins for treating hemophilia B (WO2005001025)

The application (WO2005001025) details recombinant fusion proteins attached to the constant region of heavy chains of immunoglobulins. They are found to be particularly useful for the treatment of hemostatic disorders, such as hemophilia B. It aims at engineering chimeric proteins comprising of a single molecule of human factor IX (FIX) and the constant region (Fc domain) of one or two heavy chain(s) of human IgG (rFIXFc). cDNA for rFIXFc was generated by a PCR. rFIXFc protein was isolated and purified from stably transfected mammalian cells. The concentration and clotting activity of rFIXFc were assessed in mice, rats, monkeys, and FIX-deficient mice and dogs, after intravenous administration. The half-life of rFIXFc activity is prolonged by three to fourfold, compared with rFIX, when administered intravenously in all animals. The generation of chimeric proteins, comprised of FIX fused to the Fc domain of IgG, extends the clotting activity of the recombinant molecule. rFIXFc represents a promising candidate for the treatment of patients with hemophilia B. The application claims the methods of making recombinant chimeric proteins comprising of one biologically active molecule fused to the Fc region of the heavy chain(s) of immunoglobulins and their use for therapy.     

Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins

A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the β15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of α1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the β21 and β37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.Nucleotide sequence data reported are available in the EMBL database under the accession numbers CAI79619, AM076940, and CAI79618.’     

Evaluation of an immunoassay for human-specific quantitation of therapeutic antibodies in serum samples from non-human primates

Pharmacokinetic characterization of therapeutic antibodies plays an important role during preclinical and clinical development. However, accurate pharmacokinetic evaluation of therapeutic antibodies in serum samples from non-human primates is often complicated by insufficient specificity of the assays to measure drug levels. The present paper describes the use of a murine monoclonal antibody in an immunoassay format to specifically and quantitatively measure human therapeutic antibodies in serum from non-human primates. This murine antibody is directed against a unique epitope on the constant region CH2 domain of all isotypes of human immunoglobulin G (IgG). The antibody, designated anti-human Fcγ-pan: R10Z8E9, does not cross-react with serum from mouse, rat, and the non-human primates marmoset, rhesus macaque, cynomolgus monkey and baboon when using an enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance technology (Biacore) format for measurement of the therapeutic antibody. Use of the antibody anti-human Fcγ-pan: R10Z8E9 as capturing and detection reagent allowed human-specific quantitation of total therapeutic antibody anti-IGF-1R in spiked cynomolgus monkey serum via a Sandwich ELISA format. In contrast, a commercially available polyclonal antibody (PAB) directed to the Fcγ fragment of human IgG only specifically measured the therapeutic antibody in buffer samples, but not in serum from cynomolgus monkeys. This generic human IgG assay was already applied in several pharmacokinetic studies in cynomolgus monkeys to determine serum levels of different therapeutic antibodies, including the anti-IGF-1R. Validation of the assay for a humanized IgG1 therapeutic antibody against a membrane protein revealed a lower limit of quantitation of 8 ng/mL in undiluted serum. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 15%. Dilutional linearity was evidenced by a recovery of 98.7–114% of expected concentrations. In conclusion, the monoclonal antibody anti-human Fcγ-pan: R10Z8E9 provides a standard means for human-specific quantitation of therapeutic antibodies with high sensitivity in serum samples from non-human primates in a generic human IgG assay.     

Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies

The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography–mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G–Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.     

Characterization of a technique for rapid pharmacokinetic studies of multiple co-eluting compounds by LC/MS/MS

A method for rapid pharmacokinetic screening of multiple potential drug candidates has been developed. This technique, based on the ability of liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to independently monitor multiple components, enables the quantification of substances which may or may not be chromatographically resolved. Our results indicate that the limit of quantitation and accuracy of this multiple-compound LC/MS/MRM quantitation method are comparable to a single-compound LC/MS/MRM quantitation method. No apparent ion suppression due to the existence of extraneous compounds in the analytical solution and biological matrix effect are observed in the range of the calibration curve. The issue of potential residual molecule cross-talk interference existing in the multiple-reaction monitoring mode has been discussed. This multiple-compound LC/MS/MRM quantitation method can be used for high throughput pharmacokinetic screening and to assay mixtures that have co-eluting analytes or similar m/z of precursor/product ion pairs.     

Determination of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical formulation by high performance liquid chromatography with electrochemical detection

A novel HPLC method with electrochemical detection has been developed for the determination of recombinant human epidermal growth factor (rhEGF) in pharmaceutical products. rhEGF was separated from other components in formulation on a reversed-phase C18 column with 24% acetonitrile in 0.1 M phosphate buffer (pH 4.75). The optimum electrochemical oxidation of EGF was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to electroactive tyrosine, tryptophan, methionine, and arginine residues. The quantitation range was from 1.0 to 200 ng of rhEGF with the linear correlation coefficient greater than 0.999. The method was successfully applied for the quantitation of rhEGF in a pharmaceutical preparation.     

Structural basis of the interaction between immunoglobulins and Fc receptors provided by NMR spectroscopy

Fc gamma Receptors (Fc gamma R) are membrane glycoproteins that bind the Fc portion of immunoglobulin G (IgG). The cross linking of Fc gamma R-bound IgG by multivalent antigens allows clustering of the Fc gamma R and initiates a variety of effector mechanisms which play a key role in immune defenses against pathogens. The Fc region is composed of two identical polypeptide chains, which are related to each other by a two-fold axis. Recent elucidation of the crystal structure of human Fc gamma RII provided two distinct views of modes of IgG-Fc gamma R interactions, which is controversial against each other. Nuclear magnetic resonance (NMR) spectroscopy provides a unique and irreplaceable tool to solve these issues. We recently studied the interaction between the Fc fragment of mouse IgG2b and the extracellular domain of mouse Fc gamma RII by this method. We showed that Fc gamma RII binds to a negatively charged area of the CH2 domain, corresponding to the lower hinge region, and that the binding of Fc gamma RII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. This conformational change may account for the 1:1 stoichiometry that we and others observed between Fc gamma R and Fc. We therefore propose a model that explains why the interaction between IgG molecules and Fc gamma R does not trigger cellular responses in the absence of cross linking by multivalent antigens and does not lead to spontaneous inflammatory responses that would be deleterious for the organism.     

A high-throughput UPLC method for the characterization of chemical modifications in monoclonal antibody molecules

Development of high-throughput release and characterization assays is critical for the effective support of the rapidly growing biologics pipeline for biotherapeutics. Clipping of polypeptide chains is commonly monitored during process optimization, formulation development, and stability studies. A reduced capillary electrophoresis-sodium dodecyl sulfate (rCE -SDS) method is often used as a purity release assay for monitoring clips in monoclonal antibodies (mAbs); however, it has a cycle time of approximately 40 min, which is not suited for high-throughput screening. Additionally, the characterization of clips and variants from electropherograms is not straightforward and takes significant time. Reduced reversed-phase (RP) chromatography has been a popular assay for the characterization and identification of clips and variants because it can be directly coupled with online mass spectrometric analysis. However, the high-column temperature and low pH required for RP assays can induce on-column cleavage and therefore skew the results. To minimize on-column degradation, we have developed a high-throughput method with a significantly shorter cycle time of 5 min. The short cycle time was achieved using an ultra-high-pressure liquid chromatography (UPLC) system with a 1.7 μm phenyl column. This UPLC method allowed quantitation of hinge clipping in an IgG1 molecule and acid induced aspartic acid/proline (D/P) clip in an IgG2 molecule. The results from the UPLC method were comparable to those obtained with rCE-SDS. Additionally, the phenyl column offered partial resolution of oxidation and other chemical modifications, making this technique an attractive assay for high-throughput process characterization and formulation screens.     

Structure verification of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody

Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS)and N-terminal sequencer. Molecular masses, N-terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control and structure determination of other IgG1 products.     

LC/MS联用法测定片剂中米索前列醇的含量

目的：建立用高效液相色谱-质谱联用法测定片剂中米索前列醇含量的检测方法。方法：采用Waters symmetry C18柱（2.1mm×150mm,5μm）,柱温20℃,流动相为乙腈：水（70：30）,流速为0.2mL·min^-1;质谱条件采用正离子检测的电喷雾接口（ESI＋）,选择性监测（SIM）质荷比为383（米索前列醇,M＋H）的分子离子峰。结果：米索前列醇最低定量限为1μg.mL-1,线性范围1-5μg.mL^-1,（r=0.9998）,日内和日间RSD均低于1.2%。结论：该方法灵敏度高、简便、无杂质干扰,适合于片剂中米索前列醇的含量检测。     

The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations

Glycation of a recombinant monoclonal IgG2 molecule, in sucrose containing liquid formulations, was studied using reversed-phase LC/MS analysis of the intact IgG, the F(ab')2 fragments and after complete tryptic digestion. The extent of glycation in sucrose containing formulations was monitored at different temperatures over a period of 21 months using the Hexose index (Hex(I)). Hex(I) represents the average number of hexose molecules per molecule of IgG and was calculated by using the intensity values of peaks corresponding to hexose isoforms in the deconvoluted mass spectra. The rate of glycation in mildly acidic sucrose containing formulations was proportional to the incubation temperature. No glycation was observed in sucrose containing formulations incubated at 4 degrees C even after 18 months. However, when the same formulations were incubated at 37 degrees C glycation was observed after just 1 month. The glycation sites were mapped to 10 lysine residues distributed throughout the molecule. The amino terminal end of the light chain was also shown to contain glycation. The surface accessibility of the lysine side chain could influence its susceptibility to glycation.     

Site-specific<Superscript>99m</Superscript>Tc-labeling of antibody using dihydrazinophthalazine (DHZ) conjugation to Fc region of heavy chain

The development of an antibody labeling method with 99mTc is important for cancer imaging. Most bifunctional chelate methods for 99mTc labeling of antibody incorporate a 99mTc chelator through a linkage to lysine residue. In the present study, a novel site-specific 99mTc labeling method at carbohydrate side chain in the Fc region of 2 antibodies (T101 and rabbit anti-human serum albumin antibody (RPAb)) using dihydrazinophthalazine (DHZ) which has 2 hydrazino groups was developed. The antibodies were oxidized with sodium periodate to produce aldehyde on the Fc region. Then, one hydrazine group of DHZ was conjugated with an aldehyde group of antibody through the formation of a hydrazone. The other hydrazine group was used for labeling with 99mTc. The number of conjugated DHZ was 1.7 per antibody. 99mTc labeling efficiency was 46-85% for T101 and 67-87% for RPAb. Indirect labeling with DHZ conjugated antibodies showed higher stability than direct labeling with reduced antibodies. High immunoreactivities were conserved for both indirectly and directly labeled antibodies. A biodistribution study found high blood activity related to directly labeled T101 at early time point as well as low liver activity due to indirectly labeled T101 at later time point. However, these findings do not affect practical use. No significantly different biodistribution was observed in the other organs. The research concluded that DHZ can be used as a site-specific bifunctional chelating agent for labeling antibody with 99mTc. Moreover, 99mTc labeled antibody via DHZ was found to have excellent chemical and biological properties for nuclear medicine imaging.     

The Kinetics of Relaxin Oxidation by Hydrogen Peroxide

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10–14 kcal mol^–1, with an entropy of activation of the order of –30 cal K^–1 mol^–1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.     

基于UPLC-Q-TOF-MS的双特异性抗体关键表征方法研究

目的 建立Y-Body?型双特异性抗体一级结构的超高效液相色谱-三重四级杆/飞行时间质谱联用技术(UPLC-Q-TOF-MS)研究方法。方法 以Y-Body?型双特异性抗体M808为研究对象,基于UPLC-Q-TOF-MS检测完整抗体及各亚基去糖基化处理前后的相对分子质量、肽图(氨基酸序列),并通过Unify软件进行解析。结果 完整抗体去糖基化处理前后的相对分子质量分别为128417.660 5,125 040.721 0;轻链去糖基化处理前后的相对分子质量分别为23447.232 2,23 445.560 8;重链去糖基化处理前后的相对分子质量分别为50 804.773 2,49 201.605 1;单链去糖基化处理前后的相对分子质量分别为54 189.922 4,52 415.764 4;其氨基酸序列与理论序列基本一致,肽图的互补决定区覆盖率为100%;推测N-糖基化位点位于重链及单链Fc端,糖型以G1F和G2F为主。结论 通过UPLC-Q-TOF-MS及Unify软件可有效表征双特异性抗体的一级结构,为其质量标准的制定提供依据。     

The investigation and the use of high flow column-switching LC/MS/MS as a high-throughput approach for direct plasma sample analysis of single and multiple components in pharmacokinetic studies

Recently direct plasma injection LC/MS/MS technique has been increasingly used in pharmaceutical research and development due to the demand for higher throughput of sample analyses. In this work, two on-line extraction methods including high flow LC/MS/MS and high flow column switching LC/MS/MS were investigated. The evaluations were conducted and focused on their performances with respect to peak responses, separation efficiency, and signal to-noise ratio in a multiple-component LC/MS/MS assay. Two HPLC pumps were used-with one for high flow delivery and one for gradient elution. A CTC autosampler was used to inject plasma samples. High flow LC was achieved by the use of 4 ml/min flow rate on a 1×50 mm Waters Oasis column. A 2×100 mm YMC column was coupled via a column-switching valve. The extracted analytes were analyzed in multiple-reaction-monitoring (MRM) mode using a triple quadrupole MS/MS. As a rapid and simple procedure, vortex-mixing plasma and internal standard directly in sample vials completed sample preparation. The high flow column switching method (two-column system) provided sharper peak shape than the conventional high flow method. This effect increased analyte signal-to-noise ratio and sensitivity. Narrower peak width resulted in much better separation efficiency, which was required for multiple compound (N-in-1) analysis. A 2 mm I.D. column resulted in better peak shape and resolution than using a smaller I.D. column. The selected method achieved acceptable recoveries for most of the compounds tested, and it was successfully applied to a 10-in-1 pharmacokinetic (PK) study. The results showed that the dynamic range, lower limit of quantitation, assay accuracy and precision were acceptable for all compounds. Rapid sample preparation eliminated labor intensive and time consuming processes and improved productivity. This high throughput on-line extraction high flow column switching method has been proven particularly useful for multiple component analysis in PK studies.     

Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain

We investigated the formation and stability of succinimide, an intermediate of deamidation events, in recombinant monoclonal antibodies (mAbs). During the course of an analytical development study of an IgG1 mAbs, we observed that a specific antibody population could be separated from the main product by cation-exchange (CEX) chromatography. The cell-based bioassay measured a ～70% drop in potency for this fraction. Liquid chromatography time-of-flight mass spectrometry (LC–TOF/MS) and tandem mass spectrometry (LC–MS/MS) analyses showed that the modified CEX fraction resulted from the formation of a succinimide intermediate at Asn 55 in the complementarity determining region (CDR) of the heavy chain. Biacore assay revealed a ～50% decrease in ligand binding activity for the succinimide-containing Fab with respect to the native Fab. It was found that the succinimide form existed as a stable intermediate with a half-life of ～3 h at 37°C and pH 7.6. Stress studies indicated that mildly acidic pH conditions (pH 5) favored succinimide accumulation, causing a gradual loss in potency. Hydrolysis of the succinimide resulted in a further drop in potency. The implications of the succinimide formation at Asn 55, a highly conserved residue among IgG1 (mAbs), are discussed. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3509–3521, 2009     

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure,Stability, Aggregation,and Function of a Therapeutic Monoclonal Antibody

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the C_H2 domain, whereas AAPH destabilized the variable domain in addition to C_H2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.     

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc

Monoclonal antibodies, particularly IgGs and Ig-based molecules, are a well-established and growing class of biotherapeutic drugs. In order to improve efficacy, potency and pharmacokinetics of these therapeutic drugs, pharmaceutical industries have investigated significantly in engineering fragment crystallizable (Fc) domain of these drugs to optimize the interactions of these drugs and Fc gamma receptors (FcγRs) in recent ten years. The biological function of the therapeutics with the antibody-dependent cellular cytotoxicity (ADCC) enhanced double mutation (S239D/I332E) of isotype IgG1, the ADCC reduced double mutation (L234A/L235A) of isotype IgG1, and ADCC reduced isotype IgG4 has been well understood. However, limited information regarding the effect of these mutations or isotype difference on physicochemical properties (PCP), developability, and manufacturability of therapeutics bearing these different Fc regions is available. In this report, we systematically characterize the effects of the mutations and IgG4 isotype on conformation stability, colloidal stability, solubility, and storage stability at accelerated conditions in two buffer systems using six Fc variants. Our results provide a basis for selecting appropriate Fc region during development of IgG or Ig-based therapeutics and predicting effect of the mutations on CMC development process.     

Column-switching liquid chromatographic determination of ML-1035 sulphoxide and its sulphone and sulphide metabolites in rat urine.

A fully automated column-switching LC assay has been developed for the simultaneous determination of a gastroprokinetic agent, ML-1035 sulphoxide, and its sulphone and sulphide metabolites in rat urine. ML-1035 Sulphoxide is a metoclopramide analogue. The method involved direct injection of a diluted urine sample into a CN extraction column for sample clean-up. Polar urine components, including proteins, were flushed to waste. The retained compounds were then eluted onto a C8 analytical column for further separation and analysis by fluorescence detection. After the subsequent washing and re-equilibration with a sequence of three solvent mixtures, the extraction column was ready for the next injection. The recovery of the compounds from the extraction column was 85-90%. The limit of quantitation for all compounds of interest was 25 ng ml-1 or lower, using a 100 microliters specimen of urine. Good inter-day precision (2.1-10.0%), accuracy (0.3-18.0%), and linearity were obtained for all compounds over a range of 25-1000 ng ml-1. The applicability of the LC method was validated with urine samples from rats that had received ML-1035 sulphoxide.