Lipopolysaccaride-binding peptides obtained by phage display method

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. It has strong toxicity and might cause sepsis or septic shock. Thus early detection of LPS and neutralization of LPS toxicity are required. We obtained several new LPS-binding peptides using a phage display method. We synthesized 3 of these peptides and analyzed their binding affinity and capacity to LPS. One of these peptides, named Li5-001, showed high binding affinity to LPS and lipid A; the K_d values were 10 and 1 nM, respectively. Li5-001 showed a high binding capacity to LPS, and was estimated to bind 130 ng LPS/mg, which is higher than that of polymyxin B (80 ng LPS/mg); however, its LPS-neutralizing activity was low. Li5-001 coupled with beads will be useful for eliminating endotoxin contamination from pharmaceuticals. Its low LPS-neutralizing activity allows to be used in the Limulus amebocyte lysate test without eluting LPS from the Li5-001 coupled beads.     

Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC–MS/MS: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of pantoprazole sodium (PS) in human plasma using pantoprazole D3 (PSD3) as internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 4.6 mm × 75 mm, 3.5 μm, 80 Å column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 7.10): acetonitrile (30:70, v/v), pumped at 0.6 mL/min. PS and PSD3 were detected with proton adducts at m/z 384.2 → 200.1 and 387.1 → 203.1 in multiple reaction monitoring (MRM) positive mode, respectively. Precipitation method was employed in the extraction of PS and PSD3 from the biological matrix. This method was validated over a linear concentration range of 10.00–3000.00 ng/mL with correlation coefficient (r) ≥ 0.9997. Intra- and inter-day precision of PS were found to be within the range of 1.13–1.54 and 1.76–2.86, respectively. Both analytes were stable throughout freeze/thaw cycles, bench top and postoperative stability studies. This method was successfully utilized in the analysis of blood samples following oral administration of PS (40 mg) in healthy human volunteers.     

Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use.     

Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.     

Development of high performance liquid chromatography/electrospray ionization mass spectrometry for assay of ginkgolic acid (15:1) in rat plasma and its application to pharmacokinetics study

A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C₁₈ column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r²) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters C_max, T_max, t_1/2, AUC_0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively.     

Structure of the high-molecular weight exopolysaccharide isolated from Lactobacillus pentosus LPS26

The strain Lactobacillus pentosus LPS26 produces a capsular polymer composed of a high- (2.0 × 10⁶ Da) (EPS A) and a low-molecular mass (2.4 × 10⁴ Da) (EPS B) polysaccharide when grown on semi-defined medium containing glucose as the carbon source. The structure of EPS A and its deacetylated form has been determined by monosaccharide and methylation analysis as well as by 1D/2D NMR studies (¹H and ¹³C). We conclude that EPS A is a charged heteropolymer, with a composition of d-glucose, d-glucuronic acid and l-rhamnose in a molar ratio 1:2:2. The repeating unit is a pentasaccharide with two O-acetyl groups at O-4 of the 3-substituted α-d-glucuronic acid and at O-2 of the 3-substituted β-l-rhamnose, respectively.→4)-α-d-Glcp-(1→3)-α-d-GlcpA4Ac-(1→3)-α-l-Rhap-(1→4)-α-d-GlcpA-(1→3)-β-l-Rhap2Ac-(1→This unbranched structure is not common in EPSs produced by Lactobacilli. Moreover, the presence of acetyl groups in the structure is an unusual feature which has only been reported in L. sake 0-1 [Robijn et al. Carbohydr. Res., 1995, 276, 117-136].     

Determination of casopitant and its three major metabolites in dog and rat plasma by positive ion liquid chromatography/tandem mass spectrometry

A sensitive, selective and quantitative method for the simultaneous determination of casopitant, a potent and selective antagonist of the human Neurokinin 1 (NK-1) receptor, and its three major metabolites M12, M13 and M31 was developed and validated in dog and rat plasma. Acetonitrile containing stable labeled internal standards for the four analytes was used to precipitate proteins in plasma. Chromatographic separation was obtained using a reversed phase column with multiple reaction monitoring turboionspray positive ion detection. The lower and upper limits of quantification for casopitant and its metabolites were 15 and 15,000 ng/mL, using a 50 μL of dog or rat plasma aliquot, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in dog plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 4.1% to 10.0% and −10.8% to 8.7%, respectively, for casopitant and its 3 major metabolites. The intra-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 3.9% to 6.6% and −9.6% to 8.3%, respectively, for casopitant and its three metabolites. All analytes were found to be stable in analytical solutions for at least 43 days at 4 °C, in dog and rat plasma at room temperature for at least 24 h, at the storage temperature of −20 °C for at least 6 months, and following the action of three freeze–thaw cycles from −20 °C to room temperature. All analytes were also found to be stable in processed extracts at 4 °C for at least 72 h. This assay proved to be accurate, precise, fast and was used to support long-term toxicology studies in dog and rat.     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma

We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography–tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005–5 μmol/L. The extraction recoveries for all the analytes fell in the range of 88–101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6 h at room temperature, at least three freeze–thaw cycles, in the −70 °C or −20 °C freezer for 2 months, and also in the reconstitution solution at 8 °C for 48 h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.     

Quantitative analysis of 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane,a potent synthetic steroidal liver X receptor agonist in plasma using liquid chromatography–tandem mass spectrometry

The steroidal liver X receptor agonist, 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane (ATI-829) is a potential therapeutic agent for the treatment of atherosclerosis. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the quantification of ATI-829 in mouse plasma was developed and validated. Proteins in a 25 μL aliquot of mouse plasma were precipitated, and ATI-829 was extracted from the precipitate by the addition of 125 μL methanol. The overall extraction efficiency was greater than 99%. LC–MS–MS with negative ion electrospray and selected reaction monitoring was used for the quantitative analysis of ATI-829. The lower limit of quantitation of ATI-829 corresponded to 5.0 ng/mL (9.7 nM) plasma. Interference from matrix was negligible. The calibration curve was linear over the range 5–2000 ng/mL. The intra-day precision and inter-day precision of the analyses were <4.5% and <6%, respectively, and the accuracy ranged from 92% to 103%. ATI-829 in plasma was stable for at least 6 h at room temperature, 1 week at 4 °C, and 3 weeks at −20 °C. The validated method was then utilized for pharmacokinetic studies of ATI-829 administered to mice.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

Liquid chromatography-tandem mass spectrometric assay for pravastatin and two isomeric metabolites in mouse plasma and tissue homogenates

A bioanalytical assay for pravastatin and two isomeric metabolites, 3′α-isopravastatin and 6′-epipravastatin, was developed and validated. Mouse plasma and tissue homogenates from liver, kidney, brain and heart were pre-treated using protein precipitation with acetonitrile containing deuterated internal standards of the analytes. The extract was diluted with water and injected into the chromatographic system. This system consisted of a polar embedded octadecyl silica column using isocratic elution with formic acid in a water–acetonitrile mixture. The eluate was transferred to an electrospray interface using negative ionization and the analytes were detected and quantified with the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was successfully validated in a 3.4–7100 ng/ml concentration range for pravastatin, 1.3–2200 ng/ml for 3′α-isopravastatin and 0.5–215 ng/ml for 6′-epipravastatin using only plasma for calibration. For plasma samples, subjected to full validation, within and between day precisions were 1–7% (9–18% at the LLQ level) and accuracies were between 91% and 103%. For tissue homogenates, subjected to partial validation, within and between day precisions were 2–12% (6–19% at the LLQ level) and accuracies were between 87% and 113% (81 and 113% at the LLQ level). Drug and metabolites were shown to be chemically stable under most relevant analytical conditions. Finally, the assay was successfully applied for a pilot study in mice. After intravenous administration of the drug, all isomeric compounds were found in plasma; however, in liver and kidney homogenate only the parent drug showed levels exceeding the LLQ.     

Association analysis of ERBB2 amplicon genetic polymorphisms and STARD3 expression with risk of gastric cancer in the Chinese population

The purpose of this study was to investigate whether risk of gastric cancer (GC) was associated with single nucleotide polymorphisms (SNPs) in a gene cluster on the chromosome 17q12-q21 (ERBB2 amplicon) in the Chinese Han population. We detected twenty-six SNPs in this gene cluster containing steroidogenic acute regulatory-related lipid transfer domain containing 3 (STARD3), protein phosphatase 1 regulatory subunit 1B (PPP1R1B/DARPP32), titin-cap (TCAP), per1-like domain containing 1(PERLD1/CAB2), human epidermal growth factor receptor-2 (ERBB2/HER2), zinc-finger protein subfamily 1A 3 (ZNFN1A3/IKZF3) and DNA topoisomerase 2-alpha (TOP2A) genes in 311 patients with GC and in 425 controls by Sequenom. We found no associations between genetic variations and GC risk. However, haplotype analysis implied that the haplotype CCCT of STARD3 (rs9972882, rs881844, rs11869286 and rs1877031) conferred a protective effect on the susceptibility to GC (P = 0.043, odds ratio [OR] = 0.805, 95% confidence intervals [95% CI] = 0.643–0.992). The STARD3 rs1877031 TC genotype endued histogenesis of gastric mucinous adenocarcinoma and signet-ring cell carcinoma (P = 0.021, OR = 2.882, 95% CI = 1.173–7.084). We examined the expression of STARD3 in 243 tumor tissues out of the 311 GC patients and 20 adjacent normal gastric tissues using immumohistochemical (IHC) analysis and tissue microarrays (TMA). The expression of STARD3 was observed in the gastric parietal cells and in gastric tumor tissues and significantly correlated with gender (P = 0.004), alcohol drinking (P < 0.001), tumor location (P = 0.007), histological type (P = 0.005) and differentiation (P = 0.023) in GC. We concluded that the combined effect of haplotype CCCT of STARD3 might affect GC susceptibility. STARD3 expression might be related to the tumorigenesis of GC in the Chinese population.     

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam,and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D₅) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Use of 3-hydroxy fatty acid concentrations in a murine air pouch infection model as a surrogate marker for LPS activity: a feasibility study using environmental Burkholderia cenocepacia isolates

Using a murine hypodermic air pouch infection model designed to mimic the release of bacterial products at physiological levels, 3-hydroxy fatty acid (3-OH FA) and endotoxin unit levels from Burkholderia cenocepacia isolates were assessed. The B. cenocepacia environmental isolates (n = 35) survived in the hypodermic air pouch but did not invade across the peritoneal epithelial layer during a 72-h infection. For all 35 strains, when the molar ratio of C_14:0 3-OH FA to C_16:0 3-OH FA in the air pouch fluid wash samples was between 1.4 and 2.5, the concentrations of C_14:0 3-OH FA were correlated with the endotoxin unit levels. However, both surrogate markers exhibited different correlations to the inflammatory response. The linear regression coefficient was 0.4234 for C_14:0 3-OH FA concentrations vs. NO productions, 0.223 for endotoxin unit levels vs. NO productions, 0.5008 for C_14:0 3-OH FA concentrations vs. TNF-alpha productions and 0.2869 for endotoxin unit levels vs. TNF-alpha productions. Therefore, C_14:0 3-OH FA concentrations, rather than endotoxin unit levels, acted as an immunostimulatory indicator for LPS in the B. cenocepacia isolates.     

Simultaneous determination of cytosine arabinoside,daunorubicin and etoposide in human plasma

A method for simultaneous bioanalysis of the three cytotoxic drugs cytosine arabinoside, daunorubicin and etoposide in human plasma was developed and validated. A HPLC method with ultra-violet and fluorescence detection, preceded by mixed-mode cation-exchange solid phase extraction sample preparation, was used for the quantification of the analytes. The assay was used for the simultaneous measurement of cytosine arabinoside, daunorubicin and etoposide with linearity in the ranges of 13–1500 ng/mL, 15–1000 ng/mL and 52.5–3500 ng/mL, respectively. The chromatographic run-time was 15.5 min. The overall precision (% relative standard deviation) was within 0.2–13.5% and the recovery ranged between 86.1% and 110.1% for the three drugs at all concentrations tested. Plasma samples were stable for at least two months when stored at −20 °C. The method was successfully applied to quantification of the three drugs in blood samples from patients undergoing induction treatment for acute myeloid leukaemia, thus demonstrating its suitability for clinical studies.     

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography–tandem mass spectrometry

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.     

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.