Development of an inexpensive and rapid two site microenzyme linked immunosorbent assay kit for human alpha fetoprotein

Laboratory scale development of a two site micro enzyme linked immuno assay kit is described. The kit comprises rabbit anti human alphafetoprotein (AFP), anti human AFP IgG peroxidase conjugate and standard AFP. All the above reagents were prepared in the laboratory. The kit is eminently suitable for early screening of blood sample of pregnant women for neural tube defects of their fetuses and for the quantitation of AFP as a tumor marker. The assay kit was used to determine AFP in 76 sera from women at different stages of pregnancy. During 1st trimester AFP level was 18 to 119 ng/ml, during 2nd trimester the concentration varied from 85 to 302 ng/ml and during 3rd from 103 to 580 ng/ml. No evidence for maternal antibody to AFP was found. The above data agree with AFP level in pregnant women reported by earlier workers, using RIA or ELISA. The present ELISA kit would hopefully be much cheaper than internationally available ELISA kits for human AFP.     

An ISFET-based immunosensor for the detection of beta-Bungarotoxin

An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Quantitation of salbutamol in human urine by liquid chromatography-electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) is described for quantitation of salbutamol in human urine using nadolol as the internal standard (I.S.). Urine samples were hydrolyzed with beta-glucuronidase followed by a solid-phase extraction procedure using Bond Elut-Certify cartridges. The HPLC column was an Agilent Zorbax SB-C(18) column. A mixture of 0.01 M ammonium formate buffer (pH 3.5)-acetonitrile (85:15, v/v) was used as the mobile phase. Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. Selected ion monitoring (SIM) mode was used to monitor m/z 166 for salbutamol and m/z 310 for I.S. Good linearity was obtained in the range of 10.0-2000.0 ng/ml. The limit of quantification was 10.0 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 7.3%. The accuracy as determined from QC samples was within +/-2.6%. The method was applied for determining excretion curves of salbutamol.     

An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.     

Quantitation of human anti-mouse antibody in serum by flow cytometry.

Clinical investigations utilizing murine monoclonal antibodies require techniques for the detection of the human anti-mouse antibody (HAMA) response in patient serum. We report here a flow cytometric assay for the quantitation of HAMA. Commercially available beads conjugated with goat anti-mouse antibody provide a solid phase matrix for a triple bridge immunoassay. The measurement of fluorescein labeled antibodies by flow cytometry allows accurate quantitation of the HAMA. The assay will detect antibody levels of approximately 1.0 ng/ml. Antibody recovery in serum samples with known amounts of antibody added was greater than 90% at levels greater than or equal to 10 micrograms/ml. Serum samples obtained from 41 patients prior to and after single or multidose infusions of KS1/4-Desacetyl-vinblastine were analyzed. These results were compared with HAMA titers previously determined by ELISA. With few exceptions, patients with low titers as determined by ELISA demonstrated low HAMA potencies by flow cytometry and those with highest titers the highest potencies. Patients with no detectable HAMA by ELISA were also negative by flow analysis. The results of our studies demonstrate that HAMA levels can be accurately and quantitatively determined by flow cytometry.     

Functional properties of proteins immobilized on albumin microspheres

Bovine serum albumin (BSA) microspheres with an average diameter of 12.5 micron were prepared by crosslinking of BSA molecules with glutaraldehyde in the presence of polymethylmethacrylate dissolved in chloroform-toluene. Trypsin and anti-human IgG antibody were immobilized onto their surfaces by the glutaraldehyde-activation method. The catalytic activity and storage stability of the immobilized trypsin were satisfactorily high. The enzyme immunoassay (EIA) method using BSA-microspheres as a solid phase has a high sensitivity (the minimum concentration of detectable antigen in the sample: 0.2 ng/ml) and a wide concentration range (final concentration 0.027-3000 ng/ml) for the detection of human IgG.     

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.     

Determination of cefaclor in human plasma by a sensitive and specific liquid chromatographic-tandem mass spectrometric method

A sensitive and specific liquid chromatographic-tandem mass spectrometric method is described for the determination of cefaclor in human plasma. The plasma samples were treated by two sample preparation procedures, i.e. protein precipitation (PPT) and solid-phase extraction (SPE). The pretreated samples were analyzed on a C(18) HPLC column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization (ESI) was employed as the ionization source. The analyte and internal standard ampicillin (for PPT) or cefetamet (for SPE) were detected by use of selected reaction monitoring (SRM) mode. The lower limit of quantitation obtained as a result of the PPT procedure was 100 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 12% for cefaclor. The accuracy as determined from QC samples was within +/-3% for the analyte. The SPE procedure could provide the lower limit of quantitation of 2 ng/ml. The precision and accuracy were measured to be below 7.1% and between -3.6% and 1.1%, respectively, for all QC samples. The method was applied for the evaluation of the pharmacokinetic profiles of cefaclor sustained-release formulation.     

Validation of ELISA for the determination of anti-ricin immunoglobulin G concentration in mouse sera.

An enzyme-linked immunosorbent assay (ELISA) for the determination of anti-ricin immunoglobulin G (IgG) concentration in mouse sera was systematically validated. The results obtained throughout the validation process strongly demonstrated that the ELISA was reliable, reproducible, and suitable for its intended use. The assay had a high level of precision within and between runs, was specific for the anti-ricin IgG, and showed no interference with a number of different serum matrices. The assay exhibited excellent accuracy, linearity, and stability. The mean recovery of four test samples with different known concentrations was 100.9+/-11.3%, 102.7+/-10.8%, 99.0+/-7.2%, and 95.9+/-11.3%, respectively (n=10). The mean recovery of the observed anti-ricin IgG concentration of three quality control samples run on 73 plates to their nominal concentrations was 100.1+/-7.3%, 100.2+/-5.8%, and 103.7+/-8.1%; and the coefficient of variation (CV) was 7.3%, 5.8%, and 7.8%, respectively. The back-calculated anti-ricin IgG concentration, %CV, and relative error of seven standards from the calibration curves run in the entire validation study were analyzed (n=7 x 73). The results indicated that the four-parameter logistic (4PL) equation, y=(a-d)/(1+(x/c)b)+d, provided an accurate representation of a sigmoidal relationship between the measured response and the logarithm of observed concentration of anti-ricin IgG in mouse sera for this ELISA. The lower limit of quantification and upper limit of quantification of the calibration curve were 3.3 ng/ml and 82.8 ng/ml, respectively. The measurable range of the assay would cover all possible anti-ricin IgG concentrations in mouse sera stimulated with a ricin vaccine candidate, when the test sera are measured at a 1:800 starting dilution followed by four additional fourfold serial dilutions.     

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

Improvement and validation of a liquid chromatographic method for the determination of levosulpiride in human serum and urine

A rapid, selective and highly sensitive reversed-phase high-performance liquid chromatography (HPLC) method was developed for the determination of levosulpiride, 5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy benzamide, in human serum and urine. The method involved the extraction with a dichloromethane followed by back-extraction into 0.025 M sulfuric acid. HPLC analysis was carried out using reversed-phase isocratic elution with a Luna C(18)(2) 5 microm column, a mobile phase of acetonitrile-0.01 M potassium hydrogen phosphate (30:70, v/v, adjusted to pH 8.5 with triethylamine), and a fluorescence detector with excitation at 300 nm and emission at 365 nm. The chromatograms showed good resolution and sensitivity and no interference of human serum and urine. The calibration curves were linear over the concentration range 0.25-200 ng/ml for serum and 0.2-20 microg/ml for urine with correlation coefficients greater than 0.997. Intra- and inter-day assay precision and accuracy fulfilled the international requirements. The mean absolute recovery for human serum was 89.8+/-3.7%. The lower limits of quantitation in human serum and urine were 0.25 ng/ml and 0.2 microg/ml, respectively, which were sensitive enough for pharmacokinetic studies. Stability studies showed that levosulpiride in human serum and urine was stable during storage, or during the assay procedure. This method was successfully applied to the study of pharmacokinetics of levosulpiride in human volunteers following a single oral administration of levosulpiride (25 mg) tablet.     

Measurement of endogenous uracil and dihydrouracil in plasma and urine of normal subjects by liquid chromatography-tandem mass spectrometry

A sensitive and specific HPLC-MS-MS method was developed for the determination of endogenous uracil (Ura) and its metabolite dihydrouracil (UH2) in human plasma and urine samples. Plasma samples were extracted with ethyl acetate-isopropanol (85:15, v/v) following added ammonium sulfate, and then separated on a Discovery Amide C16 column with 3% methanol solution as the mobile phase; urine samples were just centrifuged at 2500 g for detection. Quantitation was carried out by LC-MS-MS in the multiple reaction monitoring (MRM) mode. The limits of quantitation of the method for Ura and UH2 were 0.5 and 5 ng ml(-1) (for plasma), and 50 and 100 ng ml(-1) (for urine), respectively. This method can be useful to evaluate the activity of dihydropyrimidine dehydrogenase (DPD), a rate-limiting enzyme of the chemotherapy drug fluoropyrimidine, which will be helpful in investigating subject variation of DPD and adjusting clinical dosage in pyrimidine chemotherapy.     

Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.     

Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection

A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Simultaneous enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma by liquid chromatography

A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C(2) column in tandem with a chiral alpha(1)-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation <13%, accuracy <+/-20%). The method was validated for human plasma in the concentration range 10-2000 ng/ml for each of the six compounds. The method allows for the simultaneous characterisation of the metabolic capacity of two human drug-metabolising enzymes, CYP2C19 and CYP2B6, and may be used when investigating polymorphisms or changes in activity of these two enzymes.     

大鼠淋巴细胞培养上清液总IgG 含量ELISA 检测方法的建立

目的:建立一种定量测定大鼠淋巴细胞培养上清液中总IgG(免疫球蛋白G)含量的双抗体夹心ELISA(酶联免疫吸附试验)检测方法。方法:用方阵实验确定包被抗体、检测抗体的最佳工作浓度;绘制标准曲线,确定线性范围;评价标准曲线的可重复性、精密度和可应用性。结果:包被抗体和检测抗体的最佳效价分别为2μg/ml和1:4000稀释;检测的线性范围为0.25-16ng/ml。经方法学评价,可重复性和精密度较高,应用性较强。结论:该方法灵敏度高,重复性好,可作为科研过程中检测大鼠淋巴细胞培养上清液总IgG含量的一种精确、方便、可靠的方法。     

Simultaneous determination of glipizide and rosiglitazone unbound drug concentrations in plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry

Glipizide and rosiglitazone are widely used to treat Type 2 diabetes. In order to investigate drug-drug protein binding interaction between glipizide and rosiglitazone, a method was developed and validated for simultaneously determining the free (unbound) fraction of glipizide and rosiglitazone in plasma employing equilibrium dialysis for the separation of free drug and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitation. Post-dialysis human plasma or buffer samples of 0.2 ml were extracted using a liquid-liquid extraction procedure and analyzed by a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Zorbax SB-Phenyl column, ionized using an atmospheric pressure electrospray ionization source and analyzed in positive ion mode with multiple reaction monitoring. The ion transitions monitored were m/z 446-->321 for glipizide, m/z 358-->135 for rosiglitazone, and m/z 271-->155 for tolbutamide (internal standard, IS). The chromatographic run time was 5 min per injection, with retention times of 2.3, 3.4 and 2.3 min for glipizide, rosiglitazone and IS, respectively. The calibration curves of glipizide and rosiglitazone were over the range of 1-2000 ng/ml (r(2)>0.9969) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v). The inter-assay precision and accuracy of the quality control samples were <10.9% of coefficient of variability and >93.5% and 94.5% of nominal concentration for glipizide and rosiglitazone, respectively. The lower limit of quantitation of both glipizide and rosiglitazone was 1.0 ng/ml. Both glipizide and rosiglitazone bound to plasma protein extensively (>99% bound). Glipizide and rosiglitazone free fraction averaged 0.678+/-0.071 and 0.389+/-0.061%, respectively, at plasma concentration of 1000 ng/ml. This developed method proves reproducible and sensitive and its application to clinical samples is also reported.     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.