Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC

This paper deals with the chiral separation of triiodothyronine (T₃) and thyroxine (T₄) by HPLC and micro-HPLC. The separation of T₃ and T₄ is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic ™®) phase was used.

In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T₃ and T₄. l-4-Hydroxyproline was bonded to 3 μm and the glycopeptide antibiotics were bonded to 3.5 μm silica gel and separations were accomplished by microbore HPLC columns (10 cm × 1 mm I.D.). With both techniques and all chiral selectors investigated T₃ and T₄ were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.     

Optimized HPLC method for tramadol and O-desmethyl tramadol determination in human plasma

The optimized method for HPLC determination of tramadol and its metabolite O-desmethyl tramadol in human plasma using sotalol as internal standard has been developed and validated by a new approach. The determination by fluorescence detection was performed on re-eluted solution, obtained after liquid–liquid extraction with ethyl acetate of the three analytes from plasma. The chromatographic separation of tramadol under a gradient elution was achieved at a temperature of 15 °C with a RP-18 column, guarded by a C18 precolumn. The mobile phase was a mixed aqueous solution containing ortho-phosphoric acid, triethylamine, acetonitrile and methanol in a complex gradient mode. The quantitative determination of tramadol was performed at different successive pairs of excitation/emission wavelengths (200/300 nm, 200/295 nm, 212/305 nm) with lower limits of quantification: LLOQ = 4.078 ng/ml for tramadol, respectively LLOQ = 3.271 ng/ml for O-desmethyl tramadol. For the LLOQ limits, were calculated the values of the coefficient of variation and difference between mean and the nominal concentration. For tramadol analyte they were CV% = 5.147% and bias% = − 7.273% in the intra-days and CV% = 4.894% and bias% = 0.836% in the between-days assay, respectively for the metabolite O-desmethyl tramadol they were CV% = 11.517% and bias% = 0.337% in the intra-days and CV% = 6.41% and bias% = 3.259% in the between-days assay.

In addition, the stabilities of the analytes were verified in different conditions. Both, tramadol and its metabolite proved to be stable in plasma for four weeks, frozen at − 20 °C, but also for 48 h at 15 °C in the re-eluted solution after liquid–liquid extraction.     

Quantification of some active compounds in air samples at pharmaceutical workplaces by HPLC

Workers involved in the manufacture of drug substances may be exposed to active pharmaceuticals by inhalation of drug dusts or droplets which has been considered the main exposure route. The proposed HPLC method allowed to determine sulpiryde, hydroxyurea and dyprophylline in the concentration range of 0.01–0.187 mg/m³, 0.001–0.08 mg/m³ and 0.01–0.40 mg/m³ for sulpiryde, hydroxyurea and dyprophylline, respectively, when 480 L of air sample was collected on the glass fibre filters. Sulpiryde was extracted with a solvent system consisting of acetonitrile–phosphate buffer at pH 3 (85:15, v/v), while the best efficiency of extraction for hydroxyurea and dyprophylline was achieved using water. HPLC analysis of sulpiryde with fluorescence detection was more sensitive (LOD = 3.1 μg/L) in comparison with UV detection (LOD = 84.4 μg/L).     

A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems

Bacterial β-ketoacyl-ACP reductase (FabG) and the β-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its β-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The K_m and k_cat values of FabG obtained for EAA were 127 mM and 0.30 s^− 1, while those of this enzyme for NADPH were 10.0 μM and 0.59 s^− 1, respectively. The corresponding K_m and k_cat values of FAS were 126 mM and 4.63 s^− 1 for EAA; 8.7 μM and 4.09 s^− 1 for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.     

Determination of zinc in vegetal tissue microsamples by platinum-wire loop in flame atomization atomic absorption spectrometry

The zinc content of 3 μL of vegetal samples (tree leaves, lichens and grape sap) atomized from a Pt-wire in the methane–air flame has been determined by atomic absorption spectrometry. The effect of gas flow rates and the atomization height in the flame on the absorption of zinc was evaluated at 213.9 nm. The best results were obtained at a height of 5 mm and gas flow rates of 200 L/h air and 26 L/h methane, respectively. The effect of Na, K, Ca, Mg, SO₄²⁻, and PO₄³⁻ on the absorption of zinc was studied too. The detection limit of 0.40 ± 0.21 ng was obtained at a significance level of 0.05, using the two-step Neyman–Pearson criterion. The zinc content of the samples has been determined with continuous nebulization and by atomization from the Pt-wire, using both the standard calibration curve and the standard addition method. The results of the two procedures agree within the determination errors.     

High-performance liquid chromatographic methods for the determination of a new carbapenem antibiotic, L-749,345, in human plasma and urine

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of a new carbapenem antibiotic assay using ultraviolet detection has been developed for a new carbapenem antibiotic L-749,345 in human plasma and urine. A plasma sample is centrifuged and then injected onto an extraction column using 25 mM phosphate buffer, pH 6.5. After 3 min, using a column-switching valve, the analyte is back-flushed with 10.5% methanol–phosphate buffer for 3 min onto a Hypersil 5 μm C₁₈ BDS 100×4.6 mm analytical column and then detected by absorbance at 300 nm. The sample preparation and HPLC conditions for the urine assay are similar, except for a longer analytical column 150×4.6 mm. The plasma assay is specific and linear from 0.125 to 50 μg/ml; the urine assay is linear from 1.25 to 100 μg/ml.     

Role of dexamethasone on vasopressin release during endotoxemic shock

The present study was designed to assess the hypothesis that dexamethasone (DEX) through the control of nitric oxide (NO) synthesis could regulate the release of vasopressin (AVP), which plays an important role in the regulation of arterial pressure and plasma osmolality. Endotoxemic shock was induced by intravenous (i.v.) injection of 1.5 mg/kg lipopolisaccharide (LPS) in male Wistar rats weighing 250–300 g. After LPS administration, a group of animals were treated with DEX (1.0 mg/kg of body weight), whereas saline-injected rats served as controls. The LPS administration induced a significant decrease in mean arterial pressure (MAP) with a concomitant increase in heart rate (HR) (ΔVMAP: − 16.1 ± 4.2 mm Hg; ΔVHR: 47.3 ± 8.1 bpm). An increase in plasma AVP concentration occurred and was present for 2 h after LPS administration (11.1 ± 0.9 pg/mL) returning close to basal levels thereafter and remaining unchanged until the end of the experiment. When LPS was combined with i.v. administration of a low dose of DEX, we observed an attenuation in the drop of MAP (ΔVMAP: − 2.2 ± 1.9 mm Hg) and a decrease in NO plasma concentration [NO] after LPS administration (1098.1 ± 68.1 µM) compared to [NO] after DEX administration (523.4 ± 75.2 µM). However, this attenuation in the drop of MAP was accompanied by a decrease in AVP plasma concentration (3.7 ± 0.4 pg/mL). These data suggest that AVP does not participate in the recovery of MAP when DEX is administered in this endotoxemic shock model.     

Participation of the inducible nitric oxide synthase on atrial natriuretic peptide plasma concentration during endotoxemic shock

Atrial natriuretic peptide (ANP) is a hormone secreted in response to atrial or ventricular volume expansion and pressure overload, respectively. However, it has been found in studies with animals and patients an increase in ANP plasma concentration, during advanced septic shock, despite the fall in mean arterial pressure (MAP).

Several studies support the hypothesis that NO may be involved in the regulation of ANP release. Since NO may have an effect on ANP release, we hypothesized that NO pathway may participate in the control of the ANP release induced by the endotoxemic shock. Thus, the purpose of the present study was to assess the effect of the intravenous (i.v.) and intracereboventricular (i.c.v.) administration of aminoguanidine, an iNOS blocker, on plasma ANP levels and MAP during experimental endotoxemic shock.

Experiments were performed on adult male Wistar rats weighing 180–240 g. Rats were injected i.v. by bolus injection with 1.5 mg/kg of Lipopolysaccharide (LPS) or saline (0.5 mL) and were decapitated 2, 4 and 6 h after LPS injection for ANP determination by radioimmunoassay. In a separate set of experiments, rats received intravenous (i.v.) (100 mg/kg) or intracerebroventricular (i.c.v.) (250 μg in a final volume of 2 μL) injection of aminoguanidine (AG). Thirty minutes after the i.c.v. or i.v. injections, animals received LPS and were decapitated 2, 4 and 6 h later to determine plasma ANP concentration. In the two set of experiments MAP and heart rate (HR) were measured each 15 min for a period of 6 h using a polygraph.

When animals were injected with LPS, a reduction (p < 0.01) in MPA and an increase in HR occurred. A significant increase in plasma ANP concentration occurred, coinciding with the period of drop in blood pressure.

We found a significant increase in plasma ANP concentration after AG plus LPS injection, when compared to the rats treated with LPS plus saline. Further, the administration of AG plus LPS attenuated the decrease in the MAP after LPS and attenuated the increase in the HR when compared to the rats treated with LPS plus saline.

Our study suggests that inducible NOS pathway may activate an inhibitory control mechanism that attenuates ANP secretion, which is not regulated by the changes in blood pressure.     

Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1-0.6%) developed salivary H₂O₂ with C_max = 2.9-9.6 μM and AUC_0 → ∞ = 8.5-285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_0 → ∞ = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases.

After the synthesis of the 5′-carboxymethoxy derivative of enterolactone and 4′-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively.

We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 μmol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography–mass spectrometry (GC–MS). However, the assay results by the present method showed strong correlation with those obtained by GC–MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.     

Low frequency and low intensity pulsed electromagnetic field exerts its antiinflammatory effect through restoration of plasma membrane calcium ATPase activity

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting 1% of the population worldwide. Pulsed electromagnetic field (PEMF) has a number of well-documented physiological effects on cells and tissues including antiinflammatory effect. This study aims to explore the antiinflammatory effect of PEMF and its possible mechanism of action in amelioration of adjuvant induced arthritis (AIA). Arthritis was induced by a single intradermal injection of heat killed Mycobacterium tuberculosis at a concentration of 500 μg in 0.1 ml of paraffin oil into the right hind paw of rats. The arthritic animals showed a biphasic response regarding changes in the paw edema volume. During the chronic phase of the disease, arthritic animals showed an elevated level of lipid peroxides and depletion of antioxidant enzymes with significant radiological and histological changes. Besides, plasma membrane Ca²⁺ ATPase (PMCA) activity was inhibited while intracellular Ca²⁺ level as well as prostaglandin E₂ levels was noticed to be elevated in blood lymphocytes of arthritic rats. Exposure of arthritic rats to PEMF at 5 Hz × 4 μT × 90 min, produced significant antiexudative effect resulting in the restoration of the altered parameters. The antiinflammatory effect could be partially mediated through the stabilizing action of PEMF on membranes as reflected by the restoration of PMCA and intracellular Ca²⁺ levels in blood lymphocytes subsequently inhibiting PGE₂ biosynthesis. The results of this study indicated that PEMF could be developed as a potential therapy for RA in human beings.     

Enzyme-linked immunosorbent assay for 4-hydroxynonenal-histidine conjugates

Highly reactive aldehyde 4-hydroxynonenal (HNE) is the final product of lipid peroxidation, known as a second messenger of free radicals and a signaling molecule. It forms protein conjugates involved in pathology of various diseases. To determine cellular HNE-protein conjugates we developed indirect ELISA based on well-known, monoclonal antibody against HNE-histidine (HNE-His) adducts. The method was calibrated using HNE-albumin conjugates as standards (R² = 0.999) and validated on human osteosarcoma cell cultures (HOS). The ELISA showed good sensitivity (8.1 pmol HNE-His/mg of protein), precision ( ± 8% intra-assay and ± 12% inter-assay) and spiking recovery ( ± 9%). The assay revealed 60-fold increase of cellular HNE-His adducts upon copper-induced lipid peroxidation of HOS. The ELISA matched HNE-immunocytochemistry of HNE-treated HOS cells and quantified the increase of cellular HNE-His conjugates in parallel to the decrease of free HNE in culture medium. The ELISA was developed as ELISA Stress for severe lipid peroxidation and ELISA Fine for studies on HNE physiology.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Analysis of Ecteinascidin 743, a new potent marine-derived anticancer drug, in human plasma by high-performance liquid chromatography in combination with solid-phase extraction

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the quantification of the novel anticancer drug Ecteinascidin 743 in human plasma. The sample pretreatment of the plasma samples involved a solid-phase extraction (SPE) on cyano columns. Propyl-p-hydroxybenzoate was added after the sample pretreatment to correct for variability in injection volumes. The separation was performed on a Zorbax SB-C₁₈ column (75×4.6 mm I.D., particle size 3.5 μm) with acetonitrile–25 mM phosphate buffer, pH 5.0 (70:30, v/v) as the mobile phase. The flow-rate was 1.0 ml/min and the eluent was monitored at 210 nm. The accuracies and precisions of the assay fall within ±15% for all quality control samples and within ±20% for the lower limit of quantitation, which was 1.0 ng/ml using 500 μl of plasma. The overall recovery of the sample pretreatment procedure for Ecteinascidin 743 was 87.0±5.9%. The drug was found to be stable in human plasma at −30°C for at least 2 months. At room temperature Ecteinascidin 743 was stable in human plasma for 5 h at most.     

Photosensitizing action of isomeric zinc N-methylpyridylporphyrins in human carcinoma cells

The success of photodynamic therapy (PDT), as a minimally invasive approach, in treating both neoplastic and non-neoplastic diseases has stimulated the search for new compounds with potential application in PDT. We have previously reported that Zn(II) N-alkylpyridylporphyrins (ZnTM-2(3,4)-PyP⁴⁺ and ZnTE-2-PyP⁴⁺) can act as photosensitizers and kill antibiotic-resistant bacteria. This study investigated the photosensitizing effects of the isomers of ZnTMPyP⁴⁺ (ZnTM-2(3,4)-PyP⁴⁺) and respective ligands on a human colon adenocarcinoma cell line. At 10 μM and 30 min of illumination the isomeric porphyrins completely inhibited cell growth, and at 20 μM killed approximately 50% of the cancer cells. All these effects were entirely light-dependent. The isomers of the ZnTMPyP⁴⁺ and the respective ligands show high photosensitizing efficiency and no toxicity in the dark. Their efficacy as photosensitizers is comparable to that of hematoporphyrin derivative (HpD).     

Simple and sensitive method for determination of metronidazole in human serum by high-performance liquid chromatography

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml⁻¹ using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile–aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 μg ml⁻¹. Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 μg ml⁻¹ and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.     

The alternating current polarographic behavior and determination of lansoprazole and omeprazole in dosage forms and biological fluids

The alternating current (AC_t) polarographic behavior of lansoprazole (LNS) and omeprazole (OMP) was studied in Britton Robinson buffers (BRb) over the pH range 4.1–11.5. In BRb of pH 9.6 and 10.5, well-defined AC_t peaks were obtained for both LNS and OMP, respectively. The current–concentration plots were rectilinear over the ranges of 0.4–20 µg mL^− 1 and 0.2–10 µg mL^− 1 for LNS and OMP respectively. The minimum detection limits (S/N = 2) were 0.02 µg mL^− 1 (5.4 × 10^− 8 M) and 0.01 µg mL^− 1 (2.9 × 10^− 8 M) for LNS and OMP, respectively. The proposed method was successfully applied to the analysis of the two drugs in their commercial capsules. The average percent recoveries were favorably compared to those obtained by reference methods. Co-administered drugs such as naproxen and methotrexate did not interfere with the proposed method. The proposed method was further extended to the in-vitro determination of the lansoprazole in spiked plasma, the percentage recoveries was 98.47 ± 1.29 (n = 4). The pathway for the electrode reaction for both drugs involved reduction of the sulphonyl group into the corresponding thiol group at the Dropping Mercury Electrode. The advantages of the method were time saving and more sensitive than the other published voltammetric method. Yet The present study is the first report on the use of alterating current polarography (AC_t) in this respect.     

Ion-Pair Chromatography for Simultaneous Analysis of Ethionamide and Pyrazinamide from Their Porous Microparticles

Ethionamide (ETA) and pyrazinamide (PZA) are considered the drugs of choice for the treatment of multidrug-resistant tuberculosis. Current methods available in the literature for simultaneous determination of ETA and PZA have low sensitivity or involve column modifications with lipophilic cations. The aim of this study was to develop a simple and validated reversed-phase ion-pair HPLC method for simultaneous determination of ETA and PZA for the characterization of polymeric-based porous inhalable microparticles in in vitro and spiked human serum samples. Chromatographic separation was achieved on a Phenomenex C₁₈ column (250 mm × 4.6 mm) using a Shimadzu LC 10 series HPLC. The mobile phase consisted of A: 0.01% trifluoroacetic acid in distilled water and B: ACN/MeOH at 1:1 v/v. Gradient elution was run at a flow rate of 1.5 mL/min and a fixed UV wavelength of 280 nm. The validation characteristics included accuracy, precision, linearity, analytical range, and specificity. Calibration curves at seven levels for ETA and PZA were linear in the analytical range of 0.1–3.0 μg/mL with correlation coefficient of r² > 0.999. Accuracy for both ETA and PZA ranged from 94 to 106% at all quality control (QC) standards. The method was precise with relative standard deviation less than 2% at all QC levels. Limits of quantitation for ETA and PZA were 50 and 70 ng/mL, respectively. There was no interference from either the polymeric matrix ions or the biological matrix in the analysis of ETA and PZA.Key words: ethionamide, HPLC, microparticles, pyrazinamide, tuberculosis     

Phosphoinositide hydrolysis increase by angiotensin-(1--7) in neonatal rat brain

Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [³H]myoinositol, incubated with additions during 30 min and later [³H]inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala⁷]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT₁) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala⁷]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT₂) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT₂ receptors.     

Development of a sensitive and quantitative HPLC-FLD method for the determination of obestatin in human plasma

Obestatin is a gastrointestinal system peptide. The quantification of this peptide is conventionally performed using immunological techniques. In this study, a selective and sensitive HPLC method coupled with fluorescence detection for the quantitation of obestatin in human plasma was developed and validated. The separation was obtained on a C₁₈ (4.6 × 100 mm, 3.5-μm particles) column using a mobile phase composed of acetonitrile and water, both including 0.1% trifluoroacetic acid. The developed method was found to be linear in the concentration range of 20 to 1000 ng/mL, with a coefficient of determination of 0.9982. The precision results were less than 10%, and the accuracy results were between 92% and 107%. The detection and quantification limit values were obtained as 2.8 and 9.4 ng/mL, respectively. Analyte solutions were found stable for 24 h at room temperature, three freeze–thaw cycles, and 2 weeks at −20°C. The developed method was successfully used for the quantification of obestatin in human plasma samples. In conclusion, the developed method is sensitive and specific for measuring the plasma concentrations of obestatin.