Fast High-performance Liquid Chromatography Method Research Articles

Development and validation of an HPLC method for determination of quercetin

This study validated a high performance liquid chromatography (HPLC) method for the determination of quercetin (QE) in microemulsion (ME) based gel formulations. The analyses were performed on a C18 column (150x4.6 mm, 5 μm particle size) at room temperature with UV detection at 254 nm. The mobile phase was composed of methanol:water (65:35, v/v, 2% acetic acid) mixture, and flow rate was set to 1 mL/min. The method was validated according to the international guidelines with respect to linearity range, stability, limit of quantitation and detection, precision and accuracy. The method was linear within 5 – 100 μg/mL range with a correlation coefficient of 0.9995. The intra- and inter-assay precisions presented RSD values lower than 2%. The method reported is a fast and reliable HPLC method useful for QE determination in ME based gel formulations.

A Vancomycin HPLC Assay for Use in Gut Microbiome Research.

ABSTRACTThe human microbiome project has revolutionized our understanding of the interaction between commensal microbes and human health. By far, the biggest perturbation of the microbiome involves use of broad-spectrum antibiotics excreted in the gut. Thus, pharmacodynamics of microbiome changes in relation to drug exposure pharmacokinetics is an emerging field. However, reproducibility studies are necessary to develop the field. A simple and fast high-performance liquid chromatography-photodiode array detector (HPLC) method was validated for quantitative fecal vancomycin analysis. Reproducibility of results were tested based on sample storage time, homogeneity of antibiotic within stool, and concentration consistency after lyophilization. The HPLC method enabled the complete elution of vancomycin within ~4.2 min on the reversed-phase C18 column under the isocratic elution mode, with excellent recovery (85% to 110%) over a 4-log, quantitative range (0.4–100 μg/mL). Relative standard derivations (RSD) of intra-day and inter-day results ranged from 0.4% to 5.4%. Using sample stool aliquots of various weights consistently demonstrated similar vancomycin concentrations (mean RSD: 6%; range: 2–16%). After correcting for water concentrations, vancomycin concentrations obtained after lyophilization were similar to the concentrations obtained from the original samples (RSD less than 10%). These methodologies establish sample condition standards for a quantitative HPLC to enable vancomycin pharmacokinetic studies with the human microbiome.IMPORTANCE Research on antibiotic effect on the gut microbiome is an emerging field with standardization of research methods needed. In this study, a simple and fast high-performance liquid chromatography method was validated for quantitative fecal vancomycin analysis. Reproducibility of results were tested to standardize storage time, homogeneity of antibiotic within stool, and concentration consistency after lyophilization. These methodologies establish sample condition standards for a quantitative HPLC to enable vancomycin pharmacokinetic studies with the human microbiome.

Simultaneous determination of six antibiotics in human serum by high-performance liquid chromatography with UV detection.

Antibiotics are widely used in intensive care patients to treat severe infections. To avoid bacterial resistance or toxic side effects, the determination of serum concentration of ABs is advisable. Therefore, in this study, we developed and validated a simple and fast high-performance liquid chromatography method with UV detection for the simultaneous determination of four β-lactam ABs (meropenem, imipenem, ceftazidime, and piperacillin) and two coadministered substances (cilastatin and tazobactam) in human serum. Sample preparation required a simple protein precipitation by methanol. The separation of the ABs occurred within a timeframe of 17 min. For this purpose, we used a Kinetex F5 column with a linear gradient of acetonitrile and phosphate buffer (pH 6.9). The UV detector recorded two separate chromatograms at 220 and 295 nm simultaneously. Validation has demonstrated that the method is linear, accurate, and precise within the clinically relevant range for each substance.

A new RP-HPLC method as an auxiliary tool for optimization of sample preparation procedures for tracing of PPCPs of different hydrophilicities.

Recently, pharmaceutical and personal care products (PPCPs) have received considerable attention because of their increasing use. Analysis of PPCPs presents a significant analytical challenge, with high-performance liquid chromatography (HPLC) in reversed-phase mode, as the most widely used analytical technique. To facilitate the optimization of the procedures that are applied in the early stages of sample preparation, a simple and fast HPLC method is proposed in this work for the separation of some PPCPs with a wide range of hydrophilicity. Two columns were evaluated (Atlantis dC18 and Discovery HS F5); as for mobile phases: a formate buffer (40 mmol L-1, pH 4) and methanol were tested in a gradient mode. The fluorinated column allowed better separation in a shorter time and better resolution for all analytes (Rs > 1). The proposed method delivered good performance for the tracing of PPCPs and is a suitable alternative to traditional C18-based HPLC methods.

1541. A Novel and Fast Liquid Chromatography Method for Determination of Fluoroquinolones in Human Plasma

BackgroundFluoroquinolones (FQs) are frequently used antimicrobial agents. Considering that the FQs exhibit concentration-dependent bactericidal activity, concentrations of FQs in the biological fluids must be monitored to ensure treatment success. The literature search revealed that there is no method for the determination of levofloxacin (LEV), ciprofloxacin (CIP), moxifloxacin (MOX), and gemifloxacin (GEM) in plasma up to date. Consequently, the aim of this study was to develop and validate a new high-performance liquid chromatography (HPLC) method for determination of these FQs in plasma and evaluate effects of concomitant drugs on plasma FQ concentrations of patients.MethodsBlank plasma samples spiked with FQs were employed for method validation studies. Validation studies were conducted in accordance with the recommendations of the US FDA. In order to demonstrate feasibility of method, 5 patients with polypharmacy, receiving orally CIP, LEV, or MOX as part of their treatment were included in the study. Blood samples were collected at two different times, just before and 2 hours after the second drug administration.ResultsThe separation of FQs was accomplished within 7.5 minutes. The method was linear in the range of 0.1–10 µg/mL with the correlation coefficient >0.99. The RSD at four concentration levels (0.1, 0.3, 4, and 8 µg/mL) was less than 7% with accuracy in the range of 91.8–111.9%.The method was applied to the determination of CIP, LEV, and MOX levels in plasma samples of 5 patients with polypharmacy. Determined CIP and LEV levels were in accordance with literature. On the other hand, MOX concentration 2 hours after administration in plasma of one patient was found to be 6.1 ± 0.1 µg/mL which was higher than previously reported maximum plasma concentration of MOX (4.5 µg/mL). The patient had hypoalbuminemia and MOX is approximately 50% bound to serum proteins. Due to low level of albumin, the level of free MOX in plasma may be increased.ConclusionA simple, fast, and reliable HPLC method was developed and validated for the determination of LEV, CIP, MOX, and GEM in plasma. It is suitable therapeutic drug monitoring of these FQs and can be applied to other pharmacokinetic and toxicological studies.Disclosures All authors: No reported disclosures.

Simple and rapid validated HPLC-fluorescence determination of perampanel in the plasma of patients with epilepsy

We present a simple and fast high-performance liquid chromatography method with fluorescence detection for the determination of the antiepileptic drug perampanel in human plasma. The chromatographic separation was performed on a Kinetex PFP (100 × 2.6 mm, 4.6 µm) column, using a mobile phase of sodium acetate 0.03 M pH 3.7 and acetonitrile (40/60, v/v), at a flow rate of 0.8 mL/min. Total chromatography time for each run was 5 min. Sample preparation (250 µL) involved only one simple precipitation step by acetonitrile spiked with mirtazapine as internal standard. The method was validated over a concentration range of 20–1000 ng/mL and successfully applied to measure perampanel concentrations in plasma samples obtained from patients with epilepsy. This assay combines the high specificity of fluorescence detection with a very simple and fast sample pretreatment and can offer real advantages over existing methods in terms of simplicity and transferability to a therapeutic drug monitoring setting.

Stability-Indicating HPLC-UV Method for Vitamin D3 Determination in Solutions, Nutritional Supplements and Pharmaceuticals.

A simple and fast high-performance liquid chromatography method with UV detection for determination of vitamin D3 in stability studies as well as in solutions, nutritional supplements and pharmaceuticals was developed. Successful separation of vitamin D3 from its degradation products was achieved on a Gemini C18 100 × 3.0 mm column using a mixture of acetonitrile and water (99:1, v/v) as а mobile phase. The method was successfully validated according to the ICH guidelines. The described reversed-phase HPLC method is favorable compared with other published HPLC-UV methods because of its stability-indicating nature, short run time (3.3 min) and wide analytical range with outstanding linearity, accuracy and precision. The method was further applied for quantification of vitamin D3 in selected liquid and solid nutritional supplements and prescription medicines, confirming its suitability for routine analysis. Degradation products, formed under stress conditions (hydrolysis, oxidation, photolysis and thermal degradation), were additionally elucidated by suitable equipment (LC-DAD-MS) to confirm the stability-indicating nature of the developed method.

Mechanistic Approaches of PHE and PPF Columns for Separation of Rasberry Ketone and Caffeine

High-Performance Liquid Chromatography (HPLC) separation mechanisms of raspberry ketone and caffeine on phenyl columns (PhE and PPF) are described. The π–π interactions played major role in separation phenomenon. PhE column resulted into better separation than PPF due poor π–π interactions in later column. Fast, reproducible, selective, and effective HPLC method was developed for analyses of ketone and caffeine in pharmaceutical preparation. The columns used were Sunniest PhE (phenyl) (250 × 4.6 mm, 5.0 µm) and Sunshell PFP (100 × 4.6 mm, 2.6 µm) of Chromanik Japan. The mobile phase used was water–acetonitrile (60:40, v/v, at different pH values) at 1.0 mL/min flow rate. The detection was at 280 nm with 27 ± 1°C as working temperature. The ranges of capacity (k), separation (α), and resolution (Rs) factors at different pH values on both columns were 2.60–5.10, 1.32–1.60, and 3.22–4.28, respectively. The values of limit of detection (LOD) and limit of quantitation (LOQ) of raspberry ketone and caffeine on both columns ranged from 5.0 to 20.0 and 7.0 to 25.0, respectively. The developed mechanistic aspect is useful for designing separation strategies of other molecules on these phenyl columns. The reported method may be used for quality control in industries and government organizations. Besides, fast analyses may be carried out in various food products.

A rapid and sensitive method for determination of carotenoids in plant tissues by high performance liquid chromatography.

BackgroundThe dietary carotenoids serve as precursor for vitamin A and prevent several chronic-degenerative diseases. The carotenoid profiling is necessary to understand their importance on human health. However, the available high-performance liquid chromatography (HPLC) methods to resolve the major carotenoids require longer analysis times and do not adequately resolve the violaxanthin and neoxanthin.ResultsA fast and sensitive HPLC method was developed using a C30 column at 20°C with a gradient consisting of methanol, methyl-tert-butyl ether and water. A total of 15 major carotenoids, including 14 all-trans forms and one cis form were resolved within 20 min. The method also distinctly resolved violaxanthin and neoxanthin present in green tissues. Additionally this method also resolved geometrical isomers of the carotenoids.ConclusionThe HPLC coupled with C30 column efficiently resolved fifteen carotenoids and their isomers in shorter runtime of 20 min. Application of this method to diverse matrices such as tomato fruits and leaves, Arabidopsis leaves and green pepper fruits showed the versatility and robustness of the method. The method would be useful for high throughput analysis of large number of samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0051-0) contains supplementary material, which is available to authorized users.

Fast HPLC Method for Determination of Fenoxycarb and Permethrin in Antiparasitic Veterinary Shampoo Using Fused-Core Column

A new and fast high-performance liquid chromatography (HPLC) method using technology of fused-core columns for separation of fenoxycarb and cis-, trans-permethrin has been developed and used for their determination in antiparasitic veterinary shampoo. Separation of insecticides and internal standard sudan II was achieved on the fused-core column Ascentis Express RP-Amide (100 × 3.0 mm), particle size 2.7 μm, with mobile phase acetonitrile/water (55:45, v/v) at a flow rate of 1.0 mL min−1 and at temperature 60 °C. The detection wavelength of detector was set at 225 nm for both compounds and internal standard sudan II. Under the optimum chromatographic conditions standard calibration curves were measured with good linearity [r 2 = 0.99991 for fenoxycarb, r 2 = 0.99987 for trans-permethrin, and r 2 = 0.99984 for cis-permethrin (n = 8)]. Commercial samples of antiparasitic veterinary shampoo were extracted with ethanol in ultrasound bath for 5 min. A 2-μL sample volume of the filtered solution was directly injected into the HPLC system. Accuracy of the method defined as a mean recovery of insecticides from shampoo matrix was in the range 100.43–103.85 % for both insecticides.

First HPLC–UV method for rapid and simultaneous quantification of phenobarbital, primidone, phenytoin, carbamazepine, carbamazepine-10,11-epoxide, 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine, lamotrigine, oxcarbazepine and licarbazepine in human plasma

A sensitive and fast high-performance liquid chromatographic method coupled with ultraviolet detection is herein reported for the simultaneous determination of human plasma concentration of six antiepileptic drugs frequently used in clinical practice [phenobarbital (PB), primidone (PRM), phenytoin (PHT), carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC)] and some of their main metabolites, carbamazepine-10,11-epoxide (CBZ-E), 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine (trans-diol) and licarbazepine (Lic). Sample preparation consisted of a deproteinization step with methanol followed by a solid-phase extraction procedure. Chromatographic separation was achieved in approximately 15 min on a reversed-phase C18 column using a mobile phase composed by water-methanol-acetonitrile-triethylamine (68.7:25:6:0.3, v/v/v/v; pH 6.5) pumped isocratically at 1.0 mL/min. The detector was set at 237 nm. Calibration curves were linear with regression coefficients greater than 0.992 over the concentration ranges 0.25-100 μg/mL for PB, 0.4-50 μg/mL for PRM, 0.5-50 μg/mL for PHT, 0.1-50 μg/mL for CBZ, LTG and CBZ-E, 0.1-25 μg/mL for OXC, 0.25-10 μg/mL for trans-diol and 0.15-80 μg/mL for Lic. Inter- and intra-day imprecision did not exceed 12.15% and inaccuracy was within ±14.91%. Absolute mean recoveries ranged from 78.49 to 101.04% and no interferences were observed at the retention times of the analytes and internal standard (ketoprofen). This bioanalytical method was successfully applied to real plasma samples from epileptic patients and it seems to be a suitable tool for routine therapeutic drug monitoring and also to support other clinical pharmacokinetic-based studies.

Development of a Rapid LC/DAD/FLD/MSn Method for the Simultaneous Determination of Monacolins and Citrinin in Red Fermented Rice Products

Red fermented rice is used worldwide by many patients as an alternative therapy for hyperlipidemia; however, the discovery of a toxic fermentation byproduct, citrinin, causes much controversy about the safety of red mold rice products. A new and fast high-performance liquid chromatography method was developed and validated for simultaneous determination of cholesterol-lowering compounds monacolin K (lovastatin), monacolin K hydroxy acid, and other monacolins present in red fermented rice as well as nephrotoxic mycotoxin citrinin in a single run using connected diode array and fluorescence and mass spectrometric detectors. The proposed method was successfully applied for the analysis of red fermented rice food samples and various dietary supplements also containing other natural lipid-lowering agents. The deviations between label content and levels of active compounds found in investigated samples as well as high batch-to-batch variation found in one product indicate that the regular quality control of red fermented rice products is of great importance.

A New and Fast HPLC Method for Determination of Rutin, Troxerutin, Diosmin and Hesperidin in Food Supplements Using Fused-Core Column Technology

A new and fast high-performance liquid chromatography (HPLC) method using fused-core column for separation of rutin, troxerutin, diosmin, and hesperidin has been developed and used for determination of these flavonoids in food supplements. Efficient separation of flavonoids and internal standard methylparaben was achieved on the fused-core column Ascentis Express RP-Amide (100 × 3.0 mm), particle size 2.7 μm, with mobile phase acetonitrile/water solution of acetic acid pH 3 (30:70, v/v) at a flow rate of 1.0 mL min−1 and at temperature 50 °C. The detection wavelengths were set at 283 nm for hesperidin and at 255 nm for rutin, troxerutin, diosmin, and internal standard methylparaben. Under the optimal chromatographic conditions, good linearity with correlation coefficients in the range (r = 0.9991–0.9998; n = 7) for all flavonoids was achieved. Commercial samples of food supplements were extracted with 100 % dimethyl sulfoxide using ultrasound bath for 10 min and then diluted to methanol. A 5-μL sample volume of the filtered solution was directly injected into the HPLC system. Accuracy of the method defined as a mean recovery of flavonoids from food supplement matrix was in the range 96.2–104.4 % for all flavonoids. The intraday method precision was satisfactory, and relative standard deviations of sample analysis including preparation and determination of different food supplements were in the range 0.5–3.5 % for all flavonoids. The developed method has shown high sample throughput during sample preparation process, modern separation approach, and short time (5 min) of analysis.

Development and validation of a reversed phase liquid chromatographic method for analysis of oxytetracycline and related impurities

A simple, robust and fast high-performance liquid chromatographic method is described for the analysis of oxytetracycline and its related impurities. The principal peak and impurities are all baseline separated in 20min using an Inertsil C8 (150mm×4.6mm, 5μm) column kept at 50°C. The mobile phase consists of a gradient mixture of mobile phases A (0.05% trifluoroacetic acid in water) and B (acetonitrile–methanol–tetrahydrofuran, 80:15:5, v/v/v) pumped at a flow rate of 1.3ml/min. UV detection was performed at 254nm. The developed method was validated for its robustness, sensitivity, precision and linearity in the range from limit of quantification (LOQ) to 120%. The limits of detection (LOD) and LOQ were found to be 0.08μg/ml and 0.32μg/ml, respectively. This method allows the separation of oxytetracycline from all known and 5 unknown impurities, which is better than previously reported in the literature. Moreover, the simple mobile phase composition devoid of non-volatile buffers made the method suitable to interface with mass spectrometry for further characterization of unknown impurities. The developed method has been applied for determination of related substances in oxytetracycline bulk samples available from four manufacturers. The validation results demonstrate that the method is reliable for quantification of oxytetracycline and its impurities.

Qualitative and Quantitative Determination of COPs in Cypriot Meat Samples Using HPLC Determination of the Most Effective Sample Preparation Procedure

This research work describes the development of a fast high-performance liquid chromatography method for the simultaneous determination of five important cholesterol oxidation products (COPs). The influence of various experimental parameters, such as the composition of the mobile phase, the flow rate and the column temperature, were investigated. Baseline separation was achieved by using acetonitrile-methanol-water-isopropanol (67:27:5:1) as a mobile phase with a 10°C column temperature. The developed method demonstrated good linearity and high reproducibility, with relative standard deviation values below 1.26% for all the COPs that were examined. The method was then applied, for the first time, to Cypriot smoked-meat products (lountza and hiromeri). The presence of COPs in these products suggests that the preparation of the meat products, and particularly the smoking process, possibly favors the oxidation of cholesterol. Finally, three different sample preparation procedures were evaluated and the optimum procedure was determined based on recovery, precision and simplicity.

A Fast HPLC Method for Determination of Vitamin E Acetate in Dietary Supplements Using Monolithic Column

A simple and fast high-performance liquid chromatography (HPLC) method with rapid separation on monolithic column has been developed for quantitative determination of vitamin E acetate in dietary supplements. Commercial samples of dietary supplements were extracted with 100 % methanol with the help of ultrasound bath. A 20-μL sample volume of the filtered solution was directly injected into the HPLC system. Separation of ballast matrix, vitamin E acetate, and internal standard cholecalciferol was performed on the monolithic column Chromolith Performance RP-18e column (100 × 4.6 mm) with mobile phase methanol/water (98:2, v/v) at a flow rate of 2.0 mL min−1 and at temperature 30 °C. The detector was set at 290 nm. Under the optimum chromatographic conditions, standard calibration curve was measured with good linearity—correlation coefficient for vitamin E acetate (r = 0.9992; n = 6) between the peak areas and concentrations of vitamin E acetate. Accuracy of the method defined as a mean recovery was in the range 96.4–103.6 % for all dietary supplements. The intraday method precision was satisfactory and relative standard deviation of sample analysis including preparation and determination of different dietary supplements was in the range 1.1–3.6 %. The developed method has shown high sample throughput during sample preparation process and short time (3.5 min) of analysis.

Simple and fast HPLC method for simultaneous determination of retinol, tocopherols, coenzyme Q10 and carotenoids in complex samples

The effects of fat-soluble vitamins (such as vitamins A and E) and lipid microconstituents (such as carotenoids) on human health are now well established. However, high-performance liquid chromatography (HPLC) methods able to detect these molecules in simultaneous runs are often difficult to set up. We report here a 35-min reversed-phase HPLC method using a single C30 column kept at 35°C with a gradient system of methanol, methyl-tert-butyl ether and water at a flow-rate of 1 mL/min. This method resolves 11 carotenoids, retinol, α- and γ-tocopherol from complex matrixes such as food samples, human plasma and human adipose tissue within 35 min. The method is also able to separate coenzyme Q(10). The intra-day and inter-day coefficients of variation are suitable for routine clinical and scientific applications for the determination of lipid micronutrients from various sample types.

Novel method for simultaneous determination of p-cresylsulphate and p-cresylglucuronide: clinical data and pathophysiological implications

The uraemic retention solutes p-cresylsulphate (pCS) and p-cresylglucuronide (pCG), two conjugates of p-cresol, were never determined simultaneously. In the present paper, a high-performance liquid chromatography (HPLC) method was developed and used to quantify both compounds in parallel in an in vivo observational study and their in vitro effect was evaluated by flow cytometry. pCS and pCG were determined in serum. For the validation specificity, linearity, recovery, precision and the quantification limit were evaluated. In vivo, concentrations of both compounds were determined in 15 controls and 77 haemodialysis patients, as well as protein binding in the dialysed group and the reduction ratios during haemodiafiltration. In addition, the in vitro effect of the solutes on leucocyte free radical production at measured concentrations was assessed. A fast and accurate HPLC method was developed to simultaneously quantify pCS and pCG. Both conjugates are retained in uraemia with a substantially higher total serum pCS in comparison to pCG (31.4 ± 15.8 versus 7.3 ± 6.5 mg/L) but also a substantial difference in protein binding (92.4 ± 3.0 versus 8.3 ± 4.4%) and in reduction ratio during post-dilution haemodiafiltration (37.4 ± 7.1 versus 78.6 ± 6.4%). pCG per se has no effect on leucocyte oxidative burst activity, whereas in combination with pCS, a synergistic activating effect was observed. Serum concentrations of pCS and pCG are elevated in uraemia. Both conjugates show a different protein binding, resulting in a different dialytic behaviour. Biologically, both conjugates are synergistic in activating leucocytes.

Fast HPLC Method for the Determination of Ketoprofen in Human Plasma Using a Monolithic Column and its Application to a Comparative Bioavailability Study in Man

A fast, specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method with diode-array detector (DAD) was developed and validated for the determination of ketoprofen (CAS 22071-15-4) in human plasma using flubiprofen (CAS 5104-49-4) as an internal standard. The chromatographic separation was achieved on an onyx monolithic C18 (100 x 4.6 mm) analytical column with an isocratic mobile phase consisting of acetonitrile/potassium dihydrogen phosphate (KH2PO4) 0.01 M, (40:60, v/v) adjusted to pH 3.5. The flow was set at 5 ml x min(-1) and the wavelength at 254 nm. The total analysis time was less than 5 min. The ratio of peak area of analyte to internal standard was used for quantification. The limit of detection was defined as the ketoprofen concentration that produced a signal-to noise ratio greater than 3. The lower limit of quantification (LLOQ) was 10 ng x m(-1). At this level, the relative standard deviation (RSD) was lower than 13%. The calibration curve was linear over the concentration range 1-500 ng x ml(-1) with a minimum detectable limit of 10 ng x ml(-1). The coefficients of variation for the inter-day and intra-day assay were found to be less than 11%. The present method was successfully applied to the routine analysis of human plasma samples collected from healthy volunteers after dermal application of two topical formulations containing ketoprofen in order to assess the relative bioavailability and to demonstrate that the systemic bioavailability of ketoprofen administered topically is low enough to ensure a low incidence of gastrointestinal adverse events.

Development and Validation of Rapid and Sensitive HPLC Method for the Determination of Methotrexate in Human Serum

Methotrexate competitively inhibits dihydrofolic acid reductase and thereby inhibits DNA synthesis and cellular replication.This study describes a simple and fast high-performance liquid chromatography method for the determination of methotrexate [MTX] in serum.samples were collected from adult cancer patients receiving high dose MTX at Mahathma Gandhi Memorial hospital (Warangal,AP.India) at various time intervals after the end of each infusion. Serum was deproteinized with trichloroacetic acid and the supernatant was injected into a 250×4.6 mm octadecylsilane column. Mobile phase was made of TRIS-phosphate buffer (pH 5.7): methanol: acetonitrile (70:20:10) with a flow rate of 1ml/min. Ultraviolet detection was done at 313 nm and at ambient temperature. Para aminoacetophenone was used as internal standard. Methotrexate and internal standard retention times were 4.6 and 9.5 minutes, respectively. Results showed that reproducibility (precision) of method within a day was 2.6 to 6 percent and between days was 5.5 to 9.5 percent. The recovery of the method was between 61.5 and 72.7 percent. The quantitation limit of the method for methotrexate was 0.1μM. This method is suitable for quantitation of methotrexate after infusion of high doses of this drug and has good accuracy, precision and quantitation limit. Key Words: Methotrexate; HPLC; Serum Concentration. DOI: 10.3329/sjps.v2i1.1693Stamford Journal of Pharmaceutical Sciences Vol.2(1) 2009: 8-13