Isocratic Elution Mode Research Articles

Development and Validation of Stability Indicating high performance liquid chromatographic Method for Indapamide

An approach of forced degradation study was successfully applied for the development of a stability-indicating high performance liquid chromatographic method for Indapamide in the presence of its degradation products. In the present study a simple, accurate and precise reverse phase liquid chromatographic method has been developed of Olmesartan Indapamide in bulk. Developed Method was achieved on symmetry C18 (150 mm × 4.6 mm, 5 μ) column using a Acetonitrile: 0.02 M Na2HPO4(45:55 v/v) mobile phase and pH 7 adjusted with ortho phosphoric acid. Isocratic elution mode at a flow rate of 1.0 ml/min at Room temperature with a load of 20μl Injection volume. The detection was carried out at 240 nm. The linearity of the proposed method was investigated in the range of 10–50 microg/mL (r2 = 0.998) for Indapamide. The retention time of Indapamide was found to be around 4.79 min and 7.59 min respectively. The drug substances were subjected to stress conditions of acid hydrolysis, base hydrolysis, Oxidative, photolytic and thermal. The developed RP-HPLC method was validated with respect to linearity, accuracy, precision, robustness, LOD and LOQ.

Development and Validation of Stability Indicating high performance liquid chromatographic Method for Olmesartan medoxomil and Indapamide in Tablet

An approach of forced degradation study was successfully applied for the development of a stability-indicating high performance liquid chromatographic method for simultaneous determination of Olmesartan medoxomil and Indapamide in a formulation in the presence of its degradation products. In the present study a simple, accurate and precise reverse phase liquid chromatographic method has been developed and validated for simultaneous estimation of Olmesartan medoxomil and Indapamide in tablet dosage form. Developed Method was achieved on symmetry C18 (150 mm × 4.6 mm, 5 μ) column using a Acetonitrile: 0.02 M Na2HPO4 (45:55 v/v) mobile phase and pH 7 adjusted with ortho phosphoric acid. Isocratic elution mode at a flow rate of 1.0 ml/min at Room temperature with a load of 20μl Injection volume. The detection was carried out at 240 nm. The linearity of the proposed method was investigated in the range of 50–250 microg/mL (r2 = 0.998) for Olmesartan medoxomil and 10–50 microg/mL (r2 = 0.998) for Indapamide. The retention time of Olmesartan medoxomil and Indapamide were found to be around 4.79 min and 7.59 min respectively. The drug substances were subjected to stress conditions of acid hydrolysis, base hydrolysis, Oxidative, photolytic and thermal. The developed RP-HPLC method was validated with respect to linearity, accuracy, precision, robustness, LOD and LOQ.

Development and Validation of Stability Indicating High Performance Liquid Chromatographic Method for Olmesartan Medoxomil and Indapamide in Tablet Dosage Form

An approach of forced degradation study was successfully applied for the development of a stability-indicating high performance liquid chromatographic method for simultaneous determination of Olmesartan medoxomil and Indapamide in a formulation in the presence of its degradation products. In the present study a simple, accurate and precise reverse phase liquid chromatographic method has been developed and validated for simultaneous estimation of Olmesartan medoxomil and Indapamide in tablet dosage form. Developed Method was achieved on symmetry C18 (150 mm × 4.6 mm, 5 μ) column using a Acetonitrile: 0.02 M Na2HPO4(45: 55 v/v) mobile phase and pH 7 adjusted with ortho phosphoric acid. Isocratic elution mode at a flow rate of 1.0 ml/min at Room temperature with a load of 20μl Injection volume. The detection was carried out at 240 nm. The linearity of the proposed method was investigated in the range of 50–250 microg/mL (r2 = 0.998) for Olmesartan medoxomil and 10–50 microg/mL (r2 = 0.998) for Indapamide. The retention time of Olmesartan medoxomil and Indapamide were found to be around 4.79 min and 7.59 min respectively. The drug substances were subjected to stress conditions of acid hydrolysis, base hydrolysis, Oxidative, photolytic and thermal. The developed RP-HPLC method was validated with respect to linearity, accuracy, precision, robustness, LOD and LOQ.

Development of a Forced Degradation Profile of Alosetron by Single Mode Reversed-Phase HPLC, LC-MS, and its Validation

Determination of alosetron in the presence of its degradation products was studied and validated by a novel HPLC method. The separation of the drug and its degradation products was achieved with the Jones Chromatography C18 analytical column (150 mm x 4.6 mm; 3 µm) with a stationary phase in isocratic elution mode. The mobile phase used was 0.01 M ammonium acetate, pH-adjusted to 3.5 with glacial acetic acid and acetonitrile in the ratio of 75:25 (V/V) at a flow rate of 1 ml/min and UV detection was carried out at 217 nm. Further, the drug was subjected to stress studies for acidic, basic, neutral, oxidative, and thermal degradations as per ICH guidelines and the drug was found to be labile in base hydrolysis and oxidation, while stable in acid, neutral, thermal, and photolytic degradation conditions. An MS study has been performed on the major degradation products to predict the degradation pathway of alosetron. The method provided linear responses over the concentration range of 100–1500 ng/ml and regression analysis showed a correlation coefficient value (r2) of 0.994. The LOD and LOQ were found to be 1 ng/ml and 3 ng/ml, respectively. The developed LC method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness.

Determination of N-acetylglucosamine in cosmetic formulations and skin test samples by hydrophilic interaction liquid chromatography and UV detection

N-Acetylglucosamine is an ingredient in pharmaceuticals, nutritional supplements and in cosmetics. N-Acetylglucosamine in cosmetics is expected to improve skin hydration, reparation, and to contribute as anti-wrinkle agent. This study reports on the validation and application of an HPLC method based on HILIC and UV detection for determining N-acetylglucosamine in cosmetics and in samples obtained after testing the skin exposed to cosmetics formulations. The chromatographic column used is a ZIC®-pHILIC (150mm×4.6mm, 5μm particle size) on which a mobile phase containing acetonitrile–aqueous KH2PO4 (70:30, v/v) 15mM was applied in isocratic elution mode injecting 20μl of sample at 0.5ml/min constant flow-rate and 10±1°C column temperature. Under these conditions the total run time was 10min and N-acetylglucosamine eluted baseline separated from all other compounds in the samples. Calibration in the range from 40 to 80μg/ml allowed to assess the method linearity (R2>0.999) in a concentration range corresponding to about 50% to 120% of the expected levels of N-acetylglucosamine in the formulations. Precision expressed by RSD% was always better than 2% in intra-day and inter-day assays of authentic samples. Accuracy was in all cases within 95–105% of the expected concentration value in formulations containing N-acetylglucosamine. The sensitivity of the method was at the level of 10μg/ml as limit of detection, and at 40μg/ml as limit of quantitation. The application of the method to formulations containing solid lipid nanoparticles documents its usefulness in cosmetic quality control. The results witness that the method is also suitable for the determination of N-acetylglucosamine in samples obtained from skin test strips.

Use of experimental design to optimize a triple-potential waveform to develop a method for the determination of streptomycin and dihydrostreptomycin in pharmaceutical veterinary dosage forms by HPLC-PAD

An HPLC–PAD method using a gold working electrode and a triple-potential waveform was developed for the simultaneous determination of streptomycin and dihydrostreptomycin in veterinary drugs. Glucose was used as the internal standard, and the triple-potential waveform was optimized using a factorial and a central composite design. The optimum potentials were as follows: amperometric detection, E1=−0.15V; cleaning potential, E2=+0.85V; and reactivation of the electrode surface, E3=−0.65V. For the separation of the aminoglycosides and the internal standard of glucose, a CarboPac™ PA1 anion exchange column was used together with a mobile phase consisting of a 0.070molL−1 sodium hydroxide solution in the isocratic elution mode with a flow rate of 0.8mLmin−1. The method was validated and applied to the determination of streptomycin and dihydrostreptomycin in veterinary formulations (injection, suspension and ointment) without any previous sample pretreatment, except for the ointments, for which a liquid–liquid extraction was required before HPLC-PAD analysis. The method showed adequate selectivity, with an accuracy of 98–107% and a precision of less than 3.9%.

Quantitation of the immunological adjuvants, monophosphoryl lipid A and Quil A in poly (lactic-co-glycolic acid) nanoparticles using high performance liquid chromatography with evaporative light scattering detection.

Monophosphoryl lipid A (MPL) and Quil A are two immunological adjuvants commonly used in vaccines. At present no simple, validated methods for the quantification of Quil A and MPL have been previously reported therefore the aim of the current study was to develop a simple, fast and validated method to quantify MPL and Quil A using high performance liquid chromatography evaporative light scattering detection (HPLC-ELSD). The HPLC-ELSD technique was carried out using a ZORBAX Eclipse XDB-C8 column (2.1×50 mm; particle size, 3.5 μm) in an isocratic elution mode at 25 °C. MPL was eluted at a retention time of 1.8 min with methanol-water as the mobile phase and a detector temperature of 75 °C. Quil A was resolved as three peaks with retention times of 4.1, 5.5 and 6.4 min with a detector temperature of 30 °C and with water-acetonitrile and 0.01% formic acid as the mobile phase. The nebulizer pressure and gain were set at 3.5 bar and 10, respectively. Calibration curves plotted for both the adjuvants had an R(2)>0.997. Accuracy, intra- and inter-day precision were within the accepted limits. The limit of detection for MPL and Quil A were calculated as 1.343 and 2.06 μg/mL, respectively. The limit of quantification was 2.445 for MPL and 8.97 μg/mL for Quil A. This analytical method was used to quantify the entrapment and in vitro release of MPL and Quil A in a poly lactic-co-glycolic acid (PLGA) nanoparticle vaccine.

Plasma protein binding, pharmacokinetics, tissue distribution and CYP450 biotransformation studies of fidarestat by ultra high performance liquid chromatography–high resolution mass spectrometry

Fidarestat, an aldose reductase inhibitor, has been used for the treatment of the diabetic associated complications such as retinopathy, neuropathy and nephropathy. To better understand the metabolism and pharmacokinetics of fidarestat, we have evaluated plasma protein binding, pharmacokinetics, tissue distribution of the drug and its conjugated metabolites and CYP450 biotransformation by liquid chromatography–high resolution mass spectrometry. Effective chromatographic separation of fidarestat and hydrochlorothiazide (IS) in rat plasma and tissues was achieved on Hypersil gold C-18 column in an isocratic elution mode. For detection, a high-resolution Orbitrap mass spectrometer with heated electrospray ionization inlet in the negative ion mode was used. High-resolution extracted ion chromatograms for each analyte were obtained by processing the full-scan MS mode with 5ppm mass tolerance. The impact of plasma protein binding with the drug and conjugated metabolites of the drug on pharmacokinetics has been determined. The study indicated that 9.5% of free form of fidarestat may be pharmacologically active and the Cmax for free fidarestat was found to be 80.30±6.78ng/mL. The AUC0–t and AUC0–∞ were found to be 185.46±32 and 195.92±15.06ngh/mL, respectively. Among tissues, the maximum observed distribution was found to be in kidney followed by liver and heart. Docking experiments and in vitro CYP450 reaction phenotyping revealed that two CYP1A2 and CYP2D6 are involved in the phase I metabolism of fidarestat. Oxidative deamination and N/O glucuronidation are the major phase I and phase II metabolites, respectively. In vitro CYP450 inhibition assay of fidarestat for drug–drug interaction showed weak inhibition and may not alter pharmacokinetics, distribution or clearance of other co-administered drug.

High-performance liquid chromatography on glass chips using precisely defined porous polymer monoliths as particle retaining elements

A stable and permanent integration of miniature packed bed separation columns into microfluidic systems is a major issue in nano liquid chromatography. Various approaches like differently shaped retaining elements or the use of key stone effect have been investigated. We show a flexible integration of miniature packed bed separation columns into microfluidic chips utilising common HPLC material achieved by laser-assisted generation of narrow, photopolymerised frits. The generated retaining elements serve as an in- and outlet frits for the columns. An optimised pre-polymeric solution, consisting of butyl acrylates and a porogen, allows a precise fabrication of frit-type structures with lengths of less than 100μm and the capability to withstand common slurry packing pressures of more than 250bar. The separation of seven polycyclic aromatic hydrocarbons by pressure-driven, reversed-phase chromatography proves the high quality of the created chromatographic column inside a glass chip. Plate heights down to 2.9 were achieved and extremely fast separations with sub-second peak widths were performed in isocratic and gradient elution modes on very short columns (≤25mm).

A Combination of Isocratic and Gradient Elution Modes in HPLC with the Aid of Time-Overlapping Process for Rapid Determination of Methyldopa in Human Urine

A new rapid time-overlapping high-performance liquid chromatography method using coupled-column double-injection technique with fluorescence detection has been developed and validated to determine methyldopa (MTD) in human urine. The method was based on injecting a new sample onto the second column before finalizing the cleanup and the re-equilibration of the first column for the former sample. A combination of isocratic and gradient elution was employed according to a pre-set program. At the beginning, isocratic step of acetate buffer solution (0.1 M, pH 2.4) was set until 7 min. Subsequently, a gradient elution step using acetate buffer (0.1 M, pH 2.4) as mobile phase A and acetonitrile as mobile phase B was employed. After the end of each gradient step, the column was re-equilibrated with 4 mL of the starting isocratic elution system before the next analysis. The overall cycle time was 7 min per each sample. The calibration curve was linear over the concentration range of 0.1–40 µg/mL MTD. The overall mean recoveries were in the range of 98.29–101.39%. The applicability of the method was successfully evaluated by monitoring the incremental urinary excretion of MTD in human urine over 12 hr after a single oral administration of 250 mg.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR DETERMINATION OF METOPROLOL ENANTIOMERS IN PHARMACEUTICAL FORMULATIONS

A novel high performance liquid chromatography with fluorescence detector (HPLC-FL) method for determination of metoprolol enantiomers in a pharmaceutical formulation was developed and validated. This study investigated the ultrasound-assisted extraction (UAE) of metoprolol from commercial tablets using methanol as extraction solvent. The separation of enantiomers was successfully carried out with a CHIRALCEL OD-RH column, under isocratic elution mode using 0.05% trifluoroacetic acid and 0.05% diethylamine in water and acetonitrile (80:20, v/v).The chromatographic method was validated in terms of precision, accuracy, detection and quantification limits, linearity and recovery. Calibration curves were linear in the investigated range with correlation coefficient better than 0.9931 while the limit of quantifications was 0.19 µg mL-1 for S(–)-MET and 0.23 µg mL-1 for R(+)-MET. The mean recovery of S(–)-MET and R(+)-MET from tablets were found to be 78.8% and 92.1%, respectively.This method for the direct determination and quantification of metoprolol enantiomers in pharmaceutical formulations is suitable for routine analyses in quality control laboratories and was applied to evaluate for the first time, the presence and the quantities of cited analytes in commercially available formulation.

Simultaneous and enantioselective determination of cis-epoxiconazole and indoxacarb residues in various teas, tea infusion and soil samples by chiral high performance liquid chromatography coupled with tandem quadrupole-time-of-flight mass spectrometry

A novel and sensitive method for simultaneous enantiomeric analysis of two pesticides—cis-epoxiconazole and indoxacarb—in various teas, black tea infusion, and soil samples has been developed. The samples were initially subjected to acetonitrile extraction followed by cleanup using lab-made florisil/graphitized carbon black mixed solid phase extraction (SPE) column (for the different teas and soil samples) and a BondElut C18-SPE column (for the black tea infusion samples). Separation of the analytes was performed on a chiral stationary phase using high performance liquid chromatography (HPLC) under a reversed-phase isocratic elution mode followed by tandem quadrupole time-of-flight mass spectrometry (Q-TOF/MS) detection. The mobile phase components, mobile phase ratios, flow rates, column temperatures, and MS parameters were all optimized to reach high sensitivity and selectivity, good peak shape, and satisfactory resolution. The performance of the method was evaluated based on the sensitivity, linearity, accuracy, precision, and matrix effects. Under optimal conditions, for the various teas (green tea, black tea, and puer tea), fresh tea leaf, soil and black tea infusion samples spiked at low, medium, and high levels, the mean recoveries for the four enantiomers ranged from 61.0% to 129.7% with most relative standard deviations (RSDs) being 17.1% or below. Good linearity can be achieved with regression coefficients (R) of 0.9915 or above for all target enantiomers, and matrix-matched calibration concentration ranging from 5.0 to 1000μg/L. The limits of detection (LODs) for all four target enantiomers were 1.4μg/kg or below in the different teas and soil samples and 0.05μg/kg or below in the black tea infusion, whereas the limits of quantification (LOQs) for those did not exceed 5.0μg/kg and 0.2μg/L, respectively. The proposed method is convenient and reliable and has been applied to real tea samples screening. It has also been extended for studies on the degradation kinetics and environmental behaviors in the field trials, providing additional information for reliable risk assessment of these chiral pesticides.

Simultaneous determination of three dipeptides (JBP485, Gly-Sar and JBP923) in the cell lysates by liquid chromatography-tandem mass spectrometry: application to identify the function of the PEPT1 transfected cell.

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates using methanol as a deproteinization solvent was developed and validated. Detection was performed by turbo ionspray ionization in multiple reaction monitoring mode using the transitions of m/z 147.1 → m/z 90.1 for Gly-Sar, m/z 201.1 → m/z 86.1 for JBP485, m/z 219.1 → m/z 86.1 for JBP923 and m/z 152.0 → m/z 110.0 for paracetamol (internal standard). The analytes were separated on a Hypersil ODS C18 HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water-methanol (97:3, v/v) at a flow rate of 0.2 mL/min. The calibration curves were demonstrated to be linear over the concentration range of 5.00-5000 nm with coefficient of 0.9968 for Gly-Sar, 0.9975 for JBP485 and 0.9952 for JBP923. The intra- and inter-day precisions were <10.2% for each quality contro; level, and the accuracy was within ±5.6% for each analyte. The matrix effect, the extraction recovery and stabilities of LC-MS/MS analysis were also investigated. This validated method was successfully applied to the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates for identification of stably transfected HeLa cells with human PEPT1.

LC-MS/MS determination of cinacalcet enantiomers in rat plasma on Chirobiotic V column in polar ionic mode: application to a pharmacokinetic study.

A simple and selective polar ionic liquid chromatography-tandem mass spectrometric method for separation and determination of cinacalcet enantiomers in rat plasma was developed and validated. The chromatographic separation was accomplished on a Chirobiotic V column packed with vancomycin as a chiral stationary phase using 2.5 mm ammonium formate in 100% methanol as a mobile phase in an isocratic mode of elution at a flow rate of 1.0 mL/min. The analytes were extracted from rat plasma by precipitating the proteins with acetonitrile. The developed method exhibited a linear dynamic range over 0.5-500 ng/mL in rat plasma for both enantiomers. The method was successfully applied to study the pharmacokinetics after a single dose by oral administration of 10 mg/kg of cinacalcet enantiomers to healthy male Wistar rats.

Microemulsion Thin-layer Chromatographic Separation of Caffeine and Paracetamol and their Determination in Formulated Tablet and in Spiked Urine Sample by HPLC

A simple and accurate method was developed for the separation and simultaneous determination of Caffeine and Paracetamol. The separation was achieved on silica HPTLC plates with microemulsion (SDS: water: n-hexane: 1-butanol, 8 g: 8 ml: 160 ml: 25 ml) as a solvent system. The determination was achieved by HPLC after extraction of the solute from the spots. The determination was performed in isocratic elution mode with mobile phase acetonitrile-water (50 : 50, v/v) at a flow rate of 1.o mL/min in a C18 hypersil gold 150 mm x 4.6 mm, 5um particle size column. The detection was carried out at 272 by PDA detector. This method was validated for analytical parameters such as, linearity, specificity, precision, LOD and LOQ. The developed method was used for the identifiaction, separation and quantification of Caffeine and Paracetamol in formulted drug and in spiked urine sample. The % recoveries obtained in drug and in spiked urine samples are 99.2 and 98.9 (for Caffeine) and 99.8 and 99.7 (for Paracetamol), respectively.

Analysis of thiopurines using aqueous normal phase chromatography

The chromatography of several thiopurines is investigated using aqueous normal phase (ANP) conditions in conjunction with a silica hydride-based column. Both isocratic and gradient elution modes are tested. Detection of higher concentration samples is done by UV to demonstrate feasibility in this format while lower concentration samples utilize mass spectrometry (MS). Repeatability of successive runs is also tested with particular attention to gradient methods where the equilibration time of the stationary phase can be evaluated.

SIMULTANEOUS DETERMINATION OF FINASTERIDE AND TAMSULOSIN IN COMBINED DOSAGE FORM BY USING RP-HPLC METHOD

A simple, accurate, economical, and reproducible reversed-phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of finasteride and tamsulosin in bulk and pharmaceutical formulations. The separation was achieved on a phenomenex C18 column (150 × 4.6 mm i.d, particle size of 5 µ) using a mixture of 0.1% triethylamine (pH adjusted to 7.01 ± 0.05 with 0.1% ortho phosphoric acid) and methanol in the ratio of 30:70% v/v as the mobile phase in an isocratic elution mode, at a flow rate of 0.7 mL/min. The detection was monitored at 220 nm. The retention times of finasteride and tamsulosin were found to be 5.8 ± 0.12 min and 2.9 ± 0.14 min, respectively. Excellent linearity range was found between 10–100 µg/mL for finasteride and 2–8 µg/mL for tamsulosin. The method was validated with respect to linearity, precision, accuracy, specificity, robustness, and ruggedness. The method was successfully applied for the simultaneous determination of finasteride and tamsulosin from the combined dosage form.

ANALYSIS OF ACRYLAMIDE IN TRADITIONAL AND NONTRADITIONAL FOODS IN TURKEY USING HPLC–DAD WITH SPE CLEANUP

Acrylamide is a heat-generated food toxicant, which exists in various foodstuffs such as potato chips, biscuits, and coffee. It has been demonstrated that acrylamide possesses some neurotoxic and carcinogenic effects and is classified in Group 2A as a probable carcinogen to humans by International Agency for Research on Cancer. The aim of this study was to develop a rapid, robust, and sensitive high performance liquid chromatography (HPLC) method and indicate acrylamide levels of some traditional and nontraditional foods consumed in Turkey. Acrylamide content of samples was determined using HPLC coupled to photodiode array detection. Acrylamide and methacrylamide (internal standard) were separated using an ODS-3 reversed phase column in isocratic elution mode. The mobile phase (acetonitrile: water, 2:98, v/v) was pumped at 0.5 mL/min flow rate and the analytes were detected at 200 nm wavelength within 15 min. Samples were prepared by utilizing Oasis HLB (Waters) solid phase extraction cartridges for clean-up and analyzed with no further preconcentration steps. As a result, it was observed that the amount of acrylamide in traditional Turkish foods was found to be relatively low in many of the samples, especially in desserts although they are exposed to heat in their preparation.

Development and Validation of a Stability-Indicating RP-HPLC Method for Determination of Darifenacin Hydrobromide in Bulk Drugs

An isocratic stability-indicating reversed phase high performance liquid chromatographic method (RP-HPLC) was developed for determination of process related impurities and assay of darifenacin hydrobromide (DRF) in bulk drugs. DRF was subjected to various stress conditions such as hydrolysis (acid, base, and neutral), oxidation, photolysis and thermal degradation as per International Conference on Harmonization (ICH Q1A(R2) and Q1B) prescribed conditions to investigate the stability-indicating ability of the method. Significant degradation was observed during acidic hydrolysis and oxidative stress conditions. The chromatographic separation was accomplished on a Prodigy C8 column (250 × 4.6 mm, 5 μm) with mobile phase consisting of 0.05 M ammonium acetate (pH adjusted to 7.2 by using ammonia solution) and methanol (36% acetonitrile) in 35:65 v/v ratio in an isocratic elution mode at a flow rate of 1.0 mL/min at 25°C. Detection of analytes was carried out using photo diode array detector at a wavelength of 215 nm. The developed LC method was validated with respect to accuracy, linearity, precision, limits of detection and quantitation and robustness as per ICH guidelines.

Optimized Formation, Extraction, and Determination of Sulforaphane in Broccoli by Liquid Chromatography with Diode Array Detection

A new method has been developed to determine sulforaphane in broccoli florets, stems, and leaves using liquid chromatography coupled to diode array detection. The hydrolysis reaction, required to convert glucoraphanin to its breakdown product, sulforaphane, and the extraction procedure, based on solvent extraction with methyl t-butyl ether followed by solid phase extraction with silica cartridges, were optimized for the three broccoli matrices; the resulting recovery values ranged from 92 to 102 % in all cases. A fast separation was performed on a C18 analytical column with a mobile phase composed of ammonium formate (20 mM) in water and acetonitrile (55:45, v/v) in isocratic elution mode. This method was fully validated and was found to be selective, linear from 2.5 to 800 mg/kg, and precise (percent relative standard deviation values below 5 %). Moreover, the limits of detection and quantification were 0.8 and 2.5 mg/kg, respectively. Finally, the proposed method has been applied to the analysis of sulforaphane in broccoli samples of two different cultivars (Parthenon and Marathon).