Simultaneous extraction of tocotrienols and tocopherols from cereals using pressurized liquid extraction prior to LC determination

Tocopherols and tocotrienols have been simultaneously determined in food samples using a rapid and simple analytical method including pressurized liquid extraction (PLE) and LC with electrochemical detection. Separation was carried out on a Phenomenex Synergi 4 μm Hydro‐RP 80A column, using a solution of 2.5 mM acetic acid/sodium acetate in methanol/water (99:1, v/v) as mobile phase at a flow rate of 1.0 mL/min. Column temperature was maintained at 30°C. Detection was performed by coulometric detection at 500 mV except for (β+γ)‐tocotrienol, in wheat and rye samples, which was at +350 mV. A palm oil containing a relatively large amount of γ‐tocotrienol and lower concentrations of α‐ and δ‐tocotrienols and α‐ and γ‐tocopherols was used to provide reference retention times for the tocotrienols. Analyte quantification was performed using the external standard method. The calibration equations of tocopherols were used to quantify both tocopherols and their corresponding tocotrienols. The extraction recoveries obtained using the optimized PLE conditions were in the 80–114% range, with RSDs lower than 15%. The method was successfully applied to the determination of tocotrienols and tocopherols in cereal (wheat, rye, barley, maize and oat) and palm oil samples.     

On-line coupling of automatic solid-phase extraction and HPLC for determination of carotenoids in serum

The automated method developed for the determination of carotenoids uses 200 μL of serum, which was mixed with 400 μL of tetrahydrofuran, vortexed for 1 min, settled for 10 min, centrifuged for 6 min and the supernatant injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration. A 10% water-acetonitrile mobile phase at 1.5 mL min⁻¹ eluted the retained compounds and transferred them on-line to a reversed-phase analytical column for individual separation of the target analytes. Visible detection was performed at 450 and 460 nm. The detection limits for the target analytes were between 3 and 30 ng mL⁻¹; the precision (expressed as relative standard deviation) ranged between 2.83 and 5.06% for repeatability and between 3.80 and 7.40% for within laboratory reproducibility. The total analysis time was 18 min. The proposed method is reliable, robust, and has an excellent potential for high-throughput use in both clinical and research laboratories.     

Determination of tocopherols and tocotrienols in cereals by pressurized liquid extraction-liquid chromatography-mass spectrometry

A rapid analytical method including pressurized liquid extraction (PLE) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) has been developed for the determination of tocopherols and tocotrienols in cereals. The pressurized liquid extraction parameters were optimized in order to maximize the extraction efficiency. The use of methanol as extraction solvent at a temperature of 50 °C and a pressure of 110 bar, using one cycle of extraction with a static time of 5 min, provided the best results. A good LC separation was achieved using a C₁₈ column and a solution of 6.0 mM ammonia in methanol/water (97:3, v/v) as the mobile phase at a flow rate of 0.2 mL min⁻¹. MS coupling with an ESI interface in the negative ion mode was used as the detection technique. In the present work, it is shown that the addition of a base to the mobile phase is required to enhance the ionization of tocopherols and tocotrienols in negative ion mode electrospray ionization. The applicability of the method to cereal samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the 6-10% range. The recoveries of added tocopherols from cereal samples ranged from 91 to 109%.     

Development and optimization of ultra-high performance supercritical fluid chromatography mass spectrometry method for high-throughput determination of tocopherols and tocotrienols in human serum

The goal of this study was to develop an effective supercritical fluid chromatography method using single quadrupole MS for analysis of all isomeric forms of vitamin E. Finally, two fast and effective methods, the high resolution one and the high speed one, for the determination of 8 vitamin E isomers in human serum were developed.     

Development and validation of an HPLC method for the determination of benzodiazepines and tricyclic antidepressants in biological fluids after sequential SPE

A novel method for the simultaneous determination of six benzodiazepines (BZDs) and four tricyclic antidepressants (TCAs) in biological fluids by HPLC with UV detection at 240 nm has been developed. After a deproteinization step biological fluids were analyzed by direct injection. SPE on Nexus cartridges was also applied. Since two compounds, namely imipramine and diazepam, were coeluting, a sequential SPE protocol has been developed. BZDs were eluted by a mixture of methanol/ACN(1:1), followed by the elution of TCAs with methanol. Separation was performed on a Kromasil C8 column (250 x 64 mm(2) id, 5 microm) using a mobile phase of 0.05 MCH3COONH4/ACN/methanol (initial composition 55:15:30 v/v/v) at a flow rate of 1.0 mL/min delivered by a gradient program within 15 min. Colchicine was used as the internal standard (4 ng/microL). The method was linear for all analytes up to 20 ng/lL, with coefficients of regression between 0.996 and 0.99996. LODs and LOQs were 0.08-1.17 and 0.28-3.91 ng/lL, respectively. Recovery was in the range of 92.8-108.7% for within-day and 91.9-109.9% for between-day assays, with RSD values lower than 10.0% for all matrices.     

Preparative scale reversed-phase HPLC method for simultaneous separation of carotenoids and carotenoid esters

Summary A method is described for the preparative HPLC of carotenoids and carotenoid esters using a self-packing axially-compressed column. The reversed-phase system used employs an RP-18 stationary phase and a mobile phase consisting of a mixture of petroleum spirit, acetonitrile, methanol and tetrahydrofuran. The method is demonstrated for paprika fruit, rose hips and marigold flowers.     

Development and validation of a rapid and efficient method for simultaneous determination of methylxanthines and their metabolites in urine using monolithic HPLC columns

A new rapid, sensitive and validated HPLC method has been developed for the determination of methylxanthines and their metabolites in asthmatic patients. The method was initiated by using spiked urine samples on a silica monolithic column as a novel packing material. The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer/methanol (87.5:12.5 v/v), at a flow rate 1 mL/min. Detection was set at 274 nm. The LOQ for all the compounds ranged from 14 to 41 ng/mL. Excellent linearity was achieved over the studied range of concentration with correlation coefficients 0.9991–0.9998 (n = 6). The developed method was validated by precision and accuracy with RSD <2.55%. On extraction of the drugs and metabolites from the urine samples high recoveries were achieved ranging from 82.06 to 98.34% w/w on RP18 cartridges and methanol/chloroform (20:80 v/v) as the extraction solvent. This method has advantages over other methods using conventional C18 packings.     

Molecularly imprinted solid-phase extraction for the selective HPLC determination of ractopamine in pig urine

A method was developed for the determination of ractopamine in pig urine using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography. The molecularly imprinted polymer (MIP) was synthesized in acetonitrile-triethylamine system using ractopamine (RAC) as the template and acrylamide as the monomer. The binding capacity of the polymer toward RAC was found to be about 2.57 mg of ractopamine/g of polymer. The optimal procedures for MISPE consisted of conditioning with 3 mL methanol, equilibrating with 3 mL of water, loading volume of <10 mL of aqueous sample (pH 7), washing with 3 mL water and 3 mL methanol, and eluting with 5 mL of 5% ammonia in methanol. In the four spiked samples with the levels of 0.01, 0.1, 1.0 and 5.0 μg/mL, the mean recoveries of analyte on the MIP were higher than 90% with relative standard deviation <10%, and significant differences between imprinted and non-imprinted materials were observed. The MIP selectivity was evaluated by checking 11 drugs with similar and different molecular structures to that of RAC. The characteristics of three-dimensional cavities and hydrogen bond interaction were regarded as the main factors that dominated the retention of RAC on the MISPE cartridge.     

Development and validation of an HPLC method for the determination of seven penicillin antibiotics in veterinary drugs and bovine blood plasma

Herein a quantitative method for the determination of seven penicillins in bovine plasma and veterinary drugs has been developed. Amoxicillin (AMO), ampicillin (AMP), penicillin G (PENG), penicillin V (PENV), oxacillin (OXA), cloxacillin (CLO) and dicloxacillin (DICLO) were separated on a Perfectsil ODS‐2 (250×4 mm, 5 μm) column, using gradient elution, with a mobile phase of 0.1% v/v TFA and ACN–methanol (90:10 v/v). PDA detection was used at 240 nm. Penicillins were isolated from bovine plasma by SPE on Lichrolut RP‐18 cartridges with mean recoveries from 85.7 to 113.5%. Colchicine (3 ng/μL) was used as an internal standard. The developed method was validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between‐day precision (n = 5) revealed RSD < 12%. The detection limits in the bovine plasma were estimated as 18 ng for AMO and AMP, 25 for PENG, PENV and OXA, 3 ng for CLO and 12 ng for DICLO. Spiked plasma samples were stable for 1 wk, except for AMP and CLO, which were stable for 3 wk and OXA for 4 wk. AMO, PENG and PENV were stable for two freeze–thaw cycles, OXA, CLO and DICLO for four, while AMP only for one.     

Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction

An HPLC method was developed for the simultaneous determination of seven water-soluble vitamins, viz. thiamine, riboflavin, nicotinic acid, nicotinamide, pyridoxine, cyanocobalamin, and folic acid, in multivitamin pharmaceutical formulations and biological fluids (blood serum and urine). Separation was achieved at ambient temperature on a Phenomenex Luna C18 (150 x 4.6 mm) analytical column. Gradient elution was performed starting at a 99:1 A:B v/v composition, where A: 0.05 M CH3COONH4/CH3OH (99/1) and B: H2O/CH3OH (50/50), at a flow rate of 0.8 mL/min. After a 4-min isocratic elution the composition was changed to 100% of B in 18 min and elution continued isocratically for 8 min. Detection was performed with a photodiode array detector at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples. Detection limits were in the range of 1.6-3.4 ng, per 20-microL injection, while linearity held up to 25 ng/microL. Accuracy, intra-day repeatability (n = 6), and inter-day precision (n = 7) were found to be satisfactory. Theobromine (2 ng/microL) was used as internal standard. Sample preparation of biological fluids was performed by SPE on Supelclean LC-18 cartridges with methanol-water 85/15 v/v as eluent. Extraction recoveries from biological matrices ranged from 84.6% to 103.0%.     

Development of an HPLC method for the determination of mexiletine in human plasma and urine by solid-phase extraction

A sensitive and specific high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of mexiletine (MEX) in human plasma and urine. It uses solid-phase extraction (SPE) followed by an automated reversed-phase HPLC with a pre-column derivatization with 4-chloro-7-nitrobenzofurazan (NBD-CI) and UV-vis Absorbance detection. The process was set as: the UV-vis Absorbance wavelength was set at 458 nm. Chromatographic separation was performed on a Phenomenex-C₁₈ Column (Aqua, 150 mm × 4.6 mm i.d. with 5 μm particle size) with the mobile phase consisting of acetonitrile and water (80:20, v/v), and the flow rate was set at 1.0 mL min⁻¹. Calibration of the overall analytical procedure gave a linear signal (r > 0.9998) over a MEX concentration range of 0.2-2.0 μg mL⁻¹ in human plasma and urine. The detection limit in plasma and urine was 0.1 μg mL⁻¹. Intra- and inter-day precision of the assay at three concentrations within this range were 0.31-2.50%. The high specificity and sensitivity have been achieved by this fast method (total run-time <6 min). The method has been successfully validated in human plasma and urine and it has been shown to be precise, accurate and reliable.     

A new solid-phase extraction and HPLC method for determination of patulin in apple products and hawthorn juice in China

A new solid‐phase extraction (SPE) pretreatment method using a home‐made polyvinylpolypyrrolidone‐florisil (PVPP‐F) column was developed for the analysis of patulin in apple and hawthorn products in China. Fifty samples (25 apple juices, 12 apple jams, and 13 hawthorn juices) were prepared using the new method and then analyzed by high performance liquid chromatography with diode array detection (HPLC‐DAD) on an Agela Venusil MP C₁₈ reversed‐phase column (4.6 mm × 250 mm, 5 μm). The cleanup results for all samples using home‐made PVPP‐F column were compared with those obtained using a MycoSep®228 AflaPat column. The correlation coefficient R (0.9998) fulfilled the requirement of linearity for patulin in the concentration range of 2.5–250 μg/kg. The limits of detection (LODs) and quantification (LOQs) of patulin were 3.99 and 9.64 μg/kg for PVPP‐F column, and 3.56 and 8.07 μg/kg for MycoSep®228 AflaPat column, respectively. Samples were spiked with patulin at levels ranging from 25 to 250 μg/kg, and recoveries using PVPP‐F and MycoSep®228 AflaPat columns were in the range of 81.9–100.9% and 86.4–103.9%, respectively. Naturally occurring patulin was found in 2 of 25 apple juice samples (8.0%) and 1 of 13 hawthorn juice samples (7.7%) at concentrations ranging from 12.26 to 36.81 μg/kg. The positive results were further confirmed by liquid chromatography electrospray ionization mass spectrometry (LC‐ESI‐MS).     

The simultaneous determination of capsaicinoids,tocopherols, and carotenoids in pungent pepper powder

From nutritional points of view, carotenoids, capsaicinoids, and tocopherols are valuable constituents in pungent peppers. A rapid and reliable high performance liquid chromatography (HPLC) method for the simultaneous determination of phytonutrients in spice red peppers and chili products was developed and validated. The method included simultaneous detection by fluorescence and diode-array detectors. The major capsaicinoids, two tocopherols and 43 carotenoid components, were simultaneously separated, detected, and identified in the appointed pepper powder (containing Capsicum annuum and Capsicum frutescens) for method validation. The separation was performed on a Nucleosil C18 reverse phase column and optimized gradient elution. Resolution ranged between 0.96 and 1.46 with the highest values corresponding to γ-tocopherol and α-tocopherol. The limits of detection and quantification of target compounds ranged between 18.77 and 148.08 ng mL^?1. Recoveries were between 89.83–100.26 and 79.72–88.86% when standard materials were spiked at low and high amounts, respectively. The most sufficient extraction of the different phytonutrients was achieved by mixture of methanol and acetone, although it was only slightly better than the mixture of methanol and acetonitrile. These results suggest that the developed method could be used for rapid, one-step determination of a wide range of phytonutrients in chili and pepper powders.     

Optimization of Matrix Solid-Phase Dispersion method for simultaneous extraction of aflatoxins and OTA in cereals and its application to commercial samples

A method based on Matrix Solid-Phase Dispersion (MSPD) has been developed for the determination of 5 mycotoxins (ochratoxin A and aflatoxins B and G) in different cereals. Several dispersants, eluents and ratios were tested during the optimization of the process in order to obtain the best results. Finally, samples were blended with C₁₈ and the mycotoxins were extracted with acetonitrile. Regarding to matrix effects, the results clearly demonstrated the necessity to use a matrix-matched calibration to validate the method. Analyses were performed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-QqQ-MS/MS). The recoveries of the extraction process ranged from 64% to 91% with relative standard deviation lower than 19% in all cases, when samples were fortified at two different concentrations levels. Limits of detection ranged from 0.3 ng g⁻¹ for aflatoxins to 0.8 ng g⁻¹ for OTA and the limits of quantification ranged from 1 ng g⁻¹ for aflatoxins to 2 ng g⁻¹ for OTA, which were below the limits of mycotoxins set by European Union in the matrices evaluated. Application of the method to the analysis of several samples purchased in local supermarkets revealed aflatoxins and OTA levels.     

Development and validation of a solid-phase extraction method coupled to high-performance liquid chromatography with ultraviolet-diode array detection for the determination of sulfonylurea herbicide residues in bovine milk samples

This study proposes a fast, simple and sensitive liquid chromatography diode array detector (LC/UV-DAD)-based method for the simultaneous determination of eight sulfonylurea herbicides (bensulfuron methyl, chlorsulfuron, metsulfuron methyl, primisulfuron methyl, rimsulfuron, thifensulfuron methyl, triasulfuron and tribenuron methyl) in bovine whole milk at concentrations lower than the default limit of 0.01 mg kg(-1) allowed by current legislation (Regulation EC/396/2005 and following Annexes). An effective one-step solid phase extraction (SPE) and clean up procedure was defined with use of Chem Elut cartridges, providing good recoveries for all the analytes tested and with no matrix effects affecting method accuracy. Separation of herbicides was obtained on a C(18) column by acetonitrile- water gradient elution. Method validation has been performed according to European Commission Decision 2002/657/EC criteria, in terms of linearity, recovery, precision, specificity, decision limit (CC(α)) and detection capability (CC(β)). Typical recoveries ranged between 78.4% and 99.7%, at the maximum residue limits (MRLs) levels established by Regulation EC/396/2005, with relative standard deviations (RSD) no larger than 10%.     

Development and validation of an HPLC method for the simultaneous determination of clozapine and desmethylclozapine in plasma of schizophrenic patients

Summary A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of levels of clozapine (CLZ) and its active metabolite N-desmethylclozapine (DMC) in human plasma. The analysis was performed on a 5 μm C8 reversed phase column (150×4.6 mm i.d.), with acetonitrile-phosphate buffer (pH 3.5), as the mobile phase. The detection voltage was +800 mV and the cell and column temperature were 50°C. Linear responses were obtained between 2 ng mL⁻¹ and 100 ng mL⁻¹. Absolute recovery for both clozapine and desmethylclozapine exceeded 88% and the detection limit was 1 ng mL⁻¹. Repeatability, intermediate precision and accuracy were satisfactory. The method, which is rapid, sensitive and selective, has been applied to therapeutic drug monitoring in schizophrenic patients following administration of Leponex^? tablets. In 21 patients in steady state at a mean daily clozapine dosage of 358 mg (ranging from 150 to 500 mg day⁻¹), clozapine levels averaged 379 ng mL⁻¹ (ranging from 102 to 818 ng mL⁻¹) and DMC levels averaged 233 ng mL⁻¹ (ranging from 70 to 540 ng mL⁻¹). The method requires only a very small amount of plasma (100 μL), and thus it is suitable for pharmacokinetic studies, as well as for therapeutic drug monitoring.     

Rapid and quantitative extraction method for the determination of chlorophylls and carotenoids in olive oil by high-performance liquid chromatography

Colour is an organoleptic characteristic of virgin olive oil and an important attribute that affects the consumer perception of quality. Chlorophylls and carotenoids are the main pigments responsible for the colour of virgin olive oil. A simple analytical method for the quantitative determination of chlorophylls and carotenoids in virgin olive oils has been developed. The pigments were isolated from small samples of oil (1.0 g) by solid-phase extraction using diol-phase cartridges (diol-SPE), and the extract was analysed by reverse-phase HPLC with diode-array UV detection. Chromatographic peak resolution, reproducibility (coefficient of variation (C.V.) <4.5%) and recovery (>98.4%) for each component were satisfactory. A comparative study of the proposed method was performed versus classical liquid-liquid extraction (LLE) with N,N′-dimethylformamide and solid-phase extraction using a C18 column (C18-SPE). While 96.4% of the pigments were recovered by LLE, only 51.3% were isolated by C18-SPE in comparison to diol-SPE. Likewise, a higher alteration of pigment composition was observed when such LLE and C18-SPE procedures were used. In this sense, a higher ratio of pheophytin in comparison to that isolated by the diol-SPE procedure was achieved with both extraction procedures, indicating a greater extent of the pheophytinization reaction. Therefore, quantification of pigments from virgin olive oil by diol-SPE followed by RP-HPLC was found to be rapid, simple, required only a small amount of sample, consumed only small amounts of organic solvents, and provided high recoveries, accuracy and precision.     

Simple reversed-phase liquid chromatography method for determination of tocopherols in edible plant oils

A simple and rapid reversed-phase high-performance liquid chromatography method for determination of alpha-, (beta + gamma), and delta-tocopherols in edible plant oils has been developed. Oils are diluted in 2-propanol and injected directly onto Symmetry C18 column. Methanol and acetonitrile (1:1) are used as a mobile phase. Tocopherols are detected using fluorescence detector set at excitation and emission wavelength 295 nm and 325 nm, respectively. The method is precise (R.S.D. not higher than 2.24%) and sensitive-detection limits (DL) are 8 ng/ml for gamma- and delta-tocopherols and 28 ng/ml for alpha-tocopherol; quantification limits (QL) were calculated as three times higher than DL.     

Derivative spectrum chromatographic method for the determination of trimethoprim in honey samples using an on-line solid-phase extraction technique

A simple, selective and rapid analytical method for determination of trimethoprim (TMP) in honey samples was developed and validated. This method is based on a SPE technique followed by HPLC with photodiode array detection. After dilution and filtration, aliquots of 500 μL honey samples were directly injected to an on-line SPE HPLC system. TMP was extracted on an RP SPE column, and separated on a hydrophilic interaction chromatography column during HPLC analysis. At the first detection step, the noise level of the photodiode array data was reduced with two-dimensional equalizer filtering, and then the smoothed data were subjected to derivative spectrum chromatography. On the second-derivative chromatogram at 254 nm, the limit of detection and the limit of quantification of TMP in a honey sample were 5 and 10 ng/g, respectively. The proposed method showed high accuracy (60-103%) with adequate sensitivity for TMP monitoring in honey samples.     

Development of solid-phase extraction method and its application for determination of hydrochlorothiazide in human plasma using HPLC

A high-performance liquid chromatographic method was developed, validated and applied for the determination of hydrochlorothiazide in human plasma. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of hydrochlorothiazide and internal standard were investigated. The method involves solid-phase extraction on RP-select B cartridges followed by isocratic reversed-phase chromatography on a Hibar Lichrospher 100 RP-8 column with UV detection at 230 nm. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. Limit of quantification was 10 ng mL(-1). The method has been implemented to monitor hydrochlorothiazide levels in patient samples.