Simultaneous analysis of Vitamins A and E in infant milk-based formulae by normal-phase high-performance liquid chromatography-diode array detection using a short narrow-bore column

A rapid, simple and reproducible normal-phase (NP) high-performance liquid chromatography (HPLC)-diode array detection (DAD) method for simultaneous qualitative and quantitative determination of Vitamin A (retinol acetate and retinol palmitate) and Vitamin E (alpha-tocopherol acetate, alpha-, gamma- and delta-tocopherols) in milk-based infant formulae was developed and validated. The preparation sample was based on protein precipitation and vitamin extraction with ethanol, followed by re-extraction with hexane, while the chromatographic method was based on the use of a short narrow-bore column (50 mm x 2.1 mm; 3 microm particle size), which afforded less solvent consumption and higher mass sensitivity. The method showed acceptable values for precision, recovery and sensitivity, and proved very simple for routine analysis work.     

Determination of vitamins A and E in serum and plasma using a simplified clarification method and high-performance liquid chromatography

A method of sample clarification and high-performance liquid chromatography specifically developed to permit precise and rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in serum and plasma is reported. Serum proteins were denatured by the addition of acetonitrile containing alpha-tocopherol acetate, the internal standard; the vitamins were subsequently extracted into an organic matrix consisting of ethyl acetate-butanol (1:1); no solvent evaporation step was required. The three vitamins of interest were eluted from a reversed-phase C18 column with an isocratic mobile phase methanol-water (95:5); detection was accomplished by measuring ultraviolet absorption at 280 nm. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from spiked aqueous samples averaged 100.0, 100.0 and 98.8%, respectively. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from plasma and serum relative to water were 102.6, 96.9 and 96.5%, respectively. Retinol and alpha-tocopherol were stable in the extraction matrix for up to 3.5 h, and were stable in heparinized plasma stored at room temperature for two days. Oxalate, citrate and EDTA caused significant losses of retinol and alpha-tocopherol, while vitamin levels in serum and heparinized plasma were similar. Limits of detection for retinol and alpha-tocopherol were 60 ng/ml and 0.9 micrograms/ml, respectively. Each run required 12 min. Same-day coefficients of variation were 3.5 and 3.6% for retinol and alpha-tocopherol, respectively (n = 11). Between-day coefficients of variation for retinol and alpha-tocopherol were 4.8 and 5.5%, respectively (n = 5). This method permits simple, rapid, sensitive, selective and precise determination of retinol and alpha-tocopherol using 0.5 ml of serum or heparinized plasma.     

Rapid determination by reversed-phase high-performance liquid chromatography of Vitamins A and E in infant formulas

A rapid, sensitive method has been developed for the simultaneous determination of retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate. We compare two experimental procedures for simultaneous direct solvent extraction of these vitamins without previous saponification. Method I: the fat milk sample was extracted with ethanol-hexane and injected directly into the chromatographic column. Method II: the power milk sample was extracted with ethanol-hexane and also injected directly into the column. Under optimum conditions the limits of detection for retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate were 0.33, 21.2, 32.9, 32.5 and 3.2 ng and the limits of quantification were 0.42, 25.3, 37.9, 36.8 and 6.3 ng, respectively. The precision results showed that the relative standard deviations of repeatability and reproducibility were between 0.74 and 5.7%.     

Development and validation of a liquid chromatography method for the simultaneous determination of alpha-tocopherol, retinol and retinyl esters in human serum using a monolithic column for the monitoring of anticancer therapy side effects

Among other side effects, administration of anticancer agents is accompanied by manifestations of gastrointestinal toxicity and disturbances of antioxidant balance. The monitoring of these toxic effects in clinical practice is impeded by a dearth of reliable laboratory methods. Therefore, a simple and rapid reversed-phase high-performance liquid chromatography procedure for selective and sensitive determination of retinol, a-tocopherol, and retinyl esters (retinyl-palmitate and retinyl-stearate) in blood serum has been developed and presented in this study. A Series 200 LC HPLC instrument from Perkin Elmer (Norwalk, USA) with diode-array detector (DAD) was used for the analysis. Separations of retinol, alpha-tocopherol, retinyl-palmitate, and retinyl-stearate were performed using a Chromolith Performance RP-18e, 100 x 4.6 mm monolithic column from Merck (Darmstadt, Germany). Gradient elution was used at a flow rate of 3 mL/min; the mobile phase was methanol-water (95:5, v/v) for 0-2.1 min and methanol-2-propanol (60:40, v/v) for 2.1-4.9 min. The total time of analysis was 6 min. The injection volume was 20 microL and the analysis was performed at ambient temperature. Detection of retinol, alpha-tocopherol, and retinyl esters was carried out at 325, 295, and 330 nm, respectively. For practical assessment of the method, the vitamin A absorption test was performed on seven healthy controls as well as on six patients with non-small cell lung carcinoma or head and neck carcinoma previously treated by chemotherapy and/or radiotherapy, six patients with rectal carcinoma before chemoradiotherapy, four patients with gastrointestinal stromal tumor (GIST) before treatment with imatinib, and a breast cancer patient with chemotherapy-induced diarrhea. Present data demonstrate the feasibility of large scale HPLC determination of vitamin E, vitamin A, and retinyl esters in human serum using a silica monolithic column, and this method may represent a valuable aid in the laboratory monitoring of the toxicity of anticancer therapy.     

固相萃取和高效液相色谱相结合快速测定苦瓜甙A的含量(英文)

 建立了一个快速、简单、准确的固相萃取和高效液相色谱相结合的测定苦瓜甙A含量的方法。样品经石墨碳固相萃取管 (3mL/ 2 5 0mg)纯化后以高效液相色谱检测。色谱柱为C18,流动相为V(乙腈 )∶V(甲醇 )∶V(5 0mmol/L磷酸二氢钾缓冲液 ) =2 5∶2 0∶6 0 ,流速为 0 .8mL/min ,检测波长为 2 0 8nm。标准曲线自 10mg/L到 10 0 0mg/L呈线形关系 (r2 =0 .9992 )。该方法具有很好的重现性 ,日内或日间的相对标准偏差和相对平均误差均小于 10 %。样品回收率大于 90 %。     

Preparation of functionalized magnetic nanoparticulate sorbents for rapid extraction of biphenolic pollutants from environmental samples

A new solid-phase extraction coupled with magnetic carrier technology was developed for extraction of bisphenol A (BPA) and diethylstilbestrol (DES) from water samples. The SPE sorbents, functionalized magnetic nanoparticles (Fe₃O₄@SiO₂/β-CD, core/shell), were synthesized in a two-stage system. The material was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and a vibrating sample magnetometer. SPE extraction parameters, such as volume and pH of sample, adsorption time, and desorption conditions were optimized. Under selected conditions: 250 mL of water sample, 0.1 g of sorbents and elution with methanol (3 mL with 1% acetic acid), the extraction was completed in 25 min. SPE followed by HPLC was employed to determine BPA and DES in environmental samples. The developed method provided spiked recoveries of 80–105%, relative standard deviations of less than 7%, and LOD of BPA (20.0 ng/L) and DES (23.0 ng/L), respectively. The proposed method offered easy preparation of sorbents, rapid analysis, high enrichment yields, and reliable quantitative assay.     

Sensitive determination of Fenamiphos in water samples by flow injection photoinduced chemiluminescence

In this work, a sensitive flow injection chemiluminescence (FI-CL) method for the determination of nematicide Fenamiphos in a rapid and simple way is proposed. Fenamiphos is first photodegraded in basic medium. These photofragments react with Ce(IV) providing the chemiluminescence signal. To the authors’ knowledge, no chemiluminescence method has been described in the literature for the determination of the nematicide Fenamiphos. All physical and chemical parameters in the flow injection chemiluminescence system were optimized in order to obtain the best sensitivity, selectivity and sample throughput. Before the injection of the sample in the FI-CL system, a preconcentration step with solid phase extraction C18 cartridges was performed. By applying solid phase extraction (SPE) to 250?mL of standard (final volume 10?mL), the linear dynamic range was between 3.4 and 60?µg?L^?1, and the detection limit was 1?µg?L^?1. When SPE was applied to 500?mL of standard (final volume 10?mL), the detection limit was 0.5?µg?L^?1. These detection limits are below the emission limit value established by the Spanish Regulations of the Hydraulic Public Domain for pesticides (50?µg?L^?1) and of the same order as the limit established for total pesticides (0.5?µg?L^?1) at European Directive on the quality of water for human consumption. The sample throughput was 126 hour^?1. Intraday and interday coefficients of variation were below 10% in all cases. No interference was registered in presence of usual concentrations of anions, cations and other organophosphorus pesticides. The method was successfully applied to the analysis of environmental water samples, obtaining recoveries between 96 and 107.5%.     

Flow Injection Chemiluminescence Determination of Retinol and α-Tocopherol in Blood Serum and Pharmaceuticals

A simple and sensitive flow-injection method is reported for the determination of retinol and α-tocopherol in human blood serum and pharmaceuticals. The method is based on the reduction of vanadium(V) by retinol and α-tocopherol and subsequent reaction of reduced vanadium with luminol to generate chemiluminescence signal. The optimized conditions allow a linear calibration range of 30–2850 µg L^?1 and 5–4300 µg L^?1 for retinol and α-tocopherol, with relative standard deviations of 1.2–4.6% and 1.5–5.6%, respectively. The detection limits for retinol and α-tocopherol, defined as three times the standard deviation of measured blanks were 23 µg L^?1 and 2.15 µg L^?1, respectively. The proposed method allowed up to 20 determinations h^?1. The tolerance amount of foreign ions/compounds on the determination of retinol and α-tocopherol was also examined. The method was applied to the determination of retinol and α-tocopherol in human blood serum and pharmaceutical samples using hexane extraction with recoveries in the range of 92 ± 2 to 96 ± 1%, and the results obtained were compared with HPLC reference method.     

Simple and rapid high-performance liquid chromatographic method for endogenous alpha-tocopherol determination in human plasma

A simple and rapid reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of endogenous alpha-tocopherol in human plasma. Following addition of alpha-tocopheryl acetate as the internal standard, the plasma was deproteinized using acetonitrile and isopropanol mixture prior to HPLC analysis. Methanol was used as the mobile phase and the effluent was quantitated at 292 nm. By this developed method, the concentrations of alpha-tocopherol were linearly related to their responses in the range of 0.8-30 microg/mL. The relative standard deviations intra-day and inter-day for alpha-tocopherol in plasma were less than 10%. The percentage of bias was within +/-4%, which confirmed the accuracy of the method. The method has been successfully applied for determining endogenous alpha-tocopherol in healthy Thai male volunteers.     

The Use of Internal Surface Reversed-Phase Packing for the Solid Phase Extraction of Drugs from Serum

Abstract

The utilization of Internal Surface Reversed Phase (ISRP) packing as a solid phase extraction (SPE) matrix was investigated. Evaluation of the relative retention of nineteen medicinal agents on ISRP material was monitored using HPLC. The effects of altering pH and/or buffer concentration on retention were further studied using verapamil, phenelzine and tamoxifen as model compounds. Spiked serum samples containing the model compounds plus amitriptyline were also subjected to ISRP-SPE and HPLC. Verapamil, phenelzine, and tamoxifen were all strongly retained on ISPR as pH increased. The buffer concentration of the sample was not as critical on retention of these compounds as pH. Verapamil was quantitatively recovered from spiked serum samples (103 /pm 8.5%, n = 6), with a limit of detection of 10 ng/mL using fluorescent detection (γex = 280 nm, γem = 310 nm). Amitriptyline recovery was also quantitative (99.0/pm 5.3%, n = 6), and its limit of detection was 10 ng\mL employing short wavelength UV detection (γ = 214 nm). The ISRP packing as an SPE matrix failed to adequately disrupt the binding of either phenelzine or tamoxifen from serum proteins; therefore, recoveries of these compounds were poor (<50%). A comparison of ISRP to conventional C18 and C2 SPE columns indicated that the ISRP packing eliminated interferences as well or better than the conventional columns.     

Automated online pretreatment and cleanup recycle coupled with high‐performance liquid chromatography‐mass spectrometry for determination of deca‐bromodiphenyl ether in human serum

Automated online SPE‐HPLC‐MS was established for the determination of deca‐bromodiphenyl ether in human serum. The online SPE with large volume injection was utilized to enhance the sensitivity. Online SPE with dilution line greatly decreased matrices effect, which enabled serum samples to be injected directly into pre‐column. Washing line was designed for the system to solve the serious residual phenomenon and reduce the risk of sample wastage and contamination. Under the optimized conditions, the linear of the method was in the range 0.1–10 ng/mL with the LOD of 0.026 ng/mL. The recoveries of serum samples spiked with deca‐bromodiphenyl ether at 0.5 ng/mL was in the range from 83.30 to 102.7% with RSD in interday less than 8.67%. The satisfactory results demonstrated that the method of online sample pretreatment and cleanup recycle were reliable for human serum analysis.     

Rapid determination of cyclamate in foods by solid-phase extraction and capillary electrophoresis

A method for the determination of cyclamate in food was developed using solid-phase extraction (SPE) and capillary electrophoresis (CE) with indirect ultraviolet (UV) detection. A 5-10 g sample in 0.1 mol/L hydrochloric acid was homogenized and made up to a volume of 50 mL with 0.1 mol/L hydrochloric acid. After the sample was centrifuged, 25 mL of supernatant was loaded into an Oasis HLB SPE cartridge. The cartridge was washed with 2 mL of demineralized water followed by 2 mL of 50% aqueous methanol, and cyclamate was eluted with 4.5 mL of 50% aqueous methanol. The eluate was added to a solution of sodium propionate (internal standard) for CE analysis. The cyclamate in the eluate was electrophoresed on a fused-silica capillary using 1 mmol/L hexadecyltrimethylammonium bromide and 10 mmol/L potassium sorbate as a running buffer. Detection and reference wavelengths of cyclamate determined with a UV detector were 300 and 254 nm, respectively. The calibration curves for cyclamate showed good linearity in the range of 2-1000 microg/mL and the limits of detection in beverage, fruit in syrup, jam, pickles and confectionary are sample dependent and ranged from 5-10 microg/g. The recovery of cyclamate added at a level of 200 microg/g to various kinds of foods was 93.3-108.3% and the relative standard deviation was less than 4.9% (n=3). A number of commercial samples were analyzed using the proposed method. Cyclamate was detected in one waume, two pickles, and two sunflower seeds. The quantitative values determined with CE correlated to those from high-performance liquid chromatography (HPLC) (the detected values of cyclamate in a sunflower seed measured by CE and HPLC were 3.40 g/kg and 3.51 g/kg, respectively). This analytical method for cyclamate using CE is especially suitable for use in the field.     

Liquid chromatographic determination of carotenoids and vitamins A and E in multivitamin tablets

Carotenoids and vitamins A and E in multivitamin tablets can be determined simultaneously by reversed-phased liquid chromatography (LC) with a programmable UV detector. Samples were dissolved in dimethyl sulfoxide and then extracted with hexane. A portion was injected onto a Symmetry C18, 150 x 4.6 mm id, 5 microns column and chromatographed with a mobile phase of acetonitrile--0.25% ammonium acetate in methanol and 0.05% triethylamine in dichloromethane. A step gradient was used. The system was operated at 25 degrees C with a flow rate of 1.5 mL/min. UV detection was at 325 nm for retinols, 285 nm for tocopherols, and 450 nm for carotenoids. Detection limits were less than 0.3 ng for retinol and retinyl acetate; 2 ng for alpha-tocopherol acid succinate; 10 ng for alpha-tocopherol, gamma-tocopherol, and alpha-tocopherol acetate; and 0.4 ng for alpha-carotene and beta-carotene. Intraday and interday coefficients of variation ranged from 1.40 to 5.20%. The sample preparation method and LC assay are practical for quality control and routine analysis of multivitamin tablets.     

Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues.

Two high-performance liquid chromatography (HPLC) procedures, a rapid normal-phase isocratic method for the analysis primarily of retinol and retinoic acid on a 3 mu silica column, and a reversed-phase gradient method for the simultaneous analysis of retinoids and very polar to nonpolar carotenoids on a 3 mu C18 column, are described. The normal-phase isocratic HPLC procedure is rapid (12 min), requires a sample size of 100 microl or less of serum, and is suitable for routine analysis of retinol in any serum, and of retinol and retinoic acid in serum after administration of retinoic acid. The reversed-phase gradient method is suitable for the simultaneous analysis of very polar to nonpolar carotenoids such as epoxy-xanthophylls and xanthophyll esters, along with other carotenoids and retinoids that occur normally in human serum and other plant and animal tissues. A run time of 30-70 min is necessary, depending on the presence or absence of xanthophyll esters in the sample.     

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.     

A Simple HPLC Method for the Determination of Trazodone Human Serum

Abstract

A simple HPLC method with minimal sample preparation and good reproducibility for the determination of trazodone in serum is described. Basified serum samples were extracted using ethyl acetate containing diazepam as the internal standard (IS). Chromatography was performed on a cyanopropylsilane column with 15 μL sample injection. The mobile phase consisted of 0.02 M ammonium phosphate, pH 7.5 : acetonitrile (70:30 v/v). The eluent was monitored at 220 nm. The serum standard curve was linear from 10.0 to 8000.0 ng/mL serum. The overall within-run quality control CV was 6.3% for five concentrations (20.0, 40.0, 100.0, 250.0 and 1000.0 ng/mL) and the overall recovery from serum was 85.4%. This method has been applied to the analysis of human serum samples.     

Method to determine 4-oxo-retinoic acids, retinoic acids and retinol in serum and cell extracts by liquid chromatography/diode-array detection atmospheric pressure chemical ionisation tandem mass spectrometry

Retinoic acid isomers, 4-oxo-retinoic acid isomers and retinol are present in the serum of mammals. In this study a high-performance liquid chromatography (HPLC) separation, sample preparation and tandem mass spectrometry (MS/MS) method was established for quick and easy sample preparation and sensitive determination of retinoids such as all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid and retinol in serum and cell extracts. Serum samples were simply treated with three times the volume of isopropanol, dried under vacuum, taken up in the HPLC solvent and immediately put into the autosampler for an automated single-run HPLC analysis. With this MS/MS method we were able to detect 7 pg and quantify 20 pg of all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid and retinol directly on-column and were able to determine a concentration as low as 0.2 ng/mL in ethanolic standards and in biological samples. This method allows ultra-sensitive detection, excellent selectivity and a very simple sample preparation to determine retinoic acids, 4-oxo-retinoic acids and retinol in serum and cell extracts for the study of endogenous retinoids.     

Assay of naproxen in rat serum by high-performance thin-layer chromatography/densitometry

A simple, sensitive, and reproducible high-performance thin-layer chromatographic method using densitometry is presented for the determination of naproxen in rat serum. Only 0.1 mL serum was used for extraction. Separations were performed on 10 x 10 cm plates coated with silica gel, with toluene-ethyl acetate-acetic acid (82 + 15 + 3) as the mobile phase. Benzophenone was used as the internal standard. Quantification was performed by densitometry at 260 nm. The response response for naproxen was linear (r = 0.992) over the range 2-100 mg/L. Method validation demonstrated good recoveries (92-96%), sensitivity (limit of quantitation, 1 mg/L), repeatability of sample application (4%), repeatability of the method (8%), and intermediate precision (5%). The procedure was applied to the quantitation of naproxen in rat serum.     

On-line solid-phase extraction-HPLC-fluorescence detection for simultaneous determination of puerarin and daidzein in human serum

Response surface methodology (RSM) was applied to the optimization of on-line solid-phase extraction (SPE) parameters, and an automated system of on-line SPE coupled with high-performance liquid chromatography (HPLC) with fluorescence detection was developed for the determination of puerarin and daidzein in human serum. The human serum sample of 50 μL was injected into a conditioned C18 SPE cartridge, and the matrix was washed out with acetonitrile-KH₂PO₄-triethylamine buffer (0.01 M, pH 7.4) (3:97, v/v) for 3 min at a flow rate of 0.25 mL/min. Then the target analytes were eluted and transferred to the analytical column. A chromatographic gradient elution was programmed with the mobile phase consisting of acetonitrile and KH₂PO₄-triethylamine buffer, and the analytes were determined with a fluorescence detector at excitation wavelength of 350 nm and emission wavelength of 472 nm, respectively. The proposed method presented good linear relations (0.85-170 μg/mL for puerarin and 0.2-40 μg/mL for daidzein), satisfactory precision (RSD < 8%), and accredited recovery (92.5-107.8%).     

Simultaneous determination of retinol and tocopherols by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method to determine retinol and all four tocopherols (alpha-, beta-, gamma- and delta-) simultaneously was established using a reversed-phase column (YMC-PACK A-302 S-5 120A ODS). The HPLC conditions were mobile phase 65% isopropanol, sample solvent 99.5% methanol and temperature 30 degrees C. Retinol and tocopherols were measured in rat liver.