Determination of the isoflavone genistein in soybeans by high-performance liquid chromatography following cloud point extraction

A simple and sensitive method has been developed for preconcentration and determination of genistein in soybeans. This method is based on cloud point extraction (CPE) of genistein from soybeans using ethylene glycol monoalkyl ether (Genapol X-080) as a nonionic surfactant. The concentration of extracted genistein was determined by HPLC with a UV detector. Optimum experimental conditions were established. With 5% Genapol X-080 (v/v), a liquid/solid ratio of 25:1 mL/g, and ultrasonic-assisted extraction at 40 degrees C for 45 min, the extraction percentage of genistein reached its highest value. The preconcentration factor for genistein was about 16.5. The RSD for seven replicate measurements and the LOD were +/- 4.45% and 15.0 ng/mL, respectively. CPE is simple, inexpensive, and suitable for extraction of genistein from soybean. It uses environmentally friendly surfactants and offers a convenient alternative to more conventional extraction systems.     

Study of cloud point extraction and high-performance liquid chromatographic determination of isoniazid based on the formation of isonicotinylhydrazone

Isoniazid (INH) reacted with p-dimethylaminobenzaldehyde (DABD) in the presence of trichloroacetic acid to give isonicotinylhydrazone (INZ) having λ_max 365 nm. Cloud point extraction (CPE) is carried out to extract INH and IHZ in aqueous solutions using surfactant poly(ethylene glycol) 4000 (PEG4000), respectively. Langmuir model is used to study the adsorption behaviors of the two solutes on micelles of PEG4000. A linear correlation is found between variation of PEG4000 concentration required for feed concentration of the two solutes and used to predict PEG4000 concentration required for extracting INH and IHZ in CPE procedure. The results calculated show that, for a desired recovery level of 90%, only can IHZ be sufficiently extracted by PEG4000. In this experiment, the feed concentration of PEG4000 is defined by above-mentioned correlation, and the effects of other operating parameters, e.g., concentration of salt, pH and centrifugation time on extraction of PEG4000-IHZ system have also been studied in detail. The proposed CPE method coupled with HPLC-UV system is successfully used for the determination of INH in urine sample.     

Release and extraction of letrozole in blood plasma using resorcinol functionalized multi-walled carbon nanotube coupled with high-performance liquid chromatography

In this research, carboxylated multi-walled carbon nanotube was functionalized by cyanuric chloride and modified by 1,3 dihydroxybenzene (MWCNT-resorcinol). MWCNT-resorcinol was used as an adsorbent for extraction and determination of letrozole in human blood plasma prior to high-performance liquid chromatography equipped with ultraviolet detector (HPLC-UV). Synthesized carbon nanotubes were modified and characterized using Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The efficiency of the adsorbent for adsorption and release of letrozole in the blood plasma was evaluated at the optimized condition. An adsorption efficiency of 96.9% was achieved at pH of 4 and the extraction time of 20?min. A recovery percentage of 92.7% for the drug in plasma was also obtained. Lastly, the release efficiency values for letrozole from MWCNTs-resorcinol in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) were calculated as 91% and 70%, respectively. Such results enhanced the potential ability of the MWCNTs-resorcinol as a drug delivery substrate.     

Full automation of catecholamine metabolite determination by column switching and high-performance liquid chromatography

The urinary catecholamine metabolites, vanimandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid, were extracted on a silica-bonded strong-anion-exchanger cartridge (SAX) and then injected into an high-performance liquid chromatographic (HPLC) system by column switching. Chromatography was performed on a reversed-phase analytical column with electrochemical detection. Full automation was obtained by coupling two devices: a solid-phase automatic sampler and intelligent autosampler. For each substance the recovery was greater than 95% and the coefficient of variation was ca. 3%; the analysis takes 11 min. Substance instability problems are overcome, because the samples are extracted and injected in rapid succession. The normal values and correlation with manual HPLC were established for a large number of samples.     

Use of cloud point extraction methodology for the determination of PAHs priority pollutants in water samples by high-performance liquid chromatography with fluorescence detection and wavelength programming

The cloud point extraction methodology has been used to develop a new procedure for the preconcentration of polycyclic aromatic hydrocarbons in water samples (bottled and network supply). Triton X-114 was used as extractant agent. The procedure, consisting of three steps (preconcentration into the surfactant, clean-up and concentration of the eluate), allows good detection limits to be reached (from nanograms per liter to even sub-nanograms per liter in some cases). Determination was carried out by HPLC, with fluorimetric detection and wavelength programming.     

Stability-indicating high-performance column liquid chromatography and high-performance thin-layer chromatography methods for the determination of olopatadine hydrochloride in tablet dosage form

This paper describes two simple, specific, accurate, and precise methods for estimation of olopatadine hydrochloride (OLO) in tablet dosage form. The first method is a stability-indicating isocratic RP-HPLC method. The analysis is performed on an RP-18 column using 0.1% orthophosphoric acid (adjusted to pH 4.5 with triethylamine)-acetonitrile (75 + 25, v/v) mobile phase at a flow rate of 1 mL/min. Paracetamol (PAR) was selected as the internal standard. Retention times of OLO and PAR were 11.30 +/- 0.02 and 4.70 +/- 0.03 min, respectively. For the HPTLC method, precoated silica gel 60 F254 aluminum sheets were used as the stationary phase; the mobile phase was methanol-chloroform-ammonia (8 + 2 + 0.1, v/v/v). The detection of the analyte band was carried out at 301 nm, and its Rf value was 0.46 +/- 0.03. The analytical methods were validated according to International Conference on Harmonization guidelines. Linear regression analysis data for the calibration plots showed a good linear relationship between response and concentration in the range of 0.1-1 microg/mL and 0.1-0.9 microg/band for HPLC and HPTLC, respectively.     

Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a reliable pressurized liquid extraction (PLE) and HPLC method was developed for simultaneous determination of 15 flavonoids, namely hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2'-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed by using a Zorbax SB-C18 analytical column (250 mm x 4.6 mm I.D., 5 microm) at gradient elution of water and acetonitrile with diode-array detection (270 nm). All calibration curves showed good linearity (r(2)>0.9997) within test ranges. The LOD and LOQ were lower than 1.31 ng and 2.62 ng on column, respectively. The RSD for intra- and inter-day of 15 analytes was less than 3.8% at three levels, and the recoveries were 90.5-106.8%. The validated method was successfully applied for the analysis of 15 flavonoids in different species of Epimedium which had great variation on the contents of investigated flavonoids. Hierarchical clustering analysis based on the characteristics of 15 investigated compound peaks in HPLC profiles showed that 26 samples were divided into three main clusters, which were in accordance with their flavonoid contents. Four flavonoids including epimedin A, B, C and icariin were optimized as markers for quality control of the species of Epimedium used as Yinyanghuo.     

Cold column trapping-cloud point extraction coupled to high performance liquid chromatography for preconcentration and determination of curcumin in human urine

A cold column trapping-cloud point extraction (CCT-CPE) method coupled to high performance liquid chromatography (HPLC) was developed for preconcentration and determination of curcumin in human urine. A nonionic surfactant, Triton X-100, was used as the extraction medium. In the proposed method, a low surfactant concentration of 0.4% v/v and a short heating time of only 2 min at 70 °C were sufficient for quantitative extraction of the analyte. For the separation of the extraction phase, the resulted cloudy solution was passed through a packed trapping column that was cooled to 0 °C. The temperature of the CCT column was then increased to 25 °C and the surfactant rich phase was desorbed with 400 μL ethanol to be directly injected into HPLC for the analysis. The effects of different variables such as pH, surfactant concentration, cloud point temperature and time were investigated and optimum conditions were established by a central composite design (response surface) method. A limit of detection of 0.066 mg L⁻¹ curcumin and a linear range of 0.22–100 mg L⁻¹ with a determination coefficient of 0.9998 were obtained for the method. The average recovery and relative standard deviation for six replicated analysis were 101.0% and 2.77%, respectively. The CCT-CPE technique was faster than a conventional CPE method requiring a lower concentration of the surfactant and lower temperatures with no need for the centrifugation. The proposed method was successfully applied to the analysis of curcumin in human urine samples.     

Multiwalled carbon nanotubes as adsorbents of solid-phase extraction for determination of polycyclic aromatic hydrocarbons in environmental waters coupled with high-performance liquid chromatography

Multiwalled carbon nanotubes (MWCNTs) were used as a novel kind of solid-phase extraction adsorbents in this work as well as an analytical method based on MWCNTs solid-phase extraction (SPE) combined with high-performance liquid chromatography (HPLC) was established for the determination of polycyclic aromatic hydrocarbons (PAHs), some of which belong to typical persistent organic pollutants (POPs) owing to their carcinogenicity and endocrine disrupting activity. Several conditions that probably affected the extraction efficiency including the eluent volume, sample flow rate, sample pH and the sample volume were optimized in detail. The characteristic data of analytical performance were determined to investigate the sensitivity and precision of the method, and the method was applied to the determination of PAHs in environmental water samples such as river water sample, tap water sample and wastewater sample from the constructed wetland effluent. The experimental results indicated that there were excellent linear relationship between peak area and the concentration of PAHs over the range of 0.04-100 microg L(-1), and the precisions (RSD) were 1.7-4.8% under the optimal conditions. The detection limits of proposed method for the studied PAHs were 0.005-0.058 microg L(-1) (S/N=3). The recoveries of PAHs spiked in environmental water samples ranged from 78.7 to 118.1%. It was concluded that MWCNTs packed cartridge coupled with HPLC was an excellent alternative for the routine analysis of PAHs at trace level.     

Catanionic surfactant ambient cloud point extraction and high-performance liquid chromatography for simultaneous analysis of organophosphorus pesticide residues in water and fruit juice samples

A mixed anionic–cationic surfactant cloud point extraction (CPE) has been developed using sodium dodecyl sulfate (SDS) and tetrabutylammonium bromide (TBABr) for the extraction and preconcentration of organophosphorus pesticides (OPPs) at ambient temperature before analysis by high-performance liquid chromatography. The studied OPPs were azinphos-methyl, parathion-methyl, fenitrothion, diazinon, chlorpyrifos, and prothiophos. The optimum conditions of the mixed anionic–cationic CPE were 50 mmol L⁻¹ SDS, 100 mmol L⁻¹ TBABr, and 10% (w/v) NaCl. The extracted OPPs were successfully separated within 11 min using the conditions of a Waters Symmetry C8 column, a flow rate of 0.8 mL min⁻¹, a gradient elution of methanol and water, and detection at 210 nm. Linearity was found over the range 0.05–5 μg mL⁻¹, with the correlation coefficients higher than 0.996. The enrichment factor of the target analytes was in the range 6–11, which corresponds to their limits of detection from 1 to 30 ng mL⁻¹. High precisions (intra-day and inter-day) were obtained with relative standard deviation <1.5% (t _R) and 10% (peak area). Accuracies (% recovery) of the different spiked OPP concentrations were 82.7–109.1% (water samples) and 80.3–113.3% (fruit juice samples). No contamination by the OPPs was observed in any studied samples.     

Sensitive determination of buspirone in serum by solid-phase extraction and two-dimensional high-performance liquid chromatography

A selective and sensitive determination of buspirone in serum by high-performance liquid chromatography is described. The procedure is based on separation on a C18 column. A solid-phase extraction procedure is used for sample clean-up. The retention on the first column is based on the hydrophobic interaction of buspirone with the stationary phase, and the retention on the second column is based on ionic interactions due to the presence of sodium lauryl sulphate in the mobile phase as well as hydrophobic interaction. This allows for good separation of buspirone from impurities and consequently allows lower detection limits than previously reported for liquid chromatographic methods. Detection by ultraviolet absorbance gives a detection limit of 0.2 ng/ml.     

Ion-pairing high-performance liquid chromatography determination of amitrole in apple after solid-phase extraction and precolumn derivatization

A novel method based on solid-phase extraction was studied for the extraction of amitrole (3-amino-1,2,4-triazole), and its residue determination in apples has been developed. The samples were derivatized with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). The derivatization conditions and the influence of elution composition on the separation were investigated. In pH 9.5 H₃BO₃–Na₂B₄O₇ media, the reaction of amitrole with CNBF was complete at 60°C after 30 min. The separation of derivatized amitrole was achieved at room temperature within 13 min by gradient elution mode with cetyltrimethylammonium bromide in mobile phase as ion-pair reagent. The method correlation coefficient was 0.9996, in concentrations ranging from 1.66 to 415 mg L⁻¹. The calculated recoveries of the proposed method were from 94.17% to 105.67%, and relative standard deviations were 1.57% to 6.44% in the application to the quantitative determination of amitrole in apples. The detection limit of amitrole was 0.10 mg L⁻¹ with a signal-to-noise ratio of 3. Figure Residue determination of amitrole in apple by ion-pairing high-performance liquid chromatography     

Microwave-assisted extraction of scutellarin from Erigeron breviscapus Hand-Mazz and its determination by high-performance liquid chromatography

An efficient microwave-assisted extraction (MAE) technique has been developed to extract scutellarin from Erigeron breviscapus for rapid determination by high-performance liquid chromatography (HPLC). The maximum yield of scutellarin reached 1.02% in 40 min under the optimal MAE conditions with 80 °C of extraction temperature and 1:10 (w/v) of the solid/liquid ratio. The MAE showed obvious advantages in terms of short duration and high efficiency to extract scutellarin in comparison with heat-flux extraction. The mechanism of the enhanced extraction by microwave assistance was discussed by detecting particle size and specific surface area of plant materials and observing cell destruction of plant material by light microscopy and scanning electron microscopy. The results showed that the plant materials were significantly destroyed due to the cell rupture after MAE treatment. Afterward, the method validation for HPLC-UV analysis was developed. Calibration range was 0.1-100 μg mL⁻¹ for scutellarin, and correlation coefficient R was 0.9993. Limit of detection was less than 0.01 μg mL⁻¹. The intra- and inter-day relative standard deviation (R.S.D.) of scutellarin detection ranged from 1.58% to 2.96% and from 3.32% to 4.19%, respectively. The recovery of the method for scutellarin ranged from 96.7% to 101.9%.     

Online extraction and determination of octylglucoside by reversed-phase high-performance liquid chromatography with evaporative light-scattering detection

A reversed-phase high-performance liquid chromatography method using evaporative light-scattering detection is developed for the determination of residual octylglucoside (OG) levels after a detergent exchange step for in-process samples of a vaccine antigen. The reversed-phase column not only provides separation of the OG but also functions as an extraction column to remove the vaccine antigen from the sample, thereby eliminating off-line sample manipulations. In addition to column selection, the mobile phase is optimized to enhance extraction and separation. The vaccine antigen is irreversibly bound to the column, allowing nonprotein components to interact with the column for separation and elution. The assay is linear over the range of 0.00050-0.050% OG. Precision tested at 0.0010% and 0.0050% OG is 2.9% and 7.2% relative standard deviation, respectively. The limits of quantitation and detection are determined to be 0.00050 and 0.000125% OG, respectively. Accuracy is determined to be 103 and 98%, based on spike recoveries of 0.0010% and 0.0050% OG, respectively.     

Multiwalled carbon nanotubes as sorbent for on-line coupling of solid-phase extraction to high-performance liquid chromatography for simultaneous determination of 10 sulfonamides in eggs and pork

The sulfonamides (SAs) have been widely used as effective chemotherapeutics and growth promoters in animals' feeding, but their residues could be a potential danger to human health due to their carcinogenic potency and possible antibiotic resistance. Development of a simple and sensitive method for the determination of SAs residues in food of animal origin, therefore, is of great significance. An on-line solid-phase extraction (SPE) method using multiwalled carbon nanotubes as sorbent coupled with high-performance liquid chromatography (HPLC) for simultaneous determination of 10 sulfonamides (SAs) in eggs and pork was developed. The adsorptive potential of carbon nanotubes for solid-phase extraction of sulfonamides was investigated for the first time in the present paper. To on-line interface solid-phase extraction with HPLC, a conventional sample loop on the six-port injector valve of the HPLC was replaced by a preconcentration column packed with carbon nanotubes. The analytes in water solution were preconcentrated onto the preconcentration column and subsequently eluted with mobile phase of methanol-water (22:78). The developed on-line solid-phase extraction method for HPLC permitted the current HPLC separation and the next preconcentration proceeded in parallel, and thus allows one determination finished within 35 min. The RSD of 10 SAs for nine replicate measurements of a standard mixture of 1 microgl(-1) were in the range of 2.5-7.8%. The method was applied to the determination of trace sulfadiazin (SDZ), sulfamerazine (SMR), sulfadimidine (SDMD), sulfathiazole (STZ), sulfamoxol (SMO), sulfamethizole (SMT), sulfamethoxypyridazine (SMP), sulfachlorpyridazine (SCP), sulfadoxin (SDX) and sulfisoxazole (SIA) in eggs and pork. The results indicated that the proposed method was simple, cost-effective and sensitive.     

Application of a column selection system and DryLab software for high-performance liquid chromatography method development

This paper describes a strategy for the development of chromatographic methods for drug candidates based upon the use of simple MS compatible mobile phases and optimization of the chromatographic selectivity through variations of the stationary phase and mobile phase pH. The strategy employs an automated column selection system and a series of HPLC columns, varying in hydrophobicity and silanol activity, in combination with DryLab software to develop chromatographic methods for the separation of mixtures of bupivacaine and its metabolites; acidic, basic, and neutral compounds; and atenolol, nitrendipine, and their degradation products.