A simple method for simultaneous determination of nine triazines in drinking water

A simple, effective, and economic method for determination of nine triazines (ametryn, atrazine, cyanazine, prometryn, propazine, simazine, simetryn, terbuthylazine, and terbutryn) in drinking water based on solid-phase extraction (SPE) followed by high-performance liquid chromatography-diode array detection (HPLC-DAD) was developed. A specialized solid phase (Oasis HLB) was used, and the parameters that may affect the efficiency of SPE were optimized. The limits of detection (ranged from 0.010 to 0.023 µg L^?1) were satisfactory and allow the determination of triazines at the levels required by European Union legislation. Repeatability (2.4–7.6%) and intermediate precision (0.9–11.0%) calculated at 0.1 µg L^?1 (legislation level) were adequate. The accuracy calculated as the average recovery of spiked tap and mineral waters was higher than 86% for all compounds. The developed method also could be used for undergraduate laboratory experiments because it acquaints students with solution preparation, solid phase extraction procedure, and HPLC-DAD technique.     

Basal plasma-catecholamine-level determination using HPLC-ED and different sample cleanup techniques

Summary Reversed phase HPLC with electrochemical detection was used for the determination of basal adrenaline, dopamine and noradrenaline levels in human plasma. These compounds demonstrated good stability during different stages of collection and long-term storage. Using a new electrochemical detector and improving mobile phase parameters, we obtained a detection limit of 2 pg per injection. Good separation of dihydroxyphenylacetic acid was also attained, which is important in investigations with intensive care patients. Good accuracy and precision, demonstrated in the daily quality control measurements taken over a five month period, verified the reliability of the chromatographic separation. However, there was a decrease in the recovery of very low amounts of catecholamines, added to fresh frozen plasma that had previously been made catecholamine-free. According to the widely-accepted extraction method of Anton and Sayre, it is argued that the un-known affinity of catecholamines to acid-prepared aluminium oxide (in comparison to catecholamine —protein binding constants) explains the low accuracy in measurement at very low plasma levels. We thus compared this sample preparation method to recently published extraction procedures.     

Evaluation of conditions for the HPLC determination of plasma amino acids using precolumn derivatization

For the determination of free amino acids in plasma, the conditions for precolumn derivatization of the amino acids and the chromatographic separation were examined. The isoindole products, formed by the reaction of the primary amino acids with orthophthalaldehyde (OPA), were readily separated by RPLC and detected spectrofluorometrically using an excitation wave-length of 300 nm and an emission cut-off filter of 440 nm. Since the sensitivity of this method permits determination of amino acids in the femtomole range, the analysis can be performed on samples as small as 10 μl of filtered plasma or serum. The separation is achieved in approximately 35 minutes with good precision for the majority of the amino acids.     

Simultaneous capillary GC-MS determination of triazines and amides in water

Summary A simultaneous capillary Gas Chromatographic-Mass Spectrometric (GC-MS) method is described for the determination of thirteen pesticides belonging to the triazine and amide families in water. The sample is extracted in liquid-liquid mode (dichloromethane) and then the determination of the residues is carried out by capillary gas chromatography with mass spectrometric detection in the Selected-Ion Monitoring mode (SIM). The average recoveries of spiked compounds are in the 78.4–135.4% range between the relative low level (0.100 g L^–1) and the relative high level (10.0 g L^–1). The limits of detection (LOD) are in the 0.009–0.128 g L^–1 range.     

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.     

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%.     

On-capillary sample cleanup method for the electrophoretic determination of carbohydrates in juice samples

On many occasions, sample treatment is a critical step in electrophoretic analysis. As an alternative to batch procedures, in this work, a new strategy is presented with a view to develop an on-capillary sample cleanup method. This strategy is based on the partial filling of the capillary with carboxylated single-walled carbon nanotube (c-SWNT). The nanoparticles retain interferences from the matrix allowing the determination and quantification of carbohydrates (viz glucose, maltose and fructose). The precision of the method for the analysis of real samples ranged from 5.3 to 6.4%. The proposed method was compared with a method based on a batch filtration of the juice sample through diatomaceous earth and further electrophoretic determination. This method was also validated in this work. The RSD for this other method ranged from 5.1 to 6%. The results obtained by both methods were statistically comparable demonstrating the accuracy of the proposed methods and their effectiveness. Electrophoretic separation of carbohydrates was achieved using 200 mM borate solution as a buffer at pH 9.5 and applying 15 kV. During separation, the capillary temperature was kept constant at 40 degrees C. For the on-capillary cleanup method, a solution containing 50 mg/L of c-SWNTs prepared in 300 mM borate solution at pH 9.5 was introduced for 60 s into the capillary just before sample introduction. For the electrophoretic analysis of samples cleaned in batch with diatomaceous earth, it is also recommended to introduce into the capillary, just before the sample, a 300 mM borate solution as it enhances the sensitivity and electrophoretic resolution.     

HPLC method for the determination and pharmacokinetic studies on geniposide in rat serum after oral administration of traditional Chinese medicinal preparation Yin-Zhi-Ku decoction

A new HPLC method for the determination of geniposide in rat serum with solid-phase extraction (SPE) for preconcentration is described. Geniposide and an internal standard (paeoniflorin) were extracted from serum by SPE using C18 cartridges. Analysis of the extract was then performed on a reversed-phase C18 column using acetonitrile-water (16:84, v/v) as the eluting solvent system, and UV detection at 238 nm was used to measure the analyte with a limit of quantitation about 0.1 microg/mL. The calibration curve for geniposide was linear (r = 0.9993) in the concentration range 0.1-16.0 microg/mL. The intra- and inter-day precision of the geniposide were determined and their RSD did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of geniposide from rat serum after oral administration of Yin-Zhi-Ku decoction.     

HPLC determination of Vitamin D(3) and its metabolite in human plasma with on-line sample cleanup

A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D₃ and 25-hydroxyvitamin D₃ (25-OH-D₃) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1 ml of human plasma was treated with 2 ml of a mixture of ethanol–acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250 μL of a solution of methanol 5 mmol l⁻¹ phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200 μl aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5 min with a mobile phase constituted by a solution of 6% acetonitrile in 5 mmol l⁻¹ phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile–phosphate buffer 5 mmol l⁻¹, pH 6.5; 20:80 (v/v); solvent B: methanol–acetonitrile–tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265 nm. The method was linear in the range 3.0–32.0 ng ml⁻¹ with a limit of quantification of 3.0 ng ml⁻¹. Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was ≤2.80%. The proposed method permitted the simultaneous determination of Vitamin D₃ and 25-OH-D₃ in 16 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to five samples h⁻¹. The method was successfully applied for the determination of Vitamin D₃ and 25-OH-D₃ in plasma from 46 female volunteers, ranging from 50 to 94 years old. Vitamin D₃ and 25-OH-D₃ concentrations in plasma were found from 4.30–40.70 ng ml⁻¹ (19.74 ± 9.48 ng ml⁻¹) and 3.1–36.52 ng ml⁻¹ (7.13 ± 7.80 ng ml⁻¹), respectively. These results were in good agreement with data published by other authors.     

Molecularly imprinted polymer for the determination of trace ractopamine in pork using SPE followed by HPLC with fluorescence detection

In this study, a molecularly imprinted functionalized polymer for the selective separation of ractopamine (RAC) was prepared by combining a surface molecular imprinting technique with a sol–gel method process. The polymer was evaluated by static, kinetic adsorption, and selective experiments. Results indicated that the molecularly imprinted polymer had high adsorption capacity, selective ability, and fast mass transfer rate. The polymer was applied for the determination of trace RAC through online SPE‐HPLC. With a sample loading flow rate of 2 mL/min, the enhancement factor of 516.26 and the LOD (S/N = 3) of 4.6 ng/L were achieved, respectively, and the linear range of the calibration curve was 0.04–18 μg/L with r² >0.99. The RAC in pork was determined at three spiked levels (0.5, 1, and 2 ng/g) with recoveries ranging from 55.86 to 67.28%.     

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.     

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).     

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre‐treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.     

Development of a validated HPLC method for the determination of four 1,4-benzodiazepines in human biological fluids

A simple and sensitive HPLC method was developed and validated for the determination of four frequently prescribed 1,4-benzodiazepines: alprazolam (ALP), bromazepam (BRZ), diazepam (DZP), and flunitrazepam (FNZ). Separation was achieved on an Inertsil C8 analytical (250 mm x 4 mm, 5 microm) column, after selective extraction of benzodiazepine drugs from biological matrices by means of SPE. Isocratic elution was performed with a mobile phase consisting of CH3COONH4, 0.05 M CH3OH, and CH3CN (33:57:10 by volume). Quantification was performed at 240 nm with mefenamic acid (6 ng/microL) as the internal standard. DSC-18 Supelco cartridges provided high absolute recoveries (81-115%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <12%. Recoveries from biological samples ranged from 81.2 to 115%. The detection limit of the method was calculated as 3.3-10.2 ng in blood plasma and 2.6-12.6 ng in urine for 20 microL injection volume. The method was applied to spiked biological matrices. Moreover, the method was applied to real samples of urine after an oral administration.     

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.     

Studying the interaction between triazines and humic substances--a new approach using open tubular capillary electrochromatography

The strength of the interaction between a pesticide and the soil organic matter is a key parameter to assess the risk of it reaching to groundwater with potentially harmful effects to human health. In this work, a new approach that allows measuring such interactions in a few minutes using a purified fraction of the soil organic matter (humic substances) is detailed. The strength of sorption is assessed via the normalised difference of elution (retention factor, k′) between the chemical of interest and a neutral marker transported via electroosmotic flow through an open tubular column supporting the immobilised humic substances (open tubular capillary electrochromatography). The immobilisation was achieved by incubating a capillary, pre-coated with a monolayer of humic acid, with an acidic solution of humic substances. This induces the formation of a supramolecular structure of humic substances as it occurs in soils. This aggregate can easily be removed using alkaline solutions, and a new structure assembled using other humic substances (HS) or different incubations conditions. The whole procedure takes 2 h. This approach has been tested using five triazines and three types of humic substances. The order of the strength of sorption of the triazines as expected from relevant literature and the relative standard deviation of k′ was between 1 and 6%. Good repeatability was also observed after long period of wash, between re-coating and repeating of the full coating with a new capillary.     

Preparation and characterization of magnetic chitosan microsphere sorbent for separation and determination of environmental estrogens through SPE coupled with HPLC

In this paper, a new separation sorbent of magnetic chitosan microsphere was prepared by means of suspension process using magnetic-Fe(3)O(4) nanoparticles as magnetic material and glutaraldehyde as the cross-linker. The morphology and properties of magnetic chitosan microspheres were characterized by FT-IR, SEM and adsorption test, and the results showed that they had good recognition, high selective ability and fast adsorption-desorption dynamics for estriol (E3), 17β-estradiol (E2) and diethylstilbestrol (DES). Applying this material as SPE sorbent, a method for separation, enrichment and analysis of estrogens in samples coupled with HPLC was developed. Under the optimized condition, the detection limit (S/N=3) for three estrogens was 3.2-20.1 ng/L, and the RSD for five replicate extractions of 50 μg/L E3, E2 and DES was 4.6-8.3%. The blank river water spiked with E3, E2 and DES at 0.05 and 0.2 mg/L levels were extracted and determined by the developed method with good recoveries ranging from 85.5 to 103.7%.     

Validated HPLC method for the determination of fluconazole in human plasma

A high-performance liquid chromatographic assay with UV detection was developed for the determination of fluconazole in human plasma. The method utilized solid-phase extraction for sample clean-up. The separation was performed on a C18 column by isocratic elution with a mobile phase of 10 mM acetate buffer at pH 5.0 and methanol and UV detection at 210 nm. Validation was performed according to the current recommendations of the USFDA bioanalytical method validation guidance. The method proved to be specific, accurate, precise and linear between 200 and 10,000 ng/mL with correlation coefficients greater than 0.999. The coefficient of variation was within 11% and relative deviation was less than 10%.     

Photodegradation study for assessing the environmental fate of some triazine-, urea- and thiolcarbamate-type herbicides

Various biotic or abiotic factors influence the fate of pesticides in the natural environment, of which ultraviolet (UV) component in the sunlight is one of the most powerful forces. Studies on the photodegradation of pesticides have not only significance from the point of view of environmental science, but also very important in researching and developing new, safer, and effective pesticides. Previously we have studied the photodegradation of triazine- and urea-type herbicides in details. This paper is intended to outline the photodegradation pattern of some commonly used N-containing herbicides that belong to the groups of the mentioned triazines (atrazine, cyanazine, terbuthylazine, terbutryn) and ureas (chloroxuron, methabenzthiazuron, diuron, fenuron), as well as thiolcarbamates (butylate, cycloate, EPTC, molinate, vernolate), in order to gain information about their possible fate and derivatives in the environment.The most significant processes of photodegradation of triazines are the partial or complete loss of side-chains, or rather the substitution of the heteroatom-containing side-chain to hydroxyl-group. The chemical characteristics of the side chains determines basically the speed of the degradation. It can be concluded that the C-S bond breaks down much easier than C-Cl bond, and the chlorine atom remaining on the triazine-ring promotes the loss of alkyl-chains to a higher extent than that the hydroxy-group. In the case of ureas, the chemical properties of groups in both the N and N′ position can influence the degradation process. It could be stated the proximity of large aryl-substituent and methyl-group in the N position is unfavorable and instabilizing, promoting the further degradation of compound, at the same time, loss of these groups is the preferred degradation route. In the case of thiolcarbamates, the most frequent processes are the α- and β-oxidation of alkyl-groups connecting to the nitrogen atom. In the most cases, the N-formyl and N-dealkylated products were identified in the degradation mixture. The thioalkyl-group showed fair stability under the circumstances of photodegradation. Its partly or completely degraded products cannot be detected, and represent only very small part of the mixture.