SPE Procedure Research Articles

Determination of penciclovir in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid, specific and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of penciclovir in human plasma. The method involved simple, one-step SPE procedure coupled with a C(18) , 75 × 4.mm, 3 µm column with a flow-rate of 0.5 mL/min, and acyclovir was used as the internal standard. The Quattro Micro mass spectrometry was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. Using 250 µL plasma, the methods were validated over the concentration range 52.555-6626.181 ng/mL, with a lower limit of quantification of 52.55 ng/mL. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers.

Dissipation of fuphenthiourea residues in rice and field ecosystem after seed soaking application

An analytical method with SPE or matrix solid-phase dispersion clean-up procedure on PSA followed by HPLC-DAD was established for determination of fuphenthiourea residues in rice, water and soil. At three concentration levels (0.05, 0.5 and 5mgkg−1), recoveries were in the range of 61.2–82.7%, with a RSD less than 13%. The LOQ of this method was 0.005, 0.02 and 0.01mgkg−1 for the water, soil and rice samples, respectively. Fuphenthiourea was applied in supervised field trials at GAP conditions during rice seeding. It was found that under field conditions, the dissipation half-lives of fuphenthiourea were 0.8d in the water and 24.8d in the soil. At harvest, no detectable residues (<LOD) were found in the various samples.

A very fast and simple method for the determination of sulfonamide residues in seawaters

The development of simple and reproducible analytical methods for the determination of new organic pollutants like pharmaceuticals and personal care products in seawaters is of the highest priority. In the present study, a new analytical method based on offline solid phase extraction and liquid chromatography with standard UV detection (SPE-HPLC-UV) was developed to determine the presence of sulfonamides in seawaters. Special attention was paid to the sample preparation step. Different variables affecting the extraction process, such as seawater salinity and the humic acid content were studied. As a result, the presented SPE procedure for the extraction of sulfonamides from seawaters is also applicable to other techniques like LC-MS and LC-MS/MS. This method was optimized and fully validated for its performance parameters. It has very good selectivity, linearity (R2 > 0.995), precision (RSD 75%), even in highly saline waters and in the presence of humic acids.

Analysis of polyphenols using capillary zone electrophoresis – Determination of the most effective wine sample pre‐treatment method

A simple CZE method has been developed for the simultaneous determination of eight polyphenolic compounds. The influence of several experimental conditions such as buffer (concentration and pH) and temperature were studied. Optimum separation was achieved in less than 25 min by using a BGE of 50 mM Na(2)B(4)O(7) and 10 mM Na(2)HPO(4) at pH 9.6, a temperature of 25°C and an applied voltage of 25 kV. Good linearities for all eight analytes were obtained with correlation coefficients higher than 0.99. The LODs were between 0.03 and 5.05 μg/mL and the RSD values of the migration times were found to be less than 1%. The optimal separation conditions were then used for the identification and the quantitation of polyphenolic compounds in Cypriot wine samples using six different sample preparation procedures. In particular, two direct injection methods (without any extraction step), three different liquid-liquid extraction procedures and an SPE procedure were examined. These sample pre-treatment methods were also compared in order to determine the one that is the most effective, in regard to analyte recovery, time, difficulty, and reproducibility. Liquid-liquid extraction using diethyl ether as the organic solvent proved to be the most effective.

Application of HPLC and TLC with Diode Array Detection After SPE to the Determination of Pesticides in Water Samples from the Zemborzycki Reservoir (Lublin, Southeastern Poland)

The application of TLC with a diode array detector (TLC-DAD) and HPLC-DAD after SPE for identification and quantitative analysis of pesticides in water samples is demonstrated. The procedures described for the determination of compounds are inexpensive and can be applied to routine analysis of analytes in water samples after preliminary cleanup and concentration by SPE. Average recoveries for four different cartridges and three solvents by the proposed HPLC-DAD method after SPE also are presented. The efficiency of the SPE procedure was evaluated using real water samples from the Zemborzycki Reservoir, near Lublin, southeastern Poland. The method was validated for precision, repeatability, and accuracy.

A comparison of alternative sample preparation procedures for the analysis of swainsonine using LC‐MS/MS

Swainsonine, a polyhydroxy indolizidine alkaloid and known glycosidase inhibitor, is found in a number of different plants that cause a lysosomal storage disease known as locoism in the western USA. Most recently swainsonine has been analysed by LC-MS/MS after sample extraction and preparation from ion-exchange resins. To compare previously published sample preparation procedures with several new alternative procedures to provide methods using either commercially available solid-phase extraction equipment or procedures which significantly reduce sample preparation time. A previously reported and validated sample preparation method using ion-exchange resin was compared with methods using a commercially available solid-phase extraction cartridge, a solvent partitioning procedure or a single solvent extraction procedure using one of two solvents. Twenty different plant samples of varying swainsonine concentrations were prepared in triplicate and analysed by LC-MS/MS. The measured concentration of swainsonine was then statistically compared between methods. There were no statistically significant differences found between four of the five different sample preparation methods tested. A commercially available SPE cartridge can be used to replace the previously used ion-exchange resin for swainsonine analysis. For very rapid analyses the SPE procedure can be eliminated and a simple, single solvent extraction step used for sample preparation.

Determination of pesticides in surface and ground waters by liquid chromatography–electrospray–tandem mass spectrometry

The sensitive multiresidual analytical method for simultaneous analysis of 14 most commonly used agricultural pesticides in Serbia was developed and optimized. The selected insecticides, fungicides and herbicides belong to seven chemical classes (organophosphates, neonicotinoids, carbamates, diacylhydrazines, benzimidazoles, triazines and phenylureas). The method was based on solid-phase extraction followed by liquid chromatography–tandem mass spectrometry. The following parameters that may affect the SPE procedure efficiency were optimized: the sorbent type in combination with different elution solvents, the sample pH and the sample volume. For each pesticide, MS n analysis was performed and distinctive ions and transitions were selected for identification and quantification, as well as for confirmation purposes. External matrix-matched calibration method was used to eliminate variable matrix effect and ensure precise quantification. Good recoveries (72–129%), and low limits of detection (0.4–5.5 ng L −1) and quantification (1.1–18.2 ng L −1) were achieved for all selected pesticides. The developed and optimized method was successfully applied in the analysis of several river waters, as well as ground waters in Serbia, influenced by agriculture. The most frequently detected pesticide was carbendazim. Dimethoate, carbofuran and propazine were also found in the investigated samples.

Determination of fluoroquinolone antibiotics in wastewater using ultra high‐performance liquid chromatography with mass spectrometry and fluorescence detection

A new ultra HPLC (UHPLC) method using both MS and fluorescence detection (FD) was developed for the determination of five fluoroquinolones in wastewaters. Systematic method development approach was compared with a conventional one. During the systematic approach, a possibility of automatic switching among four independent analytical columns of different chemistries has been used. Acidic as well as basic pH using ACN and methanol as organic modifiers was tested. The best separation of fluoroquinolones was obtained on phenyl analytical column at pH 10.5, which is a completely novel approach for separation of fluoroquinolones. Further, a new SPE procedure was developed for the sample preparation using basic pH as well. The sensitivity and selectivity of FD and MS detection were compared. FD at basic pH 10.5 demonstrated lower sensitivity than at acidic pH, which is conventionally performed. At basic pH, UHPLC-MS/MS was found about two orders of magnitude more sensitive than FD. Both methods were validated and subsequently UHPLC-FD method was used for the evaluation of stability of fluoroquinolones. UHPLC-MS/MS method was used for the analysis of wastewater samples. Norfloxacin and ciprofloxacin were detected in samples of influent and effluent from wastewater treatment plant. Ofloxacin was detected only in influent from wastewater treatment plant.

Simultaneous determination of UV filters and polycyclic musks in aqueous samples by solid-phase microextraction and gas chromatography–mass spectrometry

A simple, precise and accurate method for the simultaneous determination of four UV filters and five polycyclic musks (PCMs) in aqueous samples was developed by solid-phase microextraction coupled with gas chromatography–mass spectrometry (SPME-GC–MS). The operating conditions affecting the performance of SPME-GC–MS, including fiber thickness, desorption time, pH, salinity, extraction time and temperature have been carefully studied. Under optimum conditions (30 μm PDMS fiber, 7 min desorption time, pH 7, 10% NaCl, 90 min extraction time at 24 °C), the correlation coefficients ( r 2 ) of the calibration curves of target compounds ranged from 0.9993 to 0.9999. The limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.2 to 9.6 ng L −1 and 0.7 to 32.0 ng L −1, respectively. The developed procedure was applied to the determinations of four UV filters and five PCMs in river water samples and internal standard was used for calibration to compensate the matrix effect. Good relative recoveries were obtained for spiked river water at low, medium and high levels. The proposed SPME method was compared with traditional SPE procedure and the results found in river water using both methods were in the same order of magnitude and both are quite agreeable.

Determination of melamine in milk powder using zwitterionic HILIC stationary phase with UV detection

A hydrophilic interaction liquid chromatography-UV method was developed for the determination of melamine in milk powder. Sulfobetaine type zwitterionic hydrophilic interaction liquid chromatography stationary phase was used to achieve straightforward separation of melamine in milk powder without any sample derivatization or addition of ion-pair reagent. The sample preparation was simple and fast with the steps of acetonitrile/perchloric acid extraction-centrifugation-filtration. No SPE or other pre-concentration procedure was required. By using large volume sample injection and choosing low UV wavelength (210 nm), the LOD and LOQ were 0.005 and 0.015 microg/mL for melamine standards, and 0.02 and 0.06 microg/mL for the spiked milk extracts. The LOD and LOQ of the latter correspond to 0.95 and 2.2 microg/g melamine in the milk powder. The correlation coefficients for melamine standard, pre-spiked milk extracts and post-spiked milk extracts in the range of 0-0.5 microg/mL were 0.9978, 0.9976 and 0.9995, respectively. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine in tainted milk products.

Development and optimization of the SPE procedure for determination of pharmaceuticals in water samples by HPLC‐diode array detection

This paper focuses on the investigation of different types of SPE sorbents for the preconcentration of eight veterinary pharmaceuticals from water samples. The pharmaceuticals studied were sulfamethazine, sulfadiazine, sulfaguanidine, trimethoprim, oxytetracycline, enrofloxacin, norfloxacin and penicillin G/procaine. Five different SPE materials (Strata-X, Strata-X-C, Strata SDB-L, Strata C8 and Strata C18) from Phenomenex were compared with Oasis HLB with a view to obtaining the best cartridges for all pharmaceuticals investigated. Extraction efficiency was determined by HPLC with diode array detection (DAD). HPLC-DAD separation and quantification of the selected pharmaceuticals were carried out under gradient elution by a binary mixture of 0.01 M oxalic acid and ACN based on cyano modified column (LiChrosphere 100 CN) from Merck. Strata-X provided the best results in the preconcentration of 100 mL water samples, yielding average pharmaceutical recoveries of higher than 90%, except for sulfaguanidine (76.1%). The developed Strata-X-HLPC-DAD method was validated and applied, for the efficient investigation of reverse osmosis/nanofiltration membranes and for the removal of these eight pharmaceuticals from the production wastewater samples. NF90 and XLE membranes were shown to be the best for the rejection of all investigated pharmaceuticals.

DEVELOPMENT OF LLE AND SPE PROCEDURES AND ITS APPLICATIONS FOR DETERMINATION OF OLMESARTAN IN HUMAN PLASMA USING RP-HPLC AND HPTLC

Two novel simple and rapid liquid–liquid and solid phase extraction methods have been developed for determination of olmesartan in human plasma using zidovudine as an internal standard. Liquid–liquid extraction from spiked human plasma samples was done using Dichloromethane: acetic acid (5.5: 0.5, v/v) solvent, while solid phase extraction was carried out on a DSC MCAX cartridge. For HPLC, the mobile phase consisted of water acetic acid pH 4.5 and methanol (25:75) at a flow rate of 0.5 mL · min−1 in isocratic mode. The calibration curve was plotted with a concentration range 10 μgmL−1 to 60 μgmL−1. Recovery studies were carried out by LLE and SPE procedures with average recoveries 69.27% and 72.87%, respectively. For HPTLC the developing phase consisting of ethyl acetate methanol and acetic acid (8.0:2.0:0.05 v/v/v) and detection was carried out on 269 nm. The calibration curve was plotted over the concentration range of 200 ng to 600 ng. The average recoveries were 90.12% and 79.64% by LLE and SPE, respectively. The proposed method was validated as per US-FDA guidance.

The Development of SPE Procedures and an UHPLC Method for the Simultaneous Determination of Ten Drugs in Water Samples

Analytical procedures for the determination of pharmaceuticals from different therapeutic groups were proposed. These groups included the corticosteroids prednisolone and dexamethasone; the β-blockers sotalol, metoprolol, propranolol, and carvedilol; and the analgesic nonsteroidal anti-inflammatory drugs paracetamol, aspirin, metamizole, and ketoprofen. Reversed-phase ultrahigh performance liquid chromatography with an ultraviolet detector, different columns, different mobile phases, and gradient elution programmes were used to obtain the best separations within the shortest possible time. Solid-phase extraction was examined as a preconcentration step. The Oasis HLB column, with the highest recoveries (over 90% for most of the drugs), was chosen for the analysis of surface waters. Limits of detection ranged from 0.06 to 0.39 μg L−1 for all drugs after optimisation of all analytical steps.

Evaluation of Immunoassays as an Alternative for the Rapid Determination of Pesticides in Wine and Grape Samples

The main objective of this paper is to address the performance of immunochemical assays for the detection of the residues of three pesticides [atrazine, bromopropylate, and 2,4,6-trichlorophenol (TCP)] in real winery samples, such as wine, grapes, and grape juice. Different approaches have been evaluated to minimize interferences from the matrixes, and suitable working protocols have been established in order to achieve the necessary LODs, accuracy, and precision for real samples. A simple dilution of the sample proved to be sufficient for the determination of atrazine and bromopropylate in red and white wine and grape juice at the required levels of concentration. However, for TCP, an SPE procedure has been optimized using amino cartridges. The recoveries were above 85% in all cases, and the LOD values were below the parts per billion level, except for bromopropylate, which ranged between 2 and 50 microg/L, depending on the matrix. The grape matrix effect could be resolved by a simple extraction with methanol. Complete recoveries were obtained, and the final measurement procedures were able to determine selected pesticides below their maximum residue levels. The newly developed methods have been compared with standard chromatographic methods.

Study on the molecularly imprinted polymers with methyl-testosterone as the template

Molecularly imprinted polymers (MIPs) using methyl-testosterone as the template, methacrylic acid (MAA) as the monomer and ethylene glycol dimethacrylate (EDMA) as the crosslinker were prepared by precipitation polymerization. The morphology of the obtained particles was characterized by scanning electron microscopy (SEM) and the pore size was measured by BET. Then, the specificity and selectivity of the MIPs were evaluated using the equilibrium rebinding experiments. Besides, the MIPs were also used as the stationary phase of HPLC column and the retention behaviour to the template and analogues was confirmed using HPLC-MS-MS. Finally, the real application of the methyl-testosterone imprinted polymers was evaluated using SPE procedure with the spiked tap water and lake water. The results indicated that the prepared methyl-testosterone imprinted polymer showed specific rebinding ability to its template and could retain the template strongly compared with other structural analogues. At the same time, the MIPs could be used as SPE column to enrich methyl-testosterone in the lake water and show broad prospects in real samples.

Determination of Tricyclazole in Water Using Solid Phase Extraction and Liquid Chromatography

A method for determination of tricyclazole in water using solid phase extraction and high performance liquid chromatography (HPLC) with UV detection at 230 nm and a mobile phase of acetonitrile:water (20:80, v/v) was developed. A performance comparison between two types of solid phase sorbents, the C18 sorbent of Supelclean ENVI-18 cartridge and the styrene-divinyl benzene copolymer sorbent of Sep-Pak PS2-Plus cartridge was conducted. The Sep-Pak PS2-Plus cartridges were found more suitable for extracting tricyclazole from water samples than the Supelclean ENVI-18 cartridges. For this cartridge, both methanol and ethyl acetate produced good results. The method was validated with good linearity and with a limit of detection of 0.008 µg L−1 for a 500-fold concentration through the SPE procedure. The recoveries of the method were stable at ∼80% and the precision was from 1.1 − 6.0% within the range of fortified concentrations. The validated method was also applied to measure the concentrations of tricyclazole in real paddy water.

Automated Solid-Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry Analysis of 11-nor- 9-Tetrahydrocannabinol-9-Carboxylic Acid in Human Urine Specimens: Application to a High-Throughput Urine Analysis Laboratory

An automated solid-phase extraction coupled with liquid chromatography and tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in human urine specimens was developed. The method was linear (R(2) = 0.9986) to 1000 ng/mL with no carryover evidenced at 2000 ng/mL. Limits of quantification and detection were found to be 2 ng/mL. Interrun precision was evaluated at the 15 ng/mL level over nine batches spanning 15 days (n = 45). The coefficient of variation (%CV) was found to be 5.5% over the course of the validation. Intrarun precision of a 15 ng/mL control (n = 5) ranged from 0.58% CV to 7.4% CV for the same set of analytical batches. Interference was tested using (+/-)-11-hydroxy-Delta(9)-tetrahydrocannabinol, cannabidiol, (-)-Delta(8)-tetrahydrocannabinol, and cannabinol. One hundred and nineteen specimens previously found to contain THC-COOH by a previously validated gas chromatographic mass spectrometry (GC-MS) procedure were compared to the SPE-LC-MS-MS method. Excellent agreement was found (R(2) = 0.9925) for the parallel comparison study. The automated SPE procedure eliminates the human factors of specimen handling, extraction, and derivatization, thereby reducing labor costs and rework resulting from human error or technique issues. Additionally, method runtime is greatly reduced (e.g., during parallel studies the SPE-LC-MS-MS instrument was often finished with analysis by the time the technician finished the offline SPE and derivatization procedure prior to the GC-MS analysis).

Multiresidue determination of penicillins in environmental waters and chicken muscle samples by means of capillary electrophoresis‐tandem mass spectrometry

A new analytical method based on CZE coupled with tandem MS detection (CE-MS/MS) has been developed for the simultaneous determination of nine penicillins of human and veterinary use (nafcillin, dicloxacillin, cloxacillin, oxacillin, ampicillin, penicillin G, amoxicillin, penicillin V and piperacillin), whose levels are regulated by the EU Council regulation No. 2377/90 in animal edible tissues. CE separation conditions, sheath liquid composition and electrospray parameters have been carefully optimized to reach high sensitivity and precision. Separation was carried out in a 96 cm total length fused-silica capillary (50 microm id, 360 microm od) using 60 mM ammonium acetate at pH 6.0 as running buffer. Lomefloxacin was used as internal standard. MS/MS experiments were carried out using an IT as analyzer operating in multiple reaction monitoring mode to achieve the minimum number of identification points established by the 2002/657/EC European Decision. The use of an SPE procedure in two steps, combining Oasis hydrophilic-liphophilic balance and Alumina N cartridges provide a satisfactory preconcentration and clean up treatment for meat samples after extraction of the compounds with ACN. Only Oasis hydrophilic-liphophilic balance cartridges were necessary in water samples to obtain adequate recoveries. The method has been characterized for its use in meat and water samples, using matrix-matched calibrations. For chicken muscle samples, LODs between 8 and 12 microg/kg have been obtained, in all cases lower than the maximum residue limits permitted. For water samples LODs from 0.18 to 0.26 microg/L could ensure its satisfactory application in aquatic samples.

Fully Automated Analysis of β-Lactams in Bovine Milk by Online Solid Phase Extraction-Liquid Chromatography-Electrospray-Tandem Mass Spectrometry

A fully automated method for the detection of beta-lactam antibiotics, including six penicillins (amoxicillin, ampicillin, cloxacillin, dicloxacillin, oxacillin, and penicillin G) and four cephalosporins (cefazolin, ceftiofur, cefoperazone, and cefalexin) in bovine milk samples has been developed. The outlined method is based on online solid-phase extraction-liquid chromatography/electrospray-tandem mass spectrometry (SPE-LC/ESI-MS-MS). Target compounds were concentrated from 500 microL of centrifuged milk samples using an online SPE procedure with C18 HD cartridges. Target analytes were eluted with a gradient mobile phase (water + 0.1% formic acid/methanol + 0.1% formic acid) at a flow rate of 0.7 mL/min. Chromatographic separation was achieved within 10 min using a C-12 reversed phase analytical column. For unequivocal identification and confirmation, two multiple reaction monitoring (MRM) transitions were acquired for each analyte in the positive electrospray ionization mode (ESI(+)). Method limits of detection (LODs) in milk were well below the maximum residue limits (MRLs) set by the European Union for all compounds. Limits of quantification in milk were between 0.09 ng/mL and 1.44 ng/mL. The developed method was validated according to EU's requirements, and accuracy results ranged from 80 to 116%. Finally, the method was applied to the analysis of twenty real samples previously screened by the inhibition of microbial growth test Eclipse 100. This new developed method offers high sensitivity and accuracy of results, minimum sample pre-treatment, and uses for the first time an automated online SPE offering a high throughput analysis. Because of all these characteristics, the proposed method is applicable and could be deemed necessary within the field of food control and safety.

An automated micro-solid phase extraction device involving integrated \high-pressure microvalves for genetic sample preparation

This paper presents an automated micro-SPE device for DNA extraction using monolithically integrated high-pressure microvalves. The automated micro-SPE device was fabricated through glass-to-glass thermal bonding and microfluidic system interface technologies. To increase the DNA extraction efficiency, silica beads were packed in the extraction microchannel involving two weir structures. Experimental results show that the DNA extraction efficiency using the automated micro-SPE device containing bare silica beads was 75.87% in the first 8 microl of solution eluted by automated SPE procedure. In addition, the reproducibility of the DNA extraction was evaluated by ten successive measurements. Genomic DNA extracted from human WBCs had an absorbance ratio of DNA to protein (A(260)/A(280)) of 1.56. The applicability of this automated micro-SPE device to genetic sample preparation was verified by PCR amplification of a beta-globulin gene using the genomic DNA extracted from WBCs. Consequently, we demonstrated that the proposed automatic micro-SPE device can extract nucleic acids from biological samples, thereby facilitating its integration with downstream genetic analyses in a micro format.