Restricted Access Media Research Articles

Preparation of Ofloxacin-Restricted Access Media–Molecularly Imprinted Polymers for Its Selective Recognition of Ofloxacin in Milk Samples

In this study, a novel restricted access media–molecularly imprinted polymer (RAM–MIP) was prepared by precipitation polymerization using ofloxacin (OFLX) as a template, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as the cross-linker and glycidyl methacrylate as pro-hydrophilic co-monomer for specific recognition and sensitive determination of trace ofloxacin in milk samples. The RAM–MIPs were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. Then, recognition ability and hydrophilicity of RAM–MIPs were evaluated and the results indicated that the prepared RAM–MIPs exhibited excellent recognition ability and good hydrophilicity. Enrofloxacin, gatifloxacin, and sulfamerazine were used as competitive molecules to research the selective adsorption capacity of the RAM–MIPs. The maximum adsorption capacity of the RAM–MIPs for OFLX was 17.49 mg/g, which was almost double that of the non-imprinted polymers, RAM–NIPs. Under the same conditions, the binding ability of MIP to bovine serum albumin was calculated to be 31.19%, while RAM–MIPs was only 7.6%. Furthermore, a molecularly imprinted solid-phase extraction column was prepared with RAM–MIPs material and then reused ten times with a recovery rate above 75%. Combined with high-performance liquid chromatography analysis, this material can be used to effectively purify and separate OFLX from milk. This work was beneficial to eliminate the protein deposition and increased the simplicity and accuracy of analytical method for the determination of trace OFLX in milk samples.

Automated on-line SPE determination of amisulpride in human plasma using LC coupled with restricted-access media column

An automated on-line solid-phase extraction (SPE) method coupled with high performance liquid chromatography (HPLC) was developed for the first application to rapid quantification of trace amisulpride in human plasma. A kind of restricted-access media (RAM) column (Capcell Pak MF Ph-1) was adopted for highly efficient extraction of amisulpride and meanwhile for the limited access of macromolecular matrices. It was robust and reusable for >1000 times. The utilization of two programmable pumps enabled the simultaneous execution of SPE and HPLC analysis within 10 min. Additionally, using appropriate valve-switching time was found to efficiently reduce the contamination of HPLC system caused by co-elution of impurity from an RAM column, facilitating continuous analysis of large-scale samples. The method was characterized by free of internal standard (IS), which exhibited low limit of detection (0.0035 μg·mL−1), satisfactory precision and accuracy (recoveries of 98.1%–104.6% with relative standard deviations below 4.6%). Amisulpride was successfully detected in the plasma samples originated from four patients. Notably, interindividual differences of plasma sample matrices did not yield an adverse effect on the accuracy of our IS-free method. The comparison to previous methods shows the superiority of the method in terms of sensitivity, IS requirement, speed, and automation. Thus, the method has great potential for the application to therapeutic drug monitoring as well as large-scale bioanalytical and clinical studies.

Recent trends in sorption-based sample preparation and liquid chromatography techniques for food analysis.

The accelerated rising of the world's population increased the consumption of food, thus demanding more rigors in the control of residue and contaminants in food-based products marketed for human consumption. In view of the complexity of most food matrices, including fruits, vegetables, different types of meat, beverages, among others, a sample preparation step is important to provide more reliable results when combined with HPLC separations. An adequate sample preparation step before the chromatographic analysis is mandatory in obtaining higher precision and accuracy in order to improve the extraction of the target analytes, one of the priorities in analytical chemistry. The recent discovery of new materials such as ionic liquids, graphene-derived materials, molecularly imprinted polymers, restricted access media, magnetic nanoparticles, and carbonaceous nanomaterials, provided high sensitivity and selectivity results in an extensive variety of applications. These materials, as well as their several possible combinations, have been demonstrated to be highly appropriate for the extraction of different analytes in complex samples such as food products. The main characteristics and application of these new materials in food analysis will be presented and discussed in this paper. Another topic discussed in this review covers the main advantages and limitations of sample preparation microtechniques, as well as their off-line and on-line combination with HPLC for food analysis.

Mathematical modeling of the batch adsorption of proteins on new restricted access media with poly(ethylene glycol) as a semi-permeable barrier using compact finite differences

Abstract In this work we describe a mathematical analysis of the batch adsorption process of several proteins using a new restricted access medium consisting of agarose beads grafted with poly(ethylene glycol) (PEG) as a semi-permeable barrier and immobilized metal ions or ion exchange groups as binding sites. The model was fitted to experimental data, allowing the estimation of the adsorption rate constant and the effective diffusivity for each protein. The model was solved using compact finite differences in a MATLAB® platform. According to the results, the presence of grafted PEG reduces the adsorption of all proteins to different extent; with high molecular weight proteins being affected the most. The model also establishes a reduction in the adsorption rate constant (which affects protein interaction with binding sites). The movement of the protein molecules in the adsorbent pores is also affected by the grafted PEG, but to a lesser extent.

Optimization of the matrices structural parameters for the synthesis of restricted access media (RAM) adsorbents for chromatography

The geometric parameters of a matrix for the synthesis of restricted access media adsorbents are optimized, using mathematical modeling based on the basis of a globular model with tetrahedral and octahedral pores. For calculations were used the distribution curves of pore sizes obtained on the basis of the desorption branches of isotherms obtained via low temperature nitrogen adsorption. In contrast to the model of cylindrical pores, the calculation model of globular pores gives realistic values of the surface area of the silica with grafted hexadecylsilyl groups, due to the change of ratio of the amounts of tetrahedral and octahedral pores in the model. The obtained value of the surface area of the silica with grafted hexadecylsilyl groups is in good agreement with elemental analysis data. The obtained refined size of the mouth of the pores makes it possible to choose correctly the particle diameter of the external protective coating.

Targeted quantification of lipid mediators in skeletal muscles using restricted access media-based trap-and-elute liquid chromatography-mass spectrometry

Lipid mediators (LMs) are a class of bioactive metabolites of the essential polyunsaturated fatty acids (PUFA), which are involved in many physiological processes. Their quantification in biological samples is critical for understanding their functions in lifestyle and chronic diseases, such as diabetes, as well allergies, cancers, and in aging processes. We developed a rapid, and sensitive LC-MS/MS method to quantify the concentrations of 14 lipid mediators of interest in mouse skeletal muscle tissue without time-consuming liquid-liquid or solid-phase extractions. A restricted-access media (RAM) based trap was used prior to LC-MS as cleanup process to prevent the analytical column from clogging and deterioration. The system enabled automatic removal of residual proteins and other biological interferences presented in the tissue extracts; the target analytes were retained in the trap and then eluted to an analytical column for separation. Matrix evaluation tests demonstrated that the use of the combined RAM trap and chromatographic separation efficiently eliminated the biological or chemical matrix interferences typically encountered in bioanalytical analysis. Using 14 LM standards and 12 corresponding deuterated compounds as internal standards, the five-point calibration curves, established over the concentration range of 0.031–320 ng mL−1, demonstrated good linearity of r2 > 0.9903 (0.9903–0.9983). The lower detection limits obtained were 0.016, 0.031, 0.062, and 0.31 ng mL−1 (0.5, 1, 2, and 10 pg on column), respectively, depending on the specific compounds. Good accuracy (87.1–114.5%) and precision (<13.4%) of the method were observed for low, medium, and high concentration quality control samples. The method was applied to measure the amount of 14 target LMs in mouse skeletal muscle tissues. All 14 analytes in this study were successfully detected and quantified in the gastrocnemius muscle samples, which provided crucial information for both age and gender-related aspects of LMs signaling in skeletal muscles previously unknown. This method could be applied to advance the understanding of skeletal muscle pathophysiology to study the role of LMs in health and disease. Furthermore, we will expand the application of this methodology to humans and other tissues/matrices in the near future.

Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid chromatography (2D-LC)

A new two-dimensional liquid chromatography (2D-LC) method using a column switching valve, with a restricted-access media (RAM) column in the first dimension was developed and validated for the quantification of two β-blockers in human plasma. Several parameters, such as sample collection, mobile phase composition and flow rate for sample cleanup, transference and analytical separation of the β-blockers were investigated and optimized. The developed method allowed for the simultaneous pre-treatment and quantification of alprenolol (ALP) and propranolol (PRO) in human plasma in less than 25min. The method consisted in the injection of 100μL of plasma samples on the RAM alkyl-diol-silica column (Lichrospher® RP-18 ADS, 25μm) with water/acetonitrile (98:2, v/v; at a flow rate of 2.0mL/min) and then transferred (via a six-port valve) to the analytical column (Luna PFP (2), 150×4.6mm ID, 100Å, 3μm) with 0.1% (v/v) triethylamine in water acidified with trifluoroacetic acid (pH=3)/acetonitrile (74:26, v/v) at a flow rate of 0.6mL/min in a back-flush mode. The column oven temperature was optimized to 42°C and the fluorescence detector set at 280nm and 310nm (excitation and emission, respectively). The method was validated according to the European Medicines Agency’s guidelines and was linear (r2>0.999) over a dynamic range of 5.0 – 200ng/mL. Recoveries ranged from 90.2 and 107% and the lower limit of quantification was 5.0ng/mL for both compounds. Precision was expressed as a percentage of relative standard deviation and did not exceed 11%. Finally, the method was successfully applied to determine the plasma concentration of PRO in four healthy volunteers.

A column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry method to determine anandamide and 2-arachidonoylglycerol in plasma samples.

This study reports a fast, sensitive, and selective column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the endocannabinoids (eCBs), anandamide (AEA), and 2-arachidonoylglycerol (2-AG) in plasma samples. This bidimensional system used a restricted access media column (RP-8 ADS, 25mm×4mm×25μM) in the first dimension and a core-shell Kinetex C18 (100mm×2, 1.7mm×1μM) column in the second dimension, followed by detection in a mass spectrometer triple quadrupole (multiple reactions monitoring mode) operating in the positive mode. RP-8 ADS was used for trace enrichment of eCBs (reverse phase partitioning) and macromolecular matrix size exclusion; the core-shell column was used for the chromatographic separation. The column switching UHPLC-MS/MS method presented a linear range spanning from 0.1ng mL-1 (LOQ) to 6ng mL-1 for AEA and from 0.04ng mL-1 (LOQ) to 10ng mL-1 for 2-AG. Excluding the LLOQ values, the precision assays provided coefficients of variation lower than 8% and accuracy with relative standard error values lower than 14%. Neither carryover nor matrix effects were detected. This high-throughput column switching method compared to conventional methods is time saving as it involves fewer steps, consumes less solvent, and presents lower LLOQ. The column switching UHPLC-MS/MS method was successfully applied to determine AEA and 2-AG in plasma samples obtained from Alzheimer's disease patients. Graphical abstract A column switching ultra high-performance liquid chromatography-tandem mass spectrometry method using RP-8 ADS column and core shell column to determine endocannabinoids in plasma samples.

Evaluation of efficiency and trapping capacity of restricted access media trap columns for the online trapping of small molecules.

Restricted access media are generally composed from multi-modal particles that combine a size excluding outer surface and an inner-pore retention mechanism for small molecules. Such materials can be used for either online isolation and pre-concentration of target small molecules or removal of small molecule interferences from large macromolecules, such as proteins in complex biological matrices. Thus, they are considered as enhanced online solid-phase extraction materials. We evaluated the efficiency and trapping capacity of different semi-permeable surface restricted access media columns (C18 , C8 , and C4 inner pores) for four model small molecule compounds (dopamine hydrochloride, acetaminophen, 4-hydroxybenzoic acid, and diethyl phthalate) having variable physicochemical properties. We further studied the effect of mobile phase flow rate (0.25, 0.5, 1, and 2 mL/min) and pH, using 98:2 0.5% acetic acid in water/ methanol (pH 2.88) and 5 mM ammonium acetate in 98:2 water/methanol (pH 6.61) as mobile phases. Breakthrough curves generated using frontal analysis were analyzed to determine important chromatographic parameters specific for each of the studied compounds. Experimental determination of these parameters allowed selection of the most efficient trap column and the best loading mobile phase conditions for maximal solute enrichment and pre-concentration on restricted access media trap columns.

Simultaneous enantioselective quantification of fluoxetine and norfluoxetine in human milk by direct sample injection using 2-dimensional liquid chromatography–tandem mass spectrometry

A two-dimensional liquid chromatography system coupled to triple quadrupole tandem mass spectrometer (2D LC–MS/MS) was employed for the simultaneously quantification of fluoxetine (FLX) and norfluoxetine (NFLX) enantiomers in human milk by direct injection of samples. A restricted access media of bovine serum albumin octadecyl column (RAM-BSAC18) was used in the first dimension for the milk proteins depletion, while an antibiotic-based chiral column was used in the second dimension. The results herein described show good selectivity, extraction efficiency, accuracy, and precision with limits of quantification in the order of 7.5ngmL−1for the FLX enantiomers and 10.0ngmL−1 for NFLX enantiomers. Furthermore, it represents a practical tool in terms of sustainability for the sample preparation of such a difficult matrix.

Fast clean-up and selective enrichment of florfenicol in milk by restricted access media molecularly imprinted magnetic microspheres based on surface-initiated photoiniferter-mediated polymerization

A novel RAM-MIMM with water-compatible, exclusion biomacromolecules and selective enrichment analytes was prepared using a surface-initiated iniferter technique for magnetic dispersion extraction of trace florfenicol from milk samples.

Simultaneous determination of fluoroquinolones in environmental water by liquid chromatography–tandem mass spectrometry with direct injection: A green approach

This work describes an on-line multi-residue method for simultaneous quantification of ciprofloxacin, enrofloxacin, gemifloxacin, moxifloxacin, norfloxacin and ofloxacin in superficial and wastewater samples. For that, an octyl restricted-access media bovine serum albumin column (RAM-BSA C8) was used for sample clean-up, enrichment and analysis with quantitation carried out by tandem mass spectrometry. For water samples volumes of only 500μL the method provided good selectivity, extraction efficiency, accuracy, and precision with quantification limits in the order of 20–150ngL−1. Out of the six fluoroquinolones only ciprofloxacin (195ngL−1) and norfloxacin (270ngL−1) were quantified in an influent sample of the wastewater treatment plant (WWTP) of São Carlos (SP, Brazil). None were found in the superficial water samples analyzed. The capability of injecting native sample in an automated mode provides high productivity and represents a greener approach in environmental sample analysis.

Quantitative Analysis of Chloramphenicol in Royal Jelly by Column-switching LC-MS/MS Using a Pretreatment Column with a Higher-pressure Capability.

An on-line pretreatment liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was developed for the analysis of chloramphenicol (CAP) in royal jelly. A novel methylcellulose-immobilized restricted access media column with a higher-pressure capability of 60 MPa (MC-ODS HP) was developed for the effective removal of proteins and other compounds in the sample matrix. CAP in a sample solution was extracted in 2 min by the column-switching LC-MS/MS system. The system provides a minimum sample pretreatment along with highly sensitive and reproducible analysis. As a result, the limit of quantitation of CAP was 10 pg/mL (= 0.1 μg/kg royal jelly) and the linear dynamic range was between 10 and 10000 pg/mL (correlation coefficient greater than 0.999). The proposed method meets the requirements of regulations in EU (0.3 μg/kg). The inter-day precision and accuracy of CAP at 100 pg/mL over 3 days were 4.5 and 95.4%, respectively. Compared with the conventional method with a pressure of below 25 MPa, the peak separation in the MRM chromatogram was improved by using smaller particles (1.6 μm) for the analytical ODS column. The LC-MS/MS system with an MC-ODS HP expanded the applicability of the automated pretreatment.

Determination of rifampicin in rat plasma by modified large-volume direct injection RAM-HPLC and its application to a pharmcokinetic study.

A direct large volume injection high-performance liquid chromatography (HPLC) method with homemade restricted-access media (RAM) pre-column and combined with a column-switching valve was established and developed for determination rifampicin (RIP) in rat plasma. The rat plasma samples (100 μL) were injected directly onto pre-column, where RIP was retained and pre-concentrated, while proteins were washed to waste using a methanol-water (5:95) as the mobile phase at a flow rate of 1 mL/min. Then, by rotation of the switching valve at 5 min, the RIP were eluted from the pre-column and transferred to an Luna C18 analytical column by the chromatographic mobile phase consisting of methanol-acetonitrile-10 mm ammonium format (60:5:35) at a flow rate of 1 mL/min. The total analytical run time was 15 min with UV detection wavelength at 254 nm. Carbamazepine was used as the internal standard. Excellent linear correlation (r = 0.9993) was obtained in the range of 0.25-8 µg/mL for rat plasma. The intra-day and inter-day precisions of RIP were all <5.0%. The recoveries were in the range of from 99.98-113.66% for plasma. This on-line RAM-HPLC method was successfully applied to the pharmacokinetic study of RIP in rat plasma.

Aspects of trapping efficiency and matrix effects in the development of a restricted-access-media-based trap-and-elute liquid chromatography with mass spectrometry method.

Online restricted access media with liquid chromatography and tandem mass spectrometry for the direct analysis of small molecules in biological fluids represents an interesting alternative to time-demanding traditional sample preparation techniques. In this study, important considerations concerning the development of a restricted access media with liquid chromatography and tandem mass spectrometry method for the analysis of dansylated estrogens in biological matrix are presented. Parameters influencing peak tailing and trapping efficiency were evaluated. The key factors included the ion strength of the mobile phase, a loading flow rate of the sample onto the trap column, and selection of a proper stationary phase of the trap column for a given set of analytes. These parameters have proven to be essential for minimizing any unwanted chromatographic peak tailing. The bulk derivatization of the analytes in the biological fluids and its relationship to the observed matrix effects was evaluated as well.

Restricted access media as a streamlined approach toward on‐line sample preparation: Recent advancements and applications

Restricted access media (RAM) as an alternative to traditional sample preparation strategies are reviewed. RAM comprise chromatographic packing materials that combine, typically, a restrictive outer surface to exclude the retention of large biomolecules, which are common interferences in biological fluids, with retentive inner pores or phases to capture analytes of interest. Through the years, a variety of RAM formats have been created, including internal surface phases, semipermeable phases, and molecularly imprinted polymer phases. Many phases are commercially available through a variety of manufacturers. The use of on-line sample preparation using RAM can increase throughput, recovery, and ease of use for sample preparation of complex biological matrices. The state-of-the-art with respect to production and use of these media for a variety of applications is covered.

Development of achiral and chiral 2D HPLC methods for analysis of albendazole metabolites in microsomal fractions using multivariate analysis for the in vitro metabolism

In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral-chiral and chiral-chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0×0.46cmI.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0×0.46cmI.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis.

Online enrichment ability of restricted-access column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride

The online enrichment ability of the restricted-access media (RAM) column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride in plasma was studied. The RAM-HPLC system consisted of an RAM column as enrichment column and a C18 column as analytical column coupled via the column switching technique. The effects of the injection volume on the peak area and the systematic pressure were studied. When the injection volume was less than 100 microL, the peak area increased with the increase of the injection volume. However, when the injection volume was more than 80 microL, the pressure of whole system increased obviously. In order to protect the whole system, 80 microL was chosen as the maximum injection volume. The peak areas of ordinary injection and the large volume injection showed a good linear relationship. The enrichment ability of RAM-HPLC system was satisfactory. The system was successfully used for the separation and detection of the trace benazepril hydrochloride in rat plasma after its administration. The sensitivity of HPLC can be improved by RAM pre-enrichment. It is a simple and economic measurement method.

Quantification of carbamazepine and its active metabolite by direct injection of human milk serum using liquid chromatography tandem ion trap mass spectrometry

This work reports the use of a liquid chromatography ion trap tandem mass spectrometry (LC–IT-MS/MS) system for quantification in human milk samples of both carbamazepine (CBZ) and its active metabolite, carbamazepine 10,11-epoxide (CBZE). An octadecyl restricted-access media bovine serum albumin column (RAM-BSA C18) was used in single-column mode. Selectivity, extraction efficiency, accuracy and precision were achieved employing 100μL of the sample, without preparation, with detection limits of 20.0ng/mL for CBZ and 40.0ng/mL for CBZE. The matrix effect was investigated for the compounds by post-column infusion (qualitative) and by on-line extraction (quantitative). It was observed suppression effect for CBZ and CBZE by post-column infusion, ion suppression of 0.80 for CBZ, and enhancement of 1.28 for CBZE by on-line extraction. The developed method was validated and applied to analyze breast milk samples from one nursing mother. CBZ and CBZE were quantified in the concentrations of 2.26μg/mL and 1.54μg/mL, respectively. To our knowledge, this is the first report on the simultaneous determination of CBZ and its active metabolite by direct injection of human milk serum.

Direct, simultaneous measurement of liposome-encapsulated and released drugs in plasma by on-line SPE–SPE–HPLC

A method for the simultaneous measurement of liposome-encapsulated and released drugs in mouse plasma by on-line solid phase extraction (SPE)–SPE–HPLC with direct plasma injection was developed using a doxorubicin (DXR)-containing liposome formulation as the model drug. During SPE, the released DXR was extracted on the 1st restricted-access media (RAM) SPE column, whereas the liposomes were eluted. The eluted liposomes were collapsed on-line, and the released DXR was delivered to the 2nd RAM SPE column for extraction. The retained DXR on the SPE columns was analyzed via HPLC–fluorescent detector by switching the valves. The method was validated and applied to the pharmacokinetic study of DXR in mice after intravenous injection of DXR-containing liposomes.