Restricted Access Media Column Research Articles

On-line solid-phase extraction of pharmaceutical compounds from wastewater treatment plant samples using restricted access media in column-switching liquid chromatography-tandem mass spectrometry

An on-line solid phase extraction using a lab-made restricted access media (RAM) was developed as sample preparation procedure for determination of the pharmaceutical compounds caffeine (CAF), carbamazepine (CBZ), norfloxacin (NOR), ciprofloxacin (CIP), fluoxetine (FLX) and venlafaxine in wastewater treatment plant samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is suitable for use in routine of analysis, avoiding cross-contamination and requiring only a small sample volume (50 µL), with minimal handling.The method was validated according to international guidelines. The chromatographic efficiency was evaluated using peak resolution and asymmetry parameters. Carryover was also evaluated, in order to ensure reliability of the analysis and the ability to reuse the cartridge. Satisfactory linearity (r2 > 0.99) was obtained for all the compounds. The intra- and inter-day precision values were lower than 5.79 and 14.1%, respectively. The limits of detection ranged from 0.01 to 3 µg L−1 and the limits of quantification were from 0.1 to 5 µg L−1. The method was applied to 20 environmental wastewater samples, with caffeine being the most widely detected compound, at the highest concentration of 392 µg L−1, while other compounds were detected in fewer samples at lower concentrations (up to 9.60 µg L−1). The lab-made modification is a cheaper option for on-line sample preparation, compared to commercially available on-line SPE cartridges and RAM columns. Moreover, a high-throughput procedure was achieved, with an analysis time of 16 min including sample preparation and chromatographic separation. The same RAM column was applied over 200 injections including method optimization, validation and application in wastewater samples without loss of analytical response.

BIOANALYTICAL METHOD BY COLUMN-SWITCHING WITH DIRECT INJECTION OF HUMAN PLASMA FOR DETERMINATION OF SULPHONYLUREAS

Sulphonylureas are widely used in the treatment of Diabetes mellitus, one of the main causes of death in human population. Their determination is essential in pharmacological research and in the development of new drugs. Generally, determination of sulphonylureas in biological matrices is performed using conventional sample preparation techniques, which frequently leads to an increase of analysis time and errors. In this context, a bioanalytical method for the simultaneous determination of sulphonylureas by direct injection of human plasma was developed and optimized. An automated column-switching high performance liquid chromatographic system with a restricted access media (RAM) column coupled to a fused-core column was employed. At the first dimension, a RAM column with mobile phase of ultrapure water pH 6.0 at a flow-rate of 1.0 mL min-1 was used. The valve switching time was 3 minutes. At the second dimension, a C18 guard-column coupled to a C18 fused core column with mobile phase of acetonitrile and 10 mM phosphate buffer pH 3.0 (54:46 v/v) at a flow-rate of 0.8 mL min-1 were employed. The column switching system was performed in backflush configuration with an analyte elution time of 1 minute. Flufenamic acid was used as the internal standard. The mean plasma protein exclusion percentage by the RAM-column was 104.5%. The developed and optimized method showed to be fast and simple, allowing the direct injection of biological sample into the chromatographic system and the simultaneous determination of three sulphonylureas in only 12 minutes, including the sample treatment, separation and detection.

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.

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.

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.

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.

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization-tandem mass spectrometry (2D-LC-ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D-LC-ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C(18 )column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5-10 ng/mL (r(2) > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies.

Effective on-line extraction of drugs from plasma using a restricted-access media column in column-switching HPLC equipped with a dilution system: Application to the simultaneous determination of ER-118585 and its metabolites in canine plasma

The present paper describes the on-line extraction of drugs in plasma using a restricted-access media (RAM) column in a column-switching high performance liquid chromatography (HPLC) apparatus that was equipped with an on-line dilution system. The use of a six- to eightfold on-line dilution ratio for plasma samples resulted in almost 100% recovery of both acidic and basic drugs from plasma. It was found that the relationship between the on-line dilution times and drug recovery efficiencies from plasma was explained in terms of the binding constant between the drug and albumin. The applicability of column-switching HPLC with an on-line dilution system and the effectiveness of the extraction procedure were confirmed by a simultaneous determination of the basic compound, ER-118585, and its metabolites in canine plasma.

Absolute ProGRP quantification in a clinical relevant concentration range using LC–MS/MS and a comprehensive internal standard

The objective of this study was to develop a method using liquid chromatography with tandem mass spectrometric detection for the absolute quantification of the small cell lung cancer biomarker ProGRP in human serum, using its tryptic signature peptide NLLGLIEAK. The samples were precipitated for most of its proteins using acetonitrile prior to tryptic digestion. Further sample clean-up and enrichment was achieved by the use of an on-line restricted access media column, followed by separation on a BioBasic C8 column. Detection and quantification was carried out by operating a triple quadrupole MS in the selected reaction monitoring mode. This setup allowed analysis of realistic samples and detections limits in human serum of 150 pg ProGRP on column. Using an internal standard derived from the parent ProGRP after acetylation of the lysine side chain allowed better quantification through variation correction in all sample pretreatment steps, trypsination included.

Simultaneous quantification of sulfamethoxazole and trimethoprim in whole egg samples by column-switching high-performance liquid chromatography using restricted access media column for on-line sample clean-up

This work reports the application of restricted access media (RAM) column, in a multidimensional configuration, for simultaneous analysis of sulfamethoxazole (SMX) and trimethoprim (TMP) in whole eggs with ultraviolet detection. The proteins were partially precipitated by adding 0.5 mL of acetonitrile into 1.0 mL of blended egg followed by centrifugation. The supernatant was injected (250 μL) directly into the muldimensional system. At the first dimension, a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 mm × 46 mm I.D., Luna ® silica, 10 μm particle size and 100 Å pore size), was used for extraction and concentration of the analytes and at second dimension, an octadecyl column (150 mm × 46 mm I.D., Luna ® silica, 10 μm particle size and 100 Å pore size), for analysis. The developed method showed good selectivity, accuracy and precision for quantification of these different compounds in eggs, and the limits of quantification were 80 ng/mL, for both compounds. The validated method is reliable and sensitive for monitoring residues in whole eggs samples and thus, to determine withdraw period for laying hens using veterinary medicine having SMX–TMP combination.

On‐line pretreatment using methylcellulose‐immobilized cation‐exchange restricted access media for direct liquid chromatography/mass spectrometric determination of basic drugs in plasma

A novel methylcellulose-immobilized restricted access media column with strong cation-exchange groups on an internal surface (MC-SCX) was evaluated for the direct injection analysis of basic polar drugs in plasma by column-switching liquid chromatography/mass spectrometry (LC/MS). Analytical conditions, including an automated pretreatment step and MS detection, were optimized for a series of basic drugs (doxepin, desipramine, imipramine, nortriptyline, amitriptyline, clomipramine). On-line pretreatment with the MC-SCX column followed by fast gradient analysis using a C18 column resulted in a total analysis cycle time of 7 min for each spiked plasma sample. More than 150 plasma samples spiked with target compounds were measured without compromising MS detection (relative standard deviations less than 11% for all compounds, and regression coefficients greater than 0.99).

Enantiomeric determination of the plasma levels of omeprazole by direct plasma injection using high-performance liquid chromatography with achiral–chiral column-switching

A direct injection high-performance liquid chromatographic (HPLC) method, with column-switching, for the determination of omeprazole enantiomers in human plasma is described. A restricted access media (RAM) of bovine serum albumin (BSA) octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the RAM column onto an amylose tris(3,5-dimethylphenylcarbamate) chiral column by the use of a column-switching valve and the enantioseparation was performed using acetonitrile–water (60:40 v/v) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40 mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. The assay was able to determine the cytochrome P450 2C19 phenotype of the subjects participating in this study.

Non-particulate (continuous bed or monolithic) restricted-access reversed-phase media for sample clean-up and separation by capillary-format liquid chromatography.

Restricted-access reversed-phase non-particulate (continuous bed or monolithic) stationary phases of different hydrophobicity synthesized in 100 microm i.d. fused silica capillaries have been evaluated. A specific property of restricted-access media (RAM) is that they interact with small analytes and exclude big molecules, e.g. proteins, from access to the active sites and adsorption on the surface. This dual property facilitates direct injection of biological fluids for drug or drug-metabolite analysis. Different RAM and RAM-precursor capillary columns were tested to assess the influence of chromatographic bed morphology on loadability. Inverse size-exclusion chromatography was used for investigation of pore structural properties of the capillary-format continuous beds. The data obtained were used to discuss the mechanism of separation of the biological samples using capillary columns and to propose a model for the topochemical architecture of the RAM investigated. Different morphology of the non-particulate reversed-phase precursors resulted in two types of RAM material shielded with hydrophilic polymer, classified as homogeneous or heterogeneous topochemistry stationary phases. Capillary columns were applied for chromatography of biological fluids. High resolution was obtained, without the need for column switching, when capillary columns operated in gradient conditions. Extensive evaluation of the chromatographic properties (hydrophobicity, efficiency, separation impedance, and loadability) of the non-particulate reversed-phase materials was performed before and after shielding with hydrophilic polymer to generate restricted-access properties. Minor changes of hydrophobicity, efficiency, or separation impedance were observed after the shielding.

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C 18-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 m M ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing ( E)- and ( Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C 18 column, with a mixture of 20 m M ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H] − were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H] − in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu] −, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of ( E)- and ( Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.

Strategies in Using Analytical Restricted Access Media Columns for the Removal of Humic Acid Interferences in the Trace Analysis of Acidic Herbicides in Water Samples by Coupled Column Liquid Chromatography with UV Detection

In this comprehensive study, analytical restricted access media (RAM) columns were investigated to determine their performance in decreasing the humic acid interference encountered in the trace analysis of acidic herbicides in environmental water samples when employing reversed-phase liquid chromatography with UV detection (RPLC-UV). The selected analytical column RAM materials are 5-μm Semi-Permeable-Surface (SPS, Regis), 5-μm Internal Surface Reversed Phase (ISRP, Pinkerton, Regis), and 5-μm Hisep (Supelco); a precolumn containing 25-μm alkyl-diol-silica (ADS) RAM material (Merck) was also included in this study. Different LC configurations were investigated, including the single RAM column mode and column switching (LC/LC), employing one RAM column in combination with an analytical C18 column, or two RAM columns. Concerning methodology, both the single-residue method (SRM) approach and the multiresidue method (MRM) approaches were tested by analyzing SPE C18 extracts of reference water samples spiked w...

Direct injection HPLC analysis of some non-steroidal anti-inflammatory drugs on restricted access media columns.

Direct serum injection methods are described for the HPLC determination of selected non-steroidal anti-inflammatory drugs (NSAIDS) on restricted access media (RAM) columns, using either a Pinkerton (ISRP) or semi-permeable surface (SPS) column. Twenty microlitres of a filtered serum sample was injected directly onto each column. Calibration curves were prepared for ketoprofen (1-20 micrograms/mL), fenbufen (0.8-20 micrograms/mL), sulindac (0.8-20 micrograms/mL) and probenecid (1.5-40 micrograms/mL) on the SPS column, and nabumetone (1.5-40 micrograms/mL) and indomethacin (1.5-40 micrograms/mL) on the Pinkerton column. The percentage error and imprecision of the methods were found to be less than 10%. The inter- and intra-day variability for analytes were in the range of 0.15-10%. The limits of quantitation and detection were in the range of 800-1500 and 300-800 ng/mL, respectively, for the analytes studied. A hydrophobic shielded phase (Hisep) column was also investigated and was found to be generally too retentive (> 29 min) for assay of these analytes.

Direct determination of stability of protease inhibitors in plasma by HPLC with automated column-switching

Automated column-switching HPLC methods were developed and utilized for the direct analyses of three hydroxamic acid based metalloprotease inhibitors in rat plasma. These column-switching methods involved the use of a restricted-access media (RAM) precolumn and a column-switching valve, allowing the complete automation of sample preparation and HPLC. The plasma samples were directly injected onto a precolumn packed with SPS/ODS stationary phase and then backflushed onto an ODS analytical column using a 6-port column-switching device. The drug stability in rat plasma was determined using both the automated and traditional HPLC methods. The results obtained from the automated column-switching methods were in good agreement with those from traditional methods that involve sequential protein precipitation, liquid extraction, solvent evaporation, and sample reconstitution. In addition to the elimination of labor-intensive and time-consuming sample preparation procedures, the column-switching methods allowed on-line analyte enrichment and accurate determination of drug stability in plasma with detection limits in the range of 10–20 ng ml −1. This work represents, for the first time, a drug stability study in plasma by automated column-switching HPLC technique with the use of a RAM column. Our column-switching methods can be readily adapted to any existing HPLC system with minimal hardware modification.

Direct plasma injection for high-performance liquid chromatographic–mass spectrometric quantitation of the anxiolytic agent CP-93 393

A direct plasma injection method has been developed for the rapid analysis of drugs in biological fluids. A new generation restricted access media column specifically designed to accommodate direct injection of plasma and other fluids is utilized for on-line HPLC–ESI-MS analysis. For rapid analysis the on-line extraction column is linked to a HPLC–ESI-MS system. Good results are obtained for the quantitation of CP-93 393 and deuterated internal standard over the range of 10–1000 ng/ml. The lower limit of detection for the assay was 58 pg injected on column. Accuracy and precision values are 9.0% or better over the entire range of the assay. In addition, more than 200 injections (100 μl) were performed per column with unattended, automated analysis.

Evaluation of liquid chromatographic behavior of restricted-access media precolumns in the course of direct injection of large volumes of plasma samples in column-switching systems

The chromatographic behavior of an alkyl-diol silica (ADS, 25×4 mm I.D.) and a semipermeable surface (SPS, 10×10 mm I.D.) supports two types of restricted-access media (RAM), which served as precolumns in column-switching systems for direct injection of large volumes of plasma samples (500 μl), was studied with regard to peak performance, retention and column back pressure. The adsorption of matrix proteins both on sealings (porous frits and sieves) and packings was also examined. Columns of ADS and SPS were unchanged after the injection of 10–20 ml human plasma under normal working conditions. Even when changes occurred on the precolumns (>50 ml of plasma in total), it was still possible to regenerate the column performance by replacing the column sieves, or by washing and removing columns from the system for a period, since the changes were more related to the blockage of sealings and/or the adsorption of proteins on the hydrophilic surfaces. Proteins could eventually be unspecifically adsorbed on the hydrophobic ligand of the support. It was found on one ADS column that the retention decreased by 20% and the pressure increased 30 bar after an intensive loading of 75 ml plasma (injection volume, 500 μl) without reconditioning procedure. Studies showed that the column sealings played the most important role for the lifetime of RAM columns. For ADS columns, using sieves without polytetrafluoroethylene (PTFE) nets were the best. No significant difference in column life span between SPS and ADS was found.