Simple surface modification of polypropylene pipette tips for anchoring of organic monolithic-based materials for micro-solid-phase extraction.

This paper presents a simple method for the surface modification of polypropylene pipette tips by adsorbing a photo-initiator, 2,2-dimethoxy-2-phenylacetophenone (DMPAP), to create reactive sites for the formation of a layer of ethylene dimethacrylate (EDMA) and subsequent monolith polymerization. The types of monomers and the degree of crosslinking dramatically affected the monolith shrinkage and detachment in unmodified tips. Effective surface modification for anchoring monolithic materials to pipette tips was achieved using 15 wt% DMPAP and 10 wt% EDMA in methanol with UV irradiation at 365 nm. The extraction of 5-hydroxyindoleacetic acid, serotonin, and bisphenol A (BPA) using methacrylate and activated charcoal composite monoliths was investigated in terms of breakthrough capacity. The application of monolithic pipette tip micro-solid-phase extraction followed by HPLC-UV was demonstrated for determining BPA leaching from baby-feeding bottles and canned foods. Wide linearity ranging from 0.1 to 100 ng mL-1 (R2 = 0.9998) with good repeatability (% RSD = 3.9 %) and accuracy (% recovery = 93-106 %) was obtained. The limit of detection and limit of quantification were 0.084 and 0.280 ng mL-1, respectively. By varying the sample loading volume from 0.50 to 10.00 mL with eluting volume of 150 µL, a 2-to-52-fold pre-concentration factor was observed.

Simple and fast analysis of iohexol in human serums using micro-hydrophilic interaction liquid chromatography with monolithic column.

A simple and rapid method based on micro-liquid chromatography using a synthetic monolithic capillary column was developed for determination of iohexol in human serums, a marker to evaluate the glomerular filtration rate. A hydrophilic methacrylic acid-ethylene dimethacrylate monolith provided excellent selectivity and efficiency for iohexol with separation time of 3 min using a mobile phase of 40:60 v/v 50 mM phosphate buffer pH 5/methanol. Four serum protein removal, methods using perchloric acid, 50% acetonitrile, 0.1 M zinc sulfate, and centrifuge membrane filter were examined. The method of zinc sulfate was chosen due to its simplicity, compatibility with the mobile phase system, nontoxicity, and low cost. Interday calibration curves were conducted over iohexol concentrations range of 2-500 mg/L (R(2) = 0.9997 ± 0.0001) with detection limit of 0.44 mg/L. Intra- and interday precisions for peak area and retention time were less than 2.8 and 1.4%, respectively. The method was successfully applied to serum samples with percent recoveries from 102 to 104. The method was applied to monitor released iohexol from healthy subject. Compared with the commercially available reversed-phase high-performance liquid chromatography method, the presented method provided simpler chromatogram, faster separation with higher separation efficiency and much lower sample and solvent consumption.

Simple in-house flow-injection capillary electrophoresis with capacitively coupled contactless conductivity method for the determination of colistin.

An in-house flow-injection capillary electrophoresis with capacitively coupled contactless conductivity detection method was developed for the direct measurement of colistin in pharmaceutical samples. The flow injection and capillary electrophoresis systems are connected by an acrylic interface. Capillary electrophoresis separation is achieved within 2 min using a background electrolyte solution of 5 mM 2-morpholinoethanesulfonic acid and 5 mM histidine (pH 6). The flow-injection section allows for convenient filling of the capillary and sample introduction without the use of a pressure/vacuum manifold. Capacitively coupled contactless conductivity detection is employed since colistin has no chromophore but is cationic at pH 6. Calibration curve is linear from 20 to 150 mg/L, with a correlation coefficient (r(2) ) of 0.997. The limit of quantitation is 20 mg/L. The developed method provides precision, simplicity, and short analysis time.

Direct injection of human serum and pharmaceutical formulations for glucosamine determination by CE-C(4)D method.

A simple CE-C(4)D method has been developed for the determination of glucosamine by direct injection of human serum and pharmaceutical samples. Glucosamine was electrokinetically injected and analysed in its protonated form using 20mM MES/His (pH 6) as background electrolyte in order to separate it from the matrix and to provide a better response to the C(4)D detector. Separation of glucosamine in human serum and pharmaceutical samples was performed in 3 min without the need for protein precipitation or matrix removal. Good precision in terms of %RSD for the migration time and peak area were less than 1.91% (n = 10). The conductivity signal was linear with glucosamine concentration in the range 0.10-2.50mg/mL, with a detection limit of 0.03 mg/mL. Recoveries of glucosamine in serum and pharmaceutical samples were 86.5-104.78%. The method was successfully applied for the determination of the glucosamine content in pharmaceutical formulations and validated with high performance liquid chromatography (HPLC). Good agreements were observed between the developed method, label values and the HPLC method. Glucosamine could be detected in spiked serum sample by direct injection. This was not possible by HPLC due to co-eluting interferences.

Repeatability in column preparation of a reversed-phase C18 monolith and its application to separation of tocopherol homologues.

This work investigated the repeatability of column preparation for a reversed-phase C18 monolith, namely stearyl methacrylate-co-ethylene glycol dimethacrylate (SMA-EDMA). The columns were thermally polymerised using three commonly available heating devices (GC oven, hot air oven and water bath) and their chromatographic performance evaluated using micro-liquid chromatography for separation of five test compounds. Precision in terms of %RSD of retention times were 9.0, 6.5, and 12.5 using GC oven, hot air oven and water bath, respectively. Between-batch precision for the hot air oven (n=3 days) was less than 10.4% for retention time. The SMA-EDMA monolith was applied to the separation of tocopherol homologues by capillary electrochromatography. Usually tocopherol homologues cannot be completely separated by conventional reversed-phase C8- or C18-packed bed or C18-silica based monolithic columns. Polymer monolith has been shown to give remarkable selectivity towards the tocopherols compared to the conventional microparticulate phase and silica based monolith. Successful separation of the tocopherol isomers was achieved on the SMA-EDMA monolith without any column modification.

Monolithic and packed particle materials for in-line pre-concentration in capillary electrophoresis for 4-hydroxy-3-methoxy-methamphetamine and terbutaline.

In-line solid-phase extraction (SPE) for capillary electrophoresis (CE) was investigated using a synthesized monolith and a commercial packing material. Terbutaline (TER) and 4-hydroxy-3-methoxy-methamphetamine (HMMA) with benzyl alcohol as the electroosmotic flow marker were employed as model compounds. Two types of methacrylate-based monoliths, namely methacrylic acid-ethylene dimethacrylate and butylmethacrylate-ethylene dimethacrylate were examined. Preliminary results indicated that a non-aqueous separating medium is more suitable for these methacrylate monoliths than a purely aqueous medium (non-reproducible elution). However, coupling of the methacrylic acid-ethylene dimethacrylate with non-aqueous capillary electrophoresis could not provide good precision for the three model compounds. A packed-silica C18 SPE was also adopted by simply packing the C18 particles in situ in the separation capillary. Using an aqueous running buffer (10 mM phosphate buffer (PPB), pH 7), acceptable precision could be obtained with this type of SPE material. With a 10 min loading time and 20 min total analysis time, the pre-concentration factors were 333 and 1000 for TER and HMMA, respectively. The %RSD were less than 4.5 and 0.3 for the peak areas and migration times, respectively, for both HMMA and TER (n=20).

Separation of alpha-, beta-, gamma-, delta-tocopherols and alpha-tocopherol acetate on a pentaerythritol diacrylate monostearate-ethylene dimethacrylate monolith by capillary electrochromatography.

This work reports the first use of a monolith with method development for the separation of tocopherol (TOH) compounds by CEC with UV detection. A pentaerythritol diacrylate monostearate-ethylene dimethacrylate (PEDAS-EDMA) monolithic column has been investigated for an optimised condition to separate alpha-, beta-, gamma- and delta-TOHs, and alpha-tocopherol acetate (TAc). The PEDAS-EDMA monolith showed a remarkably good selectivity for separation of the TOH isomers including the beta- and gamma-isomers which are not easily separated by standard C8 or C18 particle-packed columns. Retention studies indicated that an RP mechanism was involved in the separation on the PEDAS-EDMA column, but polar interactions with the underlying ester and hydroxyl groups enhanced the separation of the problematic beta- and gamma-isomers. Separation of all the compounds was achieved within 25 min using 3:10:87 v/v/v 100 mM Tris buffer (pH 9.3)/methanol/ACN as the mobile phase. The method was successfully applied to a pharmaceutical sample with recoveries from 93 to 99%. Intraday and interday precisions (%RSD) for peak area and retention time were less than 2.3. LODs for all four TOHs and TAc were below 1 ppm.