Polymer Monolithic Column Research Articles

The fabrication of poly (polyethylene glycol diacrylate) monolithic porous layer open tubular (mono-PLOT) columns and applications in hydrophilic interaction chromatography and capillary gas chromatography for small molecules.

The easy shrinkage and swelling of polymer monolithic column when exposed to mobile phase with different polarity is a problem that cannot be ignored. To overcome this drawback, a convenient aqueous two-phase polymerization approach was used to prepare poly (polyethylene glycol diacrylate, PEGDA) monolithic porous layer open tubular (mono-PLOT) columns (150μm). The poly(PEGDA) mono-PLOT column with homogeneous polymer porous layer was synthesized successfully. A maximum plate number of 41,500 plates per meter for allyl thiourea was obtained under a velocity of 1.8mm/s. Several kinds of polar molecule were separated on the proposed mono-PLOT column and a typical hydrophilic interaction retention mechanism was observed. High speed separation of benzoic acids was also carried out, baseline separation of five benzoic acids was successfully achievedwithin 5min with a 70cm mono-PLOT column at 50°C. Furthermore, the resulting PLOT column was also successfully applied to separate standard analytes of three DNA oxidative damage products and RNA-modified nucleosides and four chlorophenols. At last, the column could separate alcohols, alkanes, and aromatic isomers via GC. It had more than 20,000 plates per meter for butanol - higher than commercial coatings open tubular columns.

Determination of three benzodiazepines in human urine by solid-phase extraction with poly [2-(acryloyloxy) ethyl] trimethyl ammonium chloride-co-ethylene glycol dimethacrylate monolithic column coupled with high performance liquid chromatography

A polymer monolithic column was prepared in a syringe by using [2-(acryloyloxy) ethyl] trimethyl ammonium chloride (DAC) as a monomer and ethylene glycol dimethacrylate (EDMA) as a crosslinker. The obtained monolith was developed as a solid-phase extraction sorbent and used with high performance liquid chromatography (HPLC) for the analysis of three benzodiazepines (BZDs) including bromazepam (BRZ), lorazepam (LRZ) and diazepam (DZP) in urine. The effects of reaction time and the solid-phase extraction conditions (washing solution, elution solvent and volume) on the extraction efficiencies of the three BZDs were investigated. The monolithic column was successfully prepared within 4 h, and it offered 100% adsorption efficiency for the three BZDs. The urine sample (4 mL) was loaded on the monolith, washed with 4 mL of H2O, and eluted with 1 mL of ethyl acetate. Under the optimized conditions, the linear ranges were 4.0-1000 ng/mL for the three BZDs, with correlation coefficients (r) of 0.999. The limits of detection (S/N=3) and limits of quantification (S/N=10) of the three BZDs were in the range of 1.0-1.2 ng/mL and 3.3-4.0 ng/mL, respectively. The recoveries at three spiked levels (10, 25 and 50 ng/mL) of the three BZDs ranged from 81.4% to 102%, with intra-day and inter-day relative standard deviations (n=3) of 1.2%-4.5% and 2.5%-8.3%. The polymer monolithic column provided effective purification for the three BZDs in urine and the enrichment factor was 12-15. This polymer monolithic adsorbent has the advantages of easy preparation and high extraction efficiency. It is successfully applied to the determination of the three BZDs in urine samples.

Preparation of metal-organic framework MIL-101(Cr) incorporated polymer monolithic column for on-line solid-phase extraction of phenols

A metal-organic framework (MOF) MIL-101(Cr) incorporated polymer monolithic column was prepared, and a method was developed for the determination of 4-nitrophenol, 3-methylphenol, 2-nitrophenol and 2,4-dichlorophenol in water samples by on-line solid phase extraction (SPE) coupled with high performance liquid chromatography (HPLC). Effects of sample solution pH values, sample loading time, sample loading flow rates and desorption time on the on-line SPE of phenols were investigated. Under the optimized extraction conditions, large enhancement factors, wide linearity range, good precisions and low limits of detection were achieved. The results showed that the incorporation of MOFs in porous polymer monolith to fabricate MOF-incorporated monolith is one of the best ways to promote and expand the application of MOFs in on-line SPE.

Comprehensive two-dimensional monolithic liquid chromatography of polar compounds.

Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.

Preparation, characterization, and application of a porous poly (TAIC-co-MMA-co-EDMA) monolithic column for small molecules of aromatics and Chinese herbal medicine notoginseng by HPLC

A novel porous monolithic column was synthesized via in situ free-radical polymerization within a stainless steel column (50 × 4.6 mm i.d.) by using triallyl isocyanurate and methyl methacrylate as co-monomers, ethylene glycol dimethacrylate as a crosslinking agent, 1,4-butanediol, 1- dodecanol, and N, N-dimethylformamide as tri-porogens, and azobisisobutyronitrile as an initiator. The optimized monolithic columns were investigated by scanning electron microscopy, a nitrogen absorption-desorption apparatus, and mercury intrusion porosimetry. The effect of mobile phase composition and temperature on chromatographic behavior was studied in detail as well. The results showed that homemade monolithic columns not only have relative uniform porosity structure, low back pressure, and high thermal stability, but also show high column efficiency, the theoretical plates number of the new polymer monolithic column exceeded 31,000 plates m−1. Good resolution was achieved for HPLC separation of small molecules, including polycyclic aromatic hydrocarbons, isomers of phenols, and the Chinese herbal medicine notoginseng. Methodological validation of six drugs in notoginseng show that correlation coefficients of the linear regressions were in the range of 0.9989–0.9997; the value of relative standard deviation for precision were in the range of 1.37–4.21% for intra-day and 0.34–5.31% for inter-day, respectively; the values of accuracy expressed by recovery were in the range of 100.57–102.51%, 95.18–102.68%, 101.61–102.82%, 100.94–102.40%, 100.99–103.15% and 99.49–103.91% for the six drugs in order, respectively. Taking aromatic hydrocarbons as samples, the relative standard deviation values of the run-to-run (n = 5) and column-to-column (n = 5) based on peak area were < 2.01% and 2.39%, and two RSDs based on retention time were < 2.56% and 2.23%, respectively. The result showed that the polymer monolithic column has good separation ability and high permeability for small molecular substances.

Monolithic column with polymeric deep eutectic solvent as stationary phase for capillary electrochromatography

A novel monolithic column based on the copolymerization of ethylene dimethacrylate and a deep eutectic solvent (DES), which composed of chlorocholine chloride and itaconic acid, was developed for capillary electrochromatography (CEC). The structure and morphology of the obtained monolithic column was characterized by Fourier transform infrared spectra and scanning electron microscopy, which showed a porous monolithic structure with good permeability. The influence of pH value and acetonitrile content on electroosmotic flow (EOF) of DES-based monolithic column was investigated. The DES-based monolithic column was used as the separation column of CEC and exhibited excellent performance for the separation of neutral compounds, phenols, toluidines, nucleosides, nucleotide bases and alkaloids. The retention mechanism might be attributed to synergistic effect of hydrophobic interaction, hydrogen bond interaction and electronic interaction. The DES-based monolithic column showed good repeatability with relative standard deviations of migration time less than 0.80%, 2.15%, 4.26% and 3.08% for intra-day, inter-day, column-to-column and batch-to-batch, respectively. Thus, organic polymer monolithic columns with DESs as functional monomers are a promising option in chromatographic separation.

Heparin‐Immobilized Polymeric Monolithic Column with Submicron Skeletons and Well‐Defined Macropores for Highly Efficient Purification of Enterovirus 71

AbstractIn this study, glycidyl methacrylate‐based polymerized high internal phase emulsion monolithic column (polyHIPE) with submicron skeletons and well‐defined macropores is prepared and functionalized by immobilizing heparin to obtain a novel affinity chromatographic separation medium for effective purification of Enterovirus 71 (EV71). The pore structure of the obtained monoliths differs significantly from conventional polyHIPE monoliths. The prepared polyHIPE presents well‐defined 3D network skeletons of 0.2–0.5 µm, interconnected macropores of 0.5–2 µm, large specific surface area (47–61 m2 g−1), and excellent permeability (k = 1.0 × 10−13 m2). Further, successful immobilization of heparin is proved by FTIR and XPS and the monoliths display superiority in the purification of EV71 from culture supernatant. The final recovery and binding capacity of EV71 are calculated to be 60% and 152.8 ng per column, respectively. SDS‐PAGE gel electrophoresis and TEM analysis indicate a high efficiency in the removal of impurities from culture supernatant of EV71. This, together with easy fabrication, makes polyHIPE monolith a promising alternative to commercially available monolithic supports for virus purification.

Monolithic column functionalized with quinine derivative for anion-exchange capillary electrochromatography.

A novel anion-exchange organic polymer monolithic column based on monomers N-benzylquininium chloride and acrylamide were firstly prepared by in situ copolymerization for capillary electrochromatography. Moreover, N-benzylquininium was firstly introduced as a strong anion-exchange functional group. A relatively strong anodic EOF was obtained in the pH values from 4.0 to 9.0, which was in the same direction with the electrophoretic mobility of acid compounds. Hence, the anion-exchange monolithic column was very suitable for the rapid separation of acid compounds. Eight acid compounds (2-chlorobenzoic acid, mandelic acid, 4-hydroxybenzoic acid, indole-3-acetic acid, 2-aminoterephthalic acid, 3,5-pyridinedicarboxylic acid, benzoic acid, and 4-aminobenzoic acid) were successfully separated on the monolithic column. The highest column efficiency was 4.60 × 105 plates/m (theoretical plates, N) for 3,5-pyridinedicarboxylic acid. The proposed monolithic column was characterized by SEM and FT-IR. The RSDs of the acid compounds migration time for run-to-run, day-to-day, and column-to-column were all less than 5.0%.

Development of an SPE Method Using Ionic Liquid-Based Monolithic Column for On-Line Cleanup of Human Plasma and Simultaneous Determination of Five Steroid Drugs

An 1-vinyl-3-hexyl imidazolium bromide ionic liquid-based monolithic column was prepared via in situ free radical polymerization, which was used as the sorbent of on-line solid phase extraction. The prepared material was characterized by scanning electron microscopy, nitrogen adsorption–desorption instrument and mercury intrusion porosimetry, respectively, and the results showed that the homemade monolith occupied relatively uniform porous structure and high-specific surface area of 212.53 m2 g−1. On-line solid phase extraction–high-performance liquid chromatography was performed to quantitatively analyze five steroid drugs, including betamethasone, norgestrel, halcinonide, beclomethasone dipropionate and testosterone propionate in human plasma. The determination procedure was carried out following the optimization of the mobile phase composition. Methodological validation showed that correlation coefficients of the linear regressions were in the range of 0.9989–0.9992; the values of relative standard deviation for precision were in the range of 1.39–6.15% for intra-day and 0.32–6.34% for inter-day, respectively; the values of accuracy expressed by recovery were in the range of 99.02–101.98, 93.27–102.89, 102.79–104.01, 100.04–102.61 and 100.77–102.44% for the five drugs in order, respectively; the values of relative standard deviation for repeatability calculated according to retention times and peak areas of the five drugs were in the ranges of 0.28–0.76% (n = 5) and 0.84–2.63% (n = 5), respectively. The results showed that the polymer monolithic column was feasible for an on-line solid phase extraction column, exhibiting good selectivity and high permeability.

A novel one-pot strategy to prepare β-cyclodextrin functionalized capillary monoliths for enantioseparation of basic drugs

With native β-cyclodextrin (β-CD) added into the polymerization mixture directly, a novel, convenient and low-cost one-pot strategy was developed to prepare the β-CD functionalized organic polymer monolithic capillary column. Diazabicyclo[5.4.0]undec-7-ene (DBU) as a basic catalyst for the ring opening reaction between β-CD and glycidyl methacrylate (GMA) was introduced into the polymerization system for the first time. Thereby, two consecutive reactions namely the in situ methacrylation of β-CD and copolymerization reaction can be achieved in one pot. The preparation conditions including the type and composition of porogens，the ratio of functional monomer to crosslinker and amount of 2-acrylamido-2-methyl propane sulfonic acid (AMPS) were optimized. The specific surface area and morphology of the prepared monolith were characterized using scanning electron microscopy (SEM) and nitrogen adsorption analysis, respectively. Raman spectroscopy and nuclear magnetic resonance (NMR) spectroscopy confirmed that β-CD was covalently bonded onto the monolith successfully. Then, the monolithic column was applied to enantioseparation of six basic drugs in capillary electrochromatography (CEC). Under the optimal conditions, tropicamide, homatropine, homatropine methylbromide, brompheniramine, chlorpheniramine and clorprenaline were all totally separated with the resolution (Rs) values of 2.84, 4.70, 4.61, 3.01, 2.57 and 2.33, respectively. Furthermore, the column demonstrated satisfactory stability and repeatability of retention time and enantioselectivity using homatropine as model analyte.

Preparation and Characterization of Fe3O4@SiO2 Nanoparticles Incorporated Polymer Monolithic Column for Gas Chromatography

We report on the synthesis of Fe3O4@SiO2 nanoparticles incorporated poly(divinylbenzene) monolithic column via in situ polymerization. The monolith had larger specific surface area and relatively uniform porous structure and was characterized by scanning electron microscopy, nitrogen adsorption–desorption, and Fourier transform infrared spectroscopy. The monolith was then applied for the evaluation of gas chromatography separation properties. Here, an electromagnetic induction heating technique was employed to control the column temperature with the thermal effect of eddy current in ferromagnetic materials Fe3O4. The monolith offered good separation efficiency for benzene and toluene and a higher column efficiency was obtained up to 4481 and 9216 plates per meter, respectively. In addition, the injection volume and column capacity of the proposed system are as much as 5 µL and 100 ng. This makes it possible to use a UV detector. The fabricated Fe3O4@SiO2 nanoparticles incorporated poly(divinylbenzene) monolithic column has been shown to be very promising for gas chromatography separation.

Quinine-modified polymer monolithic column with reversed-phase /strong anion-exchange mixed-mode for pressurized capillary electrochromatography.

Via the facile ring-opening reaction of epoxy groups with quinine, a novel polymer monolith with quaternary ammonium for reversed-phase/strong anion-exchange mixed-mode has been fabricated for pressurized capillary electrochromatography (pCEC). Optimization on the preparation of quinine-modified monoliths has been investigated, and characteristics including morphology, permeability, mechanical stability, reproducibility, and column performance have been also studied. Active quaternary ammonium groups were conveniently produced to generate cationic action sites and stable anodic electroosmotic flow. Multiple interactions including reversed-phase, strong anion-exchange, electrostatic repulsion and π-π stacking interactions were obtained. Satisfactory separation capability of various analytes such as alkylbenzenes, polycyclic aromatic hydrocarbons, benzoic acid and its homologs, and β2 -receptor excitants has been achieved. Applied to the real sample, the good resolution of three alkaloids in Corydalis yanhusuo were achieved by pCEC with the quinine-modified monolith. The results light a potential access to facilely fabricating quaternary ammonium-functionalized polymer monolith with multiple interactions for efficient electrochromatography profiling of various compounds.

Preparation of polymer monolithic column functionalized by arsonic acid groups for mixed-mode capillary liquid chromatography

A mixed-mode polymer monolithic column functionalized by arsonic acid groups was prepared by single-step in situ copolymerization of monomers p-methacryloylaminophenylarsonic acid (p-MAPHA) and pentaerythritol triacrylate (PETA). The prepared poly(p-MAPHA-co-PETA) monolithic column has a homogeneous monolithic structure with good permeability and mechanical stability. Zeta potential measurements reveal that the monolithic stationary phase holds a negative surface charge when the mobile phase resides in the pH range of 3.0–8.0. The retention mechanisms of prepared monolithic column are explored by the separation of selected polycyclic aromatic hydrocarbons (PAHs), nucleosides, and three basic compounds. The results indicate that the column functions in three different separation modes associated with reversed-phase chromatography based on hydrophobic interaction, hydrophilic interaction chromatography, and cation-exchange chromatography. The column efficiency of prepared monolithic column is estimated to be 70,000 and 76,000 theoretical plates/m for thiourea and naphthalene, respectively, at a linear flow velocity of 0.85 mm/s using acetonitrile/H2O (85/15, v/v) as the mobile phase. Furthermore, an analysis of the retention factors obtained for the PAHs indicates that the prepared monolithic column exhibits good reproducibility with relative standard deviations of 2.9%, 4.0%, and 4.7% based on run-to-run injections, column-to-column preparation, and batch-to-batch preparation, respectively. Finally, we investigate the separation performance of the proposed monolithic column for select phenols, sulfonamides, nucleobases and nucleosides.

Fast Determination of Residual Sulfonamides in Milk by In-Tube Solid-Phase Microextraction Coupled with Capillary Electrophoresis-Laser Induced Fluorescence

Abstract A fast, feasible, eco-friendly method for determination of sulfonamides using in-tube solid-phase microextraction (IT-SPME) coupled with capillary electrophoresis-laser induced fluorescence was developed. The graphene-embedded porous polymer monolithic column prepared via in-situ UV polymerization was proposed as the IT-SPME column. The method was applied to determine residual sulfonamides in a real milk sample, and an amount of 3.7 ng mL−1 sulfamethazine was detected, which was much lower than the maximum residue limit.

Fabrication of an ionic-liquid-based polymer monolithic column and its application in the fractionation of proteins from complex biosamples.

An ionic-liquid-based polymer monolithic column was synthesized by free radical polymerization within the confines of a stainless-steel column (50mm×4.6mm id). In the processes, ionic liquid and stearyl methacrylate were used as dual monomers, ethylene glycol dimethacrylate as the cross-linking agent, and polyethylene glycol 200 and isopropanol as co-porogens. Effects of the prepolymerization solution components on the properties of the resulting monoliths were studied in detail. Scanning electron microscopy, nitrogen adsorption-desorption measurements, and mercury intrusion porosimetry were used to investigate the morphology and pore size distribution of the prepared monoliths, which showed that the homemade ionic-liquid-based monolith column possessed a relatively uniform macropore structure with a total macropore specific surface area of 44.72m2 /g. Compared to a non-ionic-liquid-based monolith prepared under the same conditions, the ionic-liquid-based monolith exhibited excellent selectivity and high performance for separating proteins from complex biosamples, such as egg white, snailase, bovine serum albumin digest solution, human plasma, etc., indicating promising applications in the fractionation and analysis of proteins from the complex biosamples in proteomics research.

Solid phase microextraction with poly(deep eutectic solvent) monolithic column online coupled to HPLC for determination of non-steroidal anti-inflammatory drugs

Polymer monolithic column based on a green deep eutectic solvent (DES) was prepared for in-tube solid phase microextraction of non-steroidal anti-inflammatory drugs (NSAIDs) in aqueous samples. DES composed of choline chloride and itaconic acid was adopted as functional monomer to synthesize a polymeric monolith inside polydopamine-functionalized poly(ether ether ketone) (PEEK) tube. The obtained monolithic column was characterized with fourier transform infrared spectrometry, X-ray photoelectron spectrometer and field emission scanning electron microscope. An online SPME-HPLC method was developed by connecting the PEEK tube to the HPLC system. Parameters including sampling rate, desorption time, sample pH value and sample volume that can affect the performance of this method were investigated. Under the optimized conditions, the proposed SPME-HPLC method showed high extraction efficiency (enrichment factors about 100), good linearity (R ≥ 0.9997) and good reproducibility (relative standard deviation < 4.32%) for determination of three NSAIDs in aqueous solution. The limits of quantification were found to be in a range of 0.05–0.25 ng mL−1. The SPME-HPLC method was also successfully applied to detect NSAIDs in lake water and human plasma samples.

Separation of proteins from complex bio-matrix samples using a double-functionalized polymer monolithic column.

A double-functionalized polymer monolithic column was fabricated within the confines of a stainless-steel column (50 mm × 4.6 mm i.d.) via a facile method using iron porphyrin, ionic liquid (1-allyl-3-methylimidazolium chloride) and 1,10-decanediol dimethacrylate as tri-monomers; ethylene dimethacrylate as a crosslinker; polyethylene glycol 400 and N,N-dimethylformamide as co-porogens; benzoyl peroxide and N,N-dimethyl aniline as the redox initiation system. Results obtained from scanning electron microscopy, nitrogen adsorption-desorption, and mercury intrusion porosimetry confirmed the uniform pore structure and the pore size distribution of macro-pores. The home-made monolith was further characterized by elemental analysis to investigate the elemental composition of Fe supplied by iron porphyrin, confirming the synthetic process. The resulting optimized monolithic column was used as the stationary phase in high performance liquid chromatography for separating proteins, such as mixture of standard proteins, egg white, and human plasma, exhibiting good selectivity and high performance. It is worth noting that the home-made double-functionalized polymer monolithic column shows excellent selectivity for fractionation separation of human plasma proteins, and it is a promising separation tool for complex bio-samples in proteomic research.

Synthesis of carbon quantum dots-doped dummy molecularly imprinted polymer monolithic column for selective enrichment and analysis of aflatoxin B1 in peanut

In this study, a novel method was proposed to sensitively determine aflatoxin B1 (AFB1) in peanut sample by using a carbon quantum dots-coated dummy molecularly imprinted polymer (CDs-DMIP) monolithic column for pretreatment coupled with high performance liquid chromatography-fluorescence detection (HPLC-FLD). The CDs-DMIP monolithic column was prepared by in-situ polymerization in a water bath using 5,7-dimethoxycoumarin as dummy template molecule. The CDs-DMIP monolithic column was applied to determine AFB1 by HPLC-FLD. Satisfactory linearity was obtained over 0.5–2000ngmL−1, with a high correlation coefficient of 0.9999. The recoveries of AFB1 in peanut sample ranged from 79.5% to 91.2%, and the intraday and interday relative standard deviation ranged from 1.2% to 4.9%. Limit of detection (S/N=3) and limit of quantitation (S/N=10) were 0.118ngmL−1 and 0.393ngmL−1, respectively. Under the optimized conditions, the enrichment factor was over 71-fold. AFB1 in peanut sample and even some other samples could be sensitively determined by CDs-DMIP-HPLC-FLD method.

Fractionation separation of human plasma proteins using HPLC with a homemade iron porphyrin based monolithic column.

In this work a polymer monolithic column was fabricated within the confines of a stainless steel column (50×4.6mm i.d.) via radical polymerization by using iron porphyrin and butyl methacrylate as co-monomers, ethylene glycol dimethacrylate as crosslinking agent, ethylene glycol, isopropyl alcohol and N, N-dimethylformamide as tri-porogens, benzoyl peroxide and N,N-dimethylaniline as initiators. The resulting monolithic column was characterized by elemental analysis, scanning electron microscopy, nitrogen adsorption BET surface area, and mercury intrusion porosimetry, respectively. Results showed that the homemade monolith occupied relatively uniform pore structure, low back pressure, and enhanced selectivity for proteins in complex bio-samples. The present work described a simple and efficient method for "fractionation separation" of human plasma proteins, and it is a promising separation method for complex bio-samples in proteomic research.

Rapid determination of 18 glucocorticoids in serum using reusable on-line SPE polymeric monolithic column coupled with LC-quadrupole/orbitrap high-resolution mass spectrometer.

A simple, rapid and sensitive method for the simultaneous determination of 18 glucocorticoids in serum was developed by coupling on-line solid-phase extraction (SPE) polymeric monolithic column to a liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometer. A simple poly(ethylene glycol dimethacrylate) monolith column (10mm×2.1mm i.d.) was fabricated, and the morphology, surface area and extraction performance of the monolithic column were characterized. Serum samples were extracted by acetonitrile (ACN). Then, online SPE was achieved on the synthesized monolithic column using a 10mmol/L ammonium acetate solution as the loading solvent. After the transfer from the monolith into analytical column (Capcell Pak ADME column) using ACN, the adsorbed analytes were separated on the analytical column and detected with a high-resolution hybrid quadrupole/orbitrap mass spectrometer with full scan/ddMS2 scan mode Under optimized conditions, the method was linear with target linear correlation coefficient (R2) higher than 0.995. Detection limits were in range of 0.1-0.6ng/mL, and the quantification limits were 0.3-1.5ng/mL. The recovery was between 71.9% and 89.2% in three spike levels with precision (n=5) of 5.40-12.1%. The serum sample was directly analyzed after a simple extraction procedure, and the on-line SPE and determination were achieved within only 16min. The method was used to analyze the dynamic contents variation of cortisone and hydrocortisone in serum before and after the surgery.