Volumetric absorptive microsampling coupled with hybridization LC-MS/MS for quantitation of antisense oligonucleotides.

Background: Volumetric absorptive microsampling has emerged as a less invasive alternative to venous sampling for small-molecule pharmacokinetic studies, but its application to novel therapeutics such as antisense oligonucleotides (ASOs) is not well-established. Results: A workflow was developed using Mitra microsampling coupled with hybridization LC-MS/MS for accurate determination of fomivirsen, a 21-mer ASO, in human blood. Quantitative recovery was achieved regardless of blood hematocrit level or microsample age by implementing impact-assisted extraction. A thorough method evaluation confirmed sensitivity, linearity, precision/accuracy, matrix effect, metabolite interference and four months of microsample stability. Conclusion: The combined impact-assisted extraction and hybridization LC-MS/MS workflow demonstrated the successful quantitation of fomivirsen, establishing the validity and applicability of the approach for ASO drug candidates.

The dioxygenase GIM2 functions in seed germination by altering gibberellin production in Arabidopsis.

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) antagonistically control seed germination. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germination ability need further investigation. Here, we identified the ABA-insensitive gain-of-function mutant germination insensitive to ABA mutant 2 (gim2) by screening a population of XVE T-DNA-tagged mutant lines. Based on two loss-of-function gim2-ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germination, as observed in transgenic lines overexpressing GIM2 (OE). We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2-ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA- and/or ABA-related genes were altered in both the gim2-ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA12 . Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis.

A facile approach for the polymer grafting of silica based on tandem reversible addition fragmentation chain transfer/click chemistry and its application in high performance liquid chromatography.

In this work, a tandem reversible addition fragmentation chain transfer (RAFT)/click chemistry method was developed to prepare amide-polystyrene-silica (NHCO-Psty-silica) stationary phase. Styrene was immobilized on amino-silica surface via an azide functionalized RAFT agent in a one-pot procedure. The resultant NHCO-Psty-silica column demonstrates better performance for shielding of residue silanols than traditional ODS column, which was ascertained by Engelhardt test (E test), Tanaka test (T test), Galushko test (G test), and Walters test (W test). Our results showed lower values of silanol activity calculated according to the formula in these standard tests for NHCO-Psty-silica column compared to the ODS column we tested. As a result, the NHCO-Psty-silica is suitable for the separation of basic compounds. The hydrophobic, anion exchanging and π-π interaction of the column toward analytes were also evaluated. Moreover, the NHCO-Psty-silica column also showed excellent stability with pure water as mobile phase.

Assessing gibberellins oxidase activity by anion exchange/hydrophobic polymer monolithic capillary liquid chromatography-mass spectrometry.

Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography--mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA1, GA4, GA9, GA20). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62-0.90 fmol. We determined the kinetic parameters (K m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA9 to GA4 in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.

Facile preparation of organic-silica hybrid monolith for capillary hydrophilic liquid chromatography based on "thiol-ene" click chemistry.

In this work, a one-step approach to facile preparation of organic-inorganic hybrid monoliths was successfully developed. After vinyl-end organic monomers and azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethoxysilane (TMOS) and 3-mercaptopropyltrimethoxysilane (MPTMS), the homogeneous mixture was introduced into a fused-silica capillary for simultaneous polycondensation and "thiol-ene" click reaction to form the organic-silica hybrid monoliths. By employing this strategy, two types of organic-silica hybrid monoliths with positively charged quaternary ammonium and amide groups were prepared, respectively. The functional groups were successfully introduced onto the monoliths during the sol-gel process with "thiol-ene" click reaction, which was demonstrated by ζ-potential assessment, energy dispersive X-ray spectroscopy (EDX), and Fourier transform infrared (FT-IR) spectroscopy. The porous structure of the prepared monolithic columns was examined by scanning electron microscopy (SEM), nitrogen adsorption-desorption measurement, and mercury intrusion porosimetry. These results indicate the prepared organic-silica hybrid monoliths possess homogeneous column bed, large specific surface area, good mechanical stability, and excellent permeability. The prepared monolithic columns were then applied for anion-exchange/hydrophilic interaction liquid chromatography. Different types of analytes, including benzoic acids, inorganic ions, nucleosides, and nucleotides, were well separated with high column efficiency around 80,000-130,000 plates/m. Taken together, we present a facile and universal strategy to prepare organic-silica hybrid monoliths with a variety of organic monomers using one-step approach.

Quantification of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from hepatocellular carcinoma tissues by capillary hydrophilic-interaction liquid chromatography/quadrupole TOF mass spectrometry.

BACKGROUND: 5-Methylcytosine (5-mC) is an important epigenetic modification involved in development and is frequently altered in cancer. 5-mC can be enzymatically converted to 5-hydroxymethylcytosine (5-hmC). 5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons, but the distribution of 5-hmC in human liver tumor and matched control tissues has not been rigorously explored. METHODS: We developed an online trapping/capillary hydrophilic-interaction liquid chromatography (cHILIC)/in-source fragmentation/tandem mass spectrometry system for quantifying 5-mC and 5-hmC in genomic DNA from hepatocellular carcinoma (HCC) tumor tissues and relevant tumor adjacent tissues. A polymer-based hydrophilic monolithic column was prepared and used for the separation of 12 nucleosides by cHILIC coupled with an online trapping system. Limits of detection and quantification, recovery, and imprecision of the method were determined. RESULTS: Limits of detection for 5-mC and 5-hmC were 0.06 and 0.19 fmol, respectively. The imprecision and recovery of the method were determined, with the relative SDs and relative errors being <14.9% and 15.8%, respectively. HCC tumor tissues had a 4- to 5-fold lower 5-hmC content compared to tumor-adjacent tissues. In addition, 5-hmC content highly correlated with tumor stage (tumor-nodes-metastasis, P = 0.0002; Barcelona Clinic liver cancer, P = 0.0003). CONCLUSIONS: The marked depletion of 5-hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC.

Preparation of a hyper-cross-linked polymer monolithic column and its application to the sensitive determination of genomic DNA methylation.

A hyper-cross-linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium-co-vinylbenzene chloride-co-divinylbenzene) (MATE-co-VBC-co-DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE-co-VBC-co-DVB) monolithic column was demonstrated to have strong anion exchange/reversed-phase (SAX/RP) mixed-mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5-methylcytosine (5-mC)) can be baseline separated within 15 min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise = 3) of 5-mC is 0.014 pmol and endogenous 5-mC can be distinctly detected by using only 10 ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5-azacytidine (5-aza-C) and 5-aza-2'-deoxycytidine (5-aza-CdR) could induce a significant decrease of genome-wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa).

Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC-ESI-Q-TOF-MS analysis.

In current study, we developed a highly sensitive method for the quantitative profiling of acidic phytohormones. Tandem solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was employed to efficiently purify acidic phytohormones, which were further derived by 3-bromoactonyltrimethylammonium bromide (BTA) to increase the ionization efficiency in electrospray ionization-mass spectrometry detection. Additionally, fifteen BTA-derived acidic phytohormones, including ten gibberellins (GAs), were well separated with a salt gradient on poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolithic column. By employing online trapping system, the signal intensities of the analytes were significantly improved. The limits of detection (LODs, Signal/Noise=3) of targeted phytohormones ranged from 1.05 to 122.4 pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. Good reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 10.9 and 11.9%, respectively. Recoveries of the target analytes from spiked rice leave samples ranged from 88.3 to 104.3%. By employing the method developed here, we were able to simultaneously determine 11 endogenous acidic phytohormones from only 5mg of rice leave sample, which dramatically decreased the required sample amount (three orders of magnitude lower) for the profiling of low abundant acidic phytohormones compared to previous reports. Taken together, the method provided a good solution for the highly sensitive and quantitative profiling of endogenous acidic phytohormones.

Preparation and characterization of methacrylate-based monolith for capillary hydrophilic interaction chromatography.

In current study, poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolith was successfully prepared for capillary hydrophilic liquid chromatography (cHILIC). The polymerization was optimized by changing the ratio of MAA to EDMA, the type and amount of porogen. The characterization indicated that "hydrophilic" monolithic column possessed homogeneous column bed, good permeability and narrow pore size distribution. Under HILIC mode, the "hydrophilic" monolith prepared with PEG and DMSO showed stronger hydrophilicity than the "hydrophobic" monolith prepared with dodecanol and toluene. Finally, the "hydrophilic" monolith was applied in the separation of tryptic digests of bovine serum albumin (BSA) with cHILIC-ESI-qTOF-MS system. Our results revealed that 49 peptides were identified with 50% sequence coverage under HILIC mode, which was much better than the peptides identified using particulate-packed commercial column with RPLC-ESI-qTOF-MS system or "hydrophobic" monolith with cHILIC-ESI-qTOF-MS system. Taken together, the "hydrophilic" monolithic column prepared in current study, demonstrated the excellent chromatographic performance on the separation of complex samples, which offers the potential application of the monolith on proteomics study.

Determination of benzimidazole residues in animal tissue samples by combination of magnetic solid-phase extraction with capillary zone electrophoresis.

Benzimidazole drugs (BZDs) comprise a large number of synthetic anthelmintics, which are widely used in food-producing animals for prophylactic and therapeutic purposes. To protect consumers from the risks related to BZDs residues, a simple, rapid, and efficient method for simultaneous determination of ten BZDs in animal tissues samples was developed. This analytical procedure involved extracting samples with magnetic solid-phase extraction (MSPE) using magnetite/silica/poly (methacrylic acid-co-ethylene glycol dimethacrylate) (Fe(3)O(4)/SiO(2)/poly (MAA-co-EGDMA)) magnetic microspheres, and determination by capillary zone electrophoresis (CZE). To improve the sensitivity of the method, we employed the electrokinetic injection with field-amplified sample stacking technique (FASS). Berbine solution was used as internal standard to minimize the fluctuation of analytical results. Under the optimized extraction conditions, good linearities were obtained for the ten BZDs with the correlation coefficients (R(2)) above 0.9920. The limits of detections (LODs) for ten BZDs were 1.05-10.42 ng/g in swine muscle and 1.06-12.61 ng/g in swine liver, respectively. The intra- and inter-day relative standard deviations (RSDs) of the developed method were less than 13.6%. The recoveries of the ten BZDs for the spiked samples ranged from 81.1% to 105.4% with RSDs less than 9.3%.

Preparation of methacrylate-based monolith for capillary hydrophilic interaction chromatography and its application in determination of nucleosides in urine.

A novel poly(N-acryloyltris(hydroxymethyl)aminomethane-co-pentaerythritol triacrylate) (NAHAM-co-PETA) monolith was prepared in the 100 µm i.d. capillary and investigated for capillary liquid chromatography (cLC). The polymer monolith was synthesized by in situ polymerization of NAHAM and PETA in the presence of polyethylene glycol (PEG) in dimethyl sulfoxide (DMSO) as the porogen. The porous structure of monolith was optimized by changing the ratio of NAHAM to PETA, the molecular weight and amount of PEG. To evaluate the separation performance of the resultant polymer monolith, several groups of model compounds (including nucleosides, benzoic acids and anilines) were selected to perform cLC separation. Our results showed that these model compounds can be baseline separated on the resultant poly(NAHAM-co-PETA) monolithic column with the optimized mobile phases. The column efficiency was estimated to be 87,000 plates/m for acrylamide. In addition, this monolithic column was coupled with on-line solid-phase microextraction (SPME) for the analysis of four nucleosides (uridine, adenosine, cytidine, guanosine) in urine. The limit of detection of the proposed method was in the range from 40 to 52 ng/mL. The method reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 8.3% and 10.2%, respectively. Recoveries of the target analytes from spiked urine samples were ranged from 86.5% to 106.8%.

Determination of eight illegal drugs in human urine by combination of magnetic solid-phase extraction with capillary zone electrophoresis.

Using magnetite/silica/poly(methacrylic acid-co-ethylene glycol dimethacrylate) (Fe(3)O(4)/SiO(2)/poly(MAA-co-EDMA)) magnetic microspheres, a rapid and high-throughput magnetic solid-phase extraction coupled with capillary zone electrophoresis (MSPE-CZE) method was developed for the determination of illegal drugs (ketamine, amphetamines, opiates, and metabolites). The MSPE of target analytes could be completed within 2 min, and the eight target analytes could be baseline separated within 15 min by CZE with 30 mM phosphate buffer solution (PBS, pH 2.0) containing 15% v/v ACN as background electrolyte. Furthermore, hydrodynamic injection with field-amplified sample stacking (FASS) was employed to enhance the sensitivity of this MSPE-CZE method. Under such optimal conditions, the limits of detection for the eight target analytes ranged from 0.015 to 0.105 µg/mL. The application feasibility of MSPE-CZE in illegal drugs monitoring was demonstrated by analyzing urine samples, and the recoveries of target drugs for the spiked sample ranging from 85.4 to 110.1%. The method reproducibility was tested by evaluating the intra- and interday precisions, and relative standard deviations of <10.3 and 12.4%, respectively, were obtained. To increase throughput of the analysis, a home-made MSPE array that has potential application to the treatment of 96 samples simultaneously was used.

Highly sensitive profiling assay of acidic plant hormones using a novel mass probe by capillary electrophoresis-time of flight-mass spectrometry.

Plant hormones play crucial roles in plant growth and development. However, up to date, identification and quantification of acidic plant hormones with trace amount in complicated plant matrix is still a challenge. In current study, we developed a high sensitive assay for the determination of acidic plant hormones in rice by combining capillary electrophoresis and electrospray ionization-time of flight-mass spectrometry (CE-ESI-TOF-MS). To improve the detection sensitivity of acidic plant hormones, 3-bromoactonyltrimethylammonium bromide (BTA) was synthesized as a new mass probe, which can react efficiently with acidic plant hormones in acetonitrile containing triethylamine (TEA). The positively charged BTA-derivatives were separated by CE using amino-coated capillary, which provided a reversed electroosmotic flow (EOF) at low pH, as well as reduced the adsorption of BTA-derivatives on the inner wall of capillary. Using the CE-ESI-TOF-MS method developed in current study, 15 acidic plant hormones, including 10 gibberellins (GAs), were identified and quantified with good linearities from 1.3 to 850 ng/mL with linear coefficient R(2) values of >0.99. The limits of detection (LODs) were in the range of 0.34-4.59 ng/mL. Recoveries of compounds from spiked beverage samples ranged from 84.6 to 112.2%. And a good reproducibility was obtained by evaluating the intra and inter-day precisions with relative standard deviations (RSDs) less than 6.7 and 9.9%, respectively.

Preparation of organic-inorganic hybrid silica monolith with octyl and sulfonic acid groups for capillary electrochromatograhpy and application in determination of theophylline and caffeine in beverage.

An organic-inorganic hybrid silica monolithic column with octyl and sulfonic acid groups has been prepared by sol-gel technique for capillary electrochromatograhpy. The structure of hybrid monolith was optimized by changing the composition of tetraethoxysilane (TEOS), octyltriethoxysilane (C(8)-TEOS) and 3-mercaptopropyltrimethoxysilane (MPTMS) in the mixture of precursors. Then, the obtained hybrid monolith was oxidized using hydrogen peroxide (30%, w/w) to yield sulfonic acid groups. The sulfonic acid group, which served as strong cation-exchanger, dominated the charge on the surface of the capillary column and generated stable electroosmotic flow (EOF) in a wide range of pH. The monolithic column was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and elemental analysis (EA), and the performance of column was evaluated in detail by separating different kinds of compounds with column efficiency up to 155,000 plates/m for thiourea. In addition, this monolithic column was also applied in the analysis of theophylline (TP) and caffeine (CA) in beverages. The detection limits were 0.39 and 0.48 microg/mL for theophylline and caffeine, respectively. The method reproducibility was tested by evaluating the intra- and inter-day precisions, and relative standard deviations of less than 3.9 and 8.4%, respectively, were obtained. Recoveries of compounds from spiked beverage samples ranged from 87.2 to 105.2%.