Online micro solid phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry for trace analysis of endogenous plant hormones in Ulva linza.

OBJECTIVE: Ulva linza (L.) is a species of green algae widely distributed in China. We aimed to establish a sensitive online analytical method for quantification of endogenous phytohormones in fresh minute seaweed samples. METHOD: The method for quantification of endogenous plant hormones in fresh minute samples was developed based on a homemade online micro solid phase extraction (m-SPE) system coupled with an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) platform. The online m-SPE instrument injected the eluent of m-SPE directly onto the LC separation column, improving the utilization of samples and saving time. The m-SPE column, of which the effective size was 9.6 × 2 mm i.d., was filled with 19 mg of C18 (10 µm). RESULTS: Under optimized conditions, the limits of detection were 0.002-0.060 ng ml-1 for five plant hormones. The actual sample recoveries of phytohormones were 76.4-103.4% and the coefficients of variance were below 14.1%. The temporal distribution of these endogenous plant hormones of U. linza during different growth periods is described. CONCLUSION: The proposed online m-SPE method was successfully applied to quantification of endogenous acidic and alkaline plant hormones in U. linza. It provides important information for the further study of the physiological and ecological effects of plant hormones in lower algal species.

Enhancement of Chemiluminescence Intensity of S2* in Non-premixed Hydrogen Microjet Flame in the Photometric Detector for Sulfur Detection.

A transparent quartz rod (q) placed vertically on top of a non-premixed hydrogen microjet flame in a flame photometric detector (qFPD) was developed and evaluated for sulfur detection. The microjet flame burned around the quartz rod because of Coanda effect, forming an extended downstream flame zone with a relatively low temperature between 550 and 650 °C, which is favorable to the formation of S2*. The emission intensity of S2* and the signal-to-noise ratio (SNR) of sulfur response were enhanced 2.6- and 2.1-fold, respectively. It was found that the quartz rod of diameter 4 mm with a tip shape of semicircle placed 6 mm above the nozzle yielded the highest SNR. The limits of detection (LOD) for seven kinds of tested sulfur-containing compounds of qFPD were 0.3-0.5 pg S s-1, which is 5-7 times better than that of commercially available FPD detectors (LOD: 1.6-2.8 pg S s-1). The selectivity of sulfur over carbon was 105 on qFPD when the SNR for the mass flow rate of S and C atoms was ∼3 times. It was the first time that a quartz rod was used vertically on top of a microjet hydrogen-rich flame in FPD to enhance the chemiluminescence of S2* and improve the LOD down to 0.3-0.5 pg S s-1.

Aqueous extraction followed by dispersive solid phase extraction with in situ derivatization for the determination of aflatoxins in traditional Chinese medicines.

A green sample preparation method based on aqueous extraction followed by dispersive solid phase extraction (d-SPE) with in situ derivatization (ISD) was developed for the determination of aflatoxins (AFs) in traditional Chinese medicines (TCMs). AFs in TCMs were extracted by alkaline aqueous solution and converted to substituted coumaric acids. Then, mixed-mode anion exchange (MAX) sorbent was used to isolate and enrich the substituted coumaric acids. During the elution by acetonitrile/trifluoroacetic acid solution, AFs were reconstructed and in situ derivatized. Several parameters affecting the procedure were evaluated. The developed preparation method coupled with high performance liquid chromatography-fluorescence detection was successfully applied for AFs determination in TCMs. The limit of detection (LOD) reached 10 pg/mL for AFs. Good linearity was obtained in three orders of magnitude with correlation coefficients ranging from 0.9996 to 0.9999. The relative recoveries of the method were between 72.7% and 114.5% with intra- and inter-day relative standard deviations (RSDs) less than 9.5% and 10.1%, respectively. The method was successfully applied to determine AFs in 15 kinds of TCMs in China, with the results verified by IAC standard method.

[Recent advances in spatio-temporal distribution of endogenous phytohormones].

Endogenous phytohormone is a kind of trace organic small molecule compound synthesized in plants. It plays important roles in regulating the growth and development of plants throughout their life cycles, and responding to external stimuli. With the development of analytical methods for the detection of phytohormone, the amount of analytical samples is gradually reduced. Moreover, the differences in the types and contents of phytohormones in different plant tissues (or organs) are constantly presented. These developments greatly promote the study of the physiological effects of plant hormones. In recent years, the temporal and spatial distribution of endogenous phytohormones in several plant samples has attracted more and more attention in plant hormone analysis. This review summarizes the research progress made in the study of the spatial and temporal distribution of endogenous phytohormones in the last five years. Mainly, the review summarizes and discusses the difficulties associated with analysis, the development of analytical methods, and the spatial and temporal distribution of major plant hormones. The sampling methods, sample preparation and detection methods for spatial and temporal distribution studies are discussed in detail with a special focus on our work in plant hormone detection. Finally, the future development of the plant hormone analysis research field analysis is prospected.

One step rapid dispersive liquid-liquid micro-extraction with in-situ derivatization for determination of aflatoxins in vegetable oils based on high performance liquid chromatography fluorescence detection.

A rapid dispersive liquid-liquid micro-extraction (DLLME) with in-situ derivatization method for extraction and purification of aflatoxins (AFs) in vegetable oils was developed and evaluated. Oil extract, dichloromethane and trifluoroacetic acid were mixed and injected into water to form a cloudy solution. AFs in the oil were extracted into the numerous liquid droplets (with diameters from a few microns to dozens of microns) of extractant, where derivatization was carried out in situ. The proposed sample preparation method was coupled with high performance liquid chromatography with fluorescence detection (HPLC-FLD) for determination of four AFs in vegetable oils. The method showed excellent linearity in three orders of magnitude, good relative recoveries, good repeatability and high sensitivity with limits of detection in range of 0.005-0.03 ng/mL. The accuracy of the method was also verified by certified reference sample. Finally, different kinds of vegetable oils from the local supermarket were analyzed.

Dispersive Matrix Solid-Phase Extraction Method Coupled with High Performance Liquid Chromatography-Tandem Mass Spectrometry for Ultrasensitive Quantification of Endogenous Brassinosteroids in Minute Plants and Its Application for Geographical Distribution Study.

An ultrasensitive analysis method for quantification of endogenous brassinosteroids in fresh minute plants was developed based on dispersive matrix solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. During the dispersive matrix solid-phase extraction, plant samples were first ground with solid sorbent (dispersant) in one microcentrifuge tube and then centrifuged after adding extraction solvent and cleanup materials (another type of sorbent). Three protocols based on dispersive matrix solid-phase extraction were compared and discussed for plant samples with different matrix complexity. The choice of any protocol was a compromise of increasing purification efficiency and decreasing sample loss. Under optimized conditions, the limits of detection were 1.38-6.75 pg mL-1 for five brassinosteroids in the oilseed rape samples. The intraday and interday precisions were in the range of 0.8%-9.8% and 4.6%-17.3%, respectively. The proposed method was successfully applied to detection of endogenous brassinosteroids in milligram oilseed rape (2.0 mg) and submilligram Arabidopsis thaliana seedlings (0.5 mg). Finally, the geographical distribution of five endogenous brassinosteroids of Brassica napus L. oilseed rape in different provinces of origin in the Yangtze River basin was described.

One-step preparation of zirconia coated silica microspheres and modification with d-fructose 1, 6-bisphosphate as stationary phase for hydrophilic interaction chromatography.

In this study, ZrO2 layer coated silica microspheres (ZrO2/SiO2) were successfully prepared by a facile one-step surfactant-free hydrothermal route under low pH condition. The synthesized ZrO2/SiO2 material was then modified with d-fructose 1, 6-bisphosphate (FDP) to improve the chromatographic separation property of the material. Fused-silica capillary columns were prepared with the modified material for evaluation. Phenolic, nucleobases and alkaloids compounds in hydrophilic interaction chromatographic (HILIC) mode showed symmetrical peaks. The FDP-ZrO2/SiO2 stationary phase showed better performance than ZrO2/SiO2 packing material and demonstrated great potential for application in HILIC mode.

Spatial Profiling of Gibberellins in a Single Leaf Based on Microscale Matrix Solid-Phase Dispersion and Precolumn Derivatization Coupled with Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

A spatial-resolved analysis method for profiling of gibberellins (GAs) in a single leaf was developed on the basis of microscale sample preparation and precolumn derivatization coupled with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The proposed microscale sample preparation was based on modified matrix solid-phase dispersion (MSPD) method, in which the plant sample (<1 mg) and C18 sorbent were ground together in one microcentrifuge tube, and then extraction solvent was added followed by centrifugation. In this protocol, the grinding, extraction, and purification were performed in one microcentrifuge tube without any sample transfer step, resulting in an obvious decrease in sample loss. Moreover, a new derivatization reagent, 3-bromopropyltrimethylammonium bromide (BPTAB), was used to further enhance the signal intensities of GAs on MS by 3-4 orders of magnitude, which was much higher than the reported derivatization reagents for GAs such as bromocholine bromide and 3-bromoactonyltrimethylammonium bromide. The present method showed high sensitivity (minimum detectable amount (MDA) of 10.1-72.3 amol for eight GAs) and low sample consumption (down to 0.30 mg FW). Under the optimized conditions, the distribution of GA19 in a single Arabidopsis thaliana leaf was profiled with a spatial resolution of 2 × 2 mm2.

Ultrasensitive quantification of endogenous brassinosteroids in milligram fresh plant with a quaternary ammonium derivatization reagent by pipette-tip solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometry.

Determination of endogenous brassinosteroids (BRs) in limited sample amount is vital to elucidating their tissue- and even local tissue-specific signaling pathway and physiological effects on plant growth and development. In this work, an ultra-sensitive quantification method was established for endogenous BRs in milligram fresh plant by using pipette-tip solid-phase extraction coupled with ultra-performance liquid chromatography tandem mass spectrometry (PT-SPE-UPLC-MS/MS), in which a quaternary ammonium phenyl boronic acid, 4-borono-N,N,N-trimethylbenzenaminium iodide (BTBA) was first developed for chemical derivatization of BRs. Due to the cationic quaternary ammonium group of BTBA, the ionization efficiencies of the BRs chelates with BTBA (BTBA-BRs) were enhanced by 1190-448785 times, which is the highest response enhancement factor among all derivatization reagents reported for BRs. In addition, PT-SPE packed with C18 sorbent was first used for purifying BRs from plant extracts, so the required sample amount was minimized, and recoveries higher than 91% were achieved. Under the optimized conditions, the minimal detectable amounts (MDA) of five target BRs were in the range of 27-94 amol, and the correlation coefficients (R(2)) were >0.9985 over four orders of magnitude. The relative recoveries of 75.8-104.9% were obtained with the intra- and inter-day relative standard deviations (RSDs) less than 18.7% and 19.6%, respectively. Finally, three BRs were successfully quantified in only 5mg fresh rice plant samples, and 24-epiBL can even be detected in only 0.5mg FW rice leaf segments. It is the first time that the BRs content in sub-milligram fresh plant sample has been quantified.

An array sensor consisting of a single indicator with multiple concentrations and its application in ion discrimination.

Optical sensor arrays typically require a large set of chemically responsive colorants to enhance discrimination capability. Conversely, we have proven that by using multiple concentrations of one indicator, the discrimination of various analytes could be realized.

Quantification of endogenous brassinosteroids in sub-gram plant tissues by in-line matrix solid-phase dispersion-tandem solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry.

A matrix solid-phase dispersion (MSPD)-tandem mixed mode anion exchange (MAX)-mixed mode cation exchange (MCX) solid phase extraction-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of six endogenous brassinosteroids (BRs) (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, dolichosterone, teasterone and typhasterol) in rice plant tissues. Non-polar interferences were removed effectively by C8 dispersant used in MSPD, while the following tandem MAX-MCX process facilitated the elimination of polar and ionizable compounds. The weak reversed-phase retention feature of MAX-MCX leaded to good compatibility of the elution solvents in the in-line coupled MSPD-MAX-MCX system. This system was optimized for extraction and purification of BRs in plant samples. The effects of the type of solid phase, the elution solvent, the extraction temperature and the clean-up material were studied. Before HPLC separation, BRs purified were derivatized by m-aminophenylboronic acid to enhance the sensitivity of MS/MS to BRs. Compared with traditional liquid-liquid extraction and solid phase extraction (LLE-SPE), the proposed MSPD-MAX-MCX method showed higher extraction efficiency, lower matrix effect, and advantages of easy manipulation and time-saving. The in-line MSPD-MAX-MCX coupled with HPLC-MS/MS method provided a linear response over two orders of magnitude of BRs concentration with correlation coefficients above 0.9982, limits of detection between 0.008 and 0.04ngmL(-1), relative standard deviations (RSDs) below 29.4%, and recoveries above 77.8%. The proposed method has been successfully applied to analysis of endogenous BRs in rice plant at booting stage and maturity stage.

[Recent advances in sample preparation methods of plant hormones].

Plant hormones are a group of naturally occurring trace substances which play a crucial role in controlling the plant development, growth and environment response. With the development of the chromatography and mass spectroscopy technique, chromatographic analytical method has become a widely used way for plant hormone analysis. Among the steps of chromatographic analysis, sample preparation is undoubtedly the most vital one. Thus, a highly selective and efficient sample preparation method is critical for accurate identification and quantification of phytohormones. For the three major kinds of plant hormones including acidic plant hormones & basic plant hormones, brassinosteroids and plant polypeptides, the sample preparation methods are reviewed in sequence especially the recently developed methods. The review includes novel methods, devices, extractive materials and derivative reagents for sample preparation of phytohormones analysis. Especially, some related works of our group are included. At last, the future developments in this field are also prospected.

[Temporal-spatial variations of reference evapotranspiration in Anhui Province and the quantification of the causes].

In this paper, daily reference evapotranspiration (ET0) was computed with the recommended FAO-56 Penman-Monteith equation for Anhui Province using data collected 60 weather stations during 1961 to 2010 and its temporal-spatial variations were characterized. The determining factors in ET0 trends were inquired into through partial derivative quantification analysis for the study region. Results showed that the mean annual ET0 was 878.58 mm x a(-1) over the whole region during the study period. ET0 was the highest in summer and the lowest in winter. The mean annual ET0 decreased from the north to the south and from low altitude regions to high altitude regions. Both sunshine duration and wind speed were the dominant factors contributing to the interannual change of ET0, with less contribution from air temperature or relative humidity. The annual ET0 showed a general decline at a rate of -1.61 mm x a(-1) owing to a more negative contribution of sunshine duration and wind speed than a positive contribution of air temperature and relative humidity. ET0 increased insignificantly in spring and decreased slightly in both autumn and winter. However, it decreased significantly at a rate of -1.37 mm x a(-1) in summer. The main impacting factor was wind speed in spring, autumn and winter, but it was sunshine duration in summer. Great differences in the determining factors of the mean annual ET0 existed from area to area in Anhui Province. The wind speed was the determining factor for 36.7% of the whole stations distributing in the southern part of the area north to the Huaihe River and the area along the Huaihe River, while the sunshine duration was the determining factor for the other regions.

Quantification of endogenous brassinosteroids in plant by on-line two-dimensional microscale solid phase extraction-on column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry.

An on-line two-dimensional microscale solid phase extraction (2DµSPE)-on column derivatization (OCD)-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of brassinosteroids (BRs) in plant tissues. Five BRs with widest distribution in plant species and high bioactivity (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, teasterone and typhastero) were selected as target analytes. 2DµSPE column packed sequentially with phenyl boronic acid silica sorbent (the first dimension) and C18 silica sorbent (the second dimension) was used to selectively extract and enrich BRs by 110-146 times. OCD was carried out on the second dimension of 2DµSPE column with m-aminophenylboronic acid (m-APBA) as a derivatization reagent, enhancing the sensitivity of MS/MS to BRs by 13-8437 times. It was also found that pre-trap of derivatization reagent on the C18 section of 2DµSPE column could increase reaction efficiency by 3-10 times. The whole process time of the on-line system was less than 30min. The detection limits of the method for five BRs were between 1.4 and 6.6pg with RSDs less than 10%. Endogeneous BRs in tomato leaves were analyzed by using this method. Owing to the high selectivity of this on-line 2DµSPE system, BRs in plant extracts could be quantified using matrix-free standard calibration method with relative recoveries in the range of 80-124%.

A fluorometric paper-based sensor array for the discrimination of heavy-metal ions.

A fluorometric paper-based sensor array has been developed for the sensitive and convenient determination of seven heavy-metal ions at their wastewater discharge standard concentrations. Combining with nine cross-reactive BODIPY fluorescent indicators and array technologies-based pattern-recognition, we have obtained the discrimination capability of seven different heavy-metal ions at their wastewater discharge standard concentrations. After the immobilization of indicators and the enrichment of analytes, identification of the heavy-metal ions was readily acquired using a standard chemometric approach. Clear differentiation among heavy-metal ions as a function of concentration was also achieved, even down to 10(-7)M. A semi-quantitative estimation of the heavy-metal ion concentration was obtained by comparing color changes with a set of known concentrations. The sensor array was tentatively investigated in spiked tap water and sea water, and showed possible feasibility for real sample testing.

BODIPY-based fluorometric sensor array for the highly sensitive identification of heavy-metal ions.

A BODIPY(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based fluorometric sensor array has been developed for the highly sensitive detection of eight heavy-metal ions at micromolar concentration. The di-2-picolyamine (DPA) derivatives combine high affinities for a variety of heavy-metal ions with the capacity to perturb the fluorescence properties of BODIPY, making them perfectly suitable for the design of fluorometric sensor arrays for heavy-metal ions. 12 cross-reactive BODIPY fluorescent indicators provide facile identification of the heavy-metal ions using a standard chemometric approach (hierarchical clustering analysis); no misclassifications were found over 45 trials. Clear differentiation among heavy-metal ions as a function of concentration was also achieved, even down to 10(-7)M. A semi-quantitative interpolation of the heavy-metal concentration is obtained by comparing the total Euclidean distance of the measurement with a set of known concentrations in the library.

Dispersive liquid-liquid microextraction of trace Hg2+ for visual and fluorescence test.

In this work, trace Hg(2+) in environmental water samples was first preconcentrated by dispersive liquid-liquid microextraction (DLLME), and then was colorated by one common rhodamine B thiolactone (RBT) probe for visual and fluorescence detection. In this way, a highly sensitive and selective method was developed for field monitoring of Hg(2+) in environmental waters. The color of RBT test solution could change clearly from colorless to pink around the level of 2 µg L(-1)Hg(2+), which just meets with the maximum residue level of Hg(2+) in drinking water recommended by U.S. EPA, so the water samples with Hg(2+) concentration over 2 µg L(-1) could be selected rapidly by naked eye. Moreover, the quantification of Hg(2+) could be obtained by fluorescence detection of test solution. The best extraction efficiency of Hg(2+) by DLLLME could be obtained by using 200 mL water sample (pH 5.0, 50°C), 150 µL extraction solvent (lauric acid, LA) and 3 mL dispersive solvent (methanol). Thus an enrichment factor of ≈ 407 and the limit of fluorescence detection of 0.03 µg L(-1) could be achieved. The method was also highly selective for Hg(2+) with tolerance to at least 1000-fold of the foreign metal ions. This method was successfully applied to detect Hg(2+) in deionized water, tap water and sea water.

Hollow fiber-based liquid-liquid-liquid micro-extraction with osmosis: II. Application to quantification of endogenous gibberellins in rice plant.

The phenomenon and benefits of osmosis in hollow fiber-based liquid-liquid-liquid micro-extraction (HF-LLLME) were theoretically discussed in part I of this study. In this work, HF-LLLME with osmosis was coupled with high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-triple quadrupole MS/MS) to analyze eight gibberellins (gibberellin A(1), gibberellin A(3), gibberellin A(4), gibberellin A(7), gibberellin A(8), gibberellin A(9), gibberellin A(19) and gibberellin A(20)) in rice plant samples. According to the theory of HF-LLLME with osmosis, single factor experiments, orthogonal design experiments and mass transfer simulation of extraction process were carried out to select the optimal conditions. Cyclohexanol - n-octanol (1:3, v/v) was selected as organic membrane. Donor phase of 12 mL was adjusted to pH 2 and 20% NaCl (w/v) was added. Acceptor phase with an initial volume of 20 µL was the solution of 0.12 mol L(-1) Na(2)CO(3)-NaHCO(3) buffer (pH 9). Temperature was chosen to be 30 °C and extraction time was selected to be 90 min. Under optimized conditions, this method provided good linearity (r, 0.99552-0.99991) and low limits of detection (0.0016-0.061 ng mL(-1)). Finally, this method was applied to the analysis of endogenous gibberellins from plant extract which was obtained with traditional solvent extraction of rice plant tissues, and the relative recoveries were from 62% to 166%.

Array capillary in-tube solid-phase microextraction: a rapid preparation technique for water samples.

A rapid sample preparation technique, namely array capillary in-tube solid-phase microextraction (ACIT-SPME) for direct extraction of organic pollutants from water samples, was developed and evaluated. The ACIT-SPME cartridge consisted of a bundle of glass capillary tubes of 0.5 mm I.D. × 30mm contained inside a quartz liner of 4 mm I.D. The high ratio of cross-section area of channel-to-wall allowed water sample flow through the cartridge just under gravity. Both the internal/external surfaces of the array capillary tubing were coated with extraction phase of 2-5 µm in thickness, which provided large extraction surface area up to 30 cm² for a cartridge containing 19 glass capillaries. The large surface area and thin extraction phase improved greatly both the mass transfer process of extraction and the thermo desorption process, leading to fast extraction and fast desorption. The extracted analytes were thermally desorbed in a homemade thermal desorption unit (TDU), which was coupled to a gas chromatograph equipped with a flame ionization detector for analysis. By using polydimethylsiloxane (PDMS) as the extraction phase and polycyclic aromatic hydrocarbons (PAHs) as the model analytes, the performance of the ACIT-SPME cartridge was systematically investigated. The extraction time was 2 min for 350 mL of water sample, and detection limits were between 0.8 and 1.7 ng/L with deviation of 2.8-9.7% RSD. Relative recoveries of analytes for real water samples were between 65.0% and 116%. The extraction time can even be further shortened to 10s for 250 mL sample by applying vacuum at the outlet of the cartridge, with detection limits of 2.2-5.3 ng/L and deviation of 4.0-12% RSD.

[Advances in miniaturization of chromatograph and detectors].

The innovation design of miniaturization of chromatographic instruments, in combination with the progress in materials and opto-electron technologies, has led to the breakthrough in key chromatography techniques. The novel design of detectors has improved the sensitivity and reduced the consumptions of power and gases. The utilization of surface thermo ionization principle yields a detector of extremely high sensitivity and selectivity towards amines. The low cost, high performance integration and miniaturization of chromatograph will lead to high standard commercial instruments in the two or three coming years. These instruments will be applied for on-line or off-line monitoring in fields and laboratories. Important progresses on the developments of detectors and miniaturization of instruments are reviewed and commented in this paper.