Shin-ichiro Fujii

Determination of phosphorus using capillary electrophoresis and micro-high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry for the quantification of nucleotides

We performed the quantification of phosphorus in deoxynucleotides using capillary electrophoresis (CE) and micro-HPLC (microHPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). DNA and its component units have conventionally been determined by photometry; however, more selective and sensitive methods are needed for small biological samples. CE and microHPLC offer the advantages of good separation and small consumption of samples, and ICP-MS is a highly sensitive technique for the determination of a chemical element. Therefore, we have developed an interface device for combining CE and microHPLC with ICP-MS for quantifying nucleotides based on phosphorus content. The interface utilizes 4.5 microL/min for nebulizing and effective introduction of the sample into ICP. The samples of nucleotides and free phosphoric acid were well separated in the CE-ICP-MS measurement, and the calibration curves (1-100 microg/mL) of the nucleotides showed a linear (R2>0.999) increase in intensity. Similarly, the samples of nucleotides were baseline separated using microHPLC-ICP-MS, and the calibration curves of the nucleotides were linear (R2>0.998). The detection limits of these species and phosphorus in nucleotides using CE-ICP-MS and microHPLC-ICP-MS were 0.77-6.5 ng/mL and 4.0-6.5 ng/mL, respectively. These values were about one or two orders lower than those in a previous report. The sample volumes of these experiments were calculated to be about 10 nL and 50 nL per analysis. Therefore, these analytical methods have the potential to be useful for the determination of biological samples, such as DNA and RNA molecules.

High performance concentric nebulizer for low-flow rate liquid sample introduction to ICP-MS

A high performance concentric nebulizer (HPCN) was developed for sample introduction to inductively coupled plasma mass spectrometer (ICP-MS) at liquid flow rates of less than 10 µL min−1. The HPCN has a triple tube concentric structure and consists of a concentric type nebulizer body and a fused silica glass capillary (i.d./o.d.: 50 µm/150 µm) mounted in the center of the nebulizer body as a sample introduction capillary. The nebulizing performance was greatly improved by tapering the liquid capillary (tip orifice i.d.: 20 µm, tip wall thickness: <8 µm) and by setting the liquid capillary tip at a proper position. When the set position was recessed by 25 µm to 50 µm with respect to the nebulizer nozzle tip, a liquid jet was produced inside the nozzle by a flow focusing effect. This phenomenon gives a fine aerosol generation. At a liquid flow rate of 5 µL min−1 and a nebulizer gas flow rate of 1 L min−1, the Sauter mean diameter D3,2 of the primary drops generated by the HPCN (2.4 µm) was almost the same as that generated by the HEN-120-A.01 (2.6 µm) and was smaller than those generated by the HEN-90-A.01 (4.6 µm), CEI-100 (5.9 µm), and AriMist-HP (6.3 µm). The sensitivity in ICP-MS with the HPCN was over two-fold higher than those with the other microflow nebulizers, except for the HEN-120-A.01. The HEN-120-A.01 provides almost the same sensitivity as the HPCN, but it requires two-fold higher gas pressure than the HPCN. The HPCN can continuously and stably nebulize 1% NaCl solution for over 8 h without clogging. The HPCN combined with an on-axis cylinder chamber was applied to capillary liquid chromatography-ICP-MS, by which the speciation of eight arsenic compounds was demonstrated. We concluded the HPCN is a useful nebulizer for less than 10 µL min−1 sample introduction to ICP-MS.

Highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry

To realise highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry (ICP-MS), we developed a modified high efficiency cell introduction system (HECIS), consisting of a large-bore high performance concentric nebulizer (LB-HPCN) with a centre capillary tube of 150 μm inner diameter and a custom-made small-volume (15 cm3) on-axis spray chamber that uses a sheath gas flow near the chamber exit to suppress cell deposition. We also assembled an external ion pulse counting unit to directly read the ion pulse current from the electron multiplier of the ICP-MS via a function generator with no dead time, in order to obtain data with sufficiently high time resolution (i.e., 0.05–1 ms). As compared to a conventional ICP-MS working at its minimum integration time (10 ms), this assembly led to more than ca. 13-fold higher signal-to-background ratios for 31P, and made higher throughput of cells to the plasma more feasible. By using the modified HECIS and the external ion pulse counting unit for determination of the cell introduction efficiencies of different-sized unicellular microbes, including yeast (Saccharomyces cerevisiae), cyanobacterium (Synechocystis sp. PCC 6803), red algae (Cyanidioschyzon merolae 10D and Galdieria sulphuraria), and green alga (Chlamydomonas reinhardtii CC-125), it was revealed that their cell introduction efficiencies ranged from 86% (for C. reinhardtii CC-125 with a mean cell diameter of 6.4 μm) to ca. 100% (for other microbes with mean cell diameters of 2.0–3.0 μm), implying that by use of the ICP-MS system, the cell introduction efficiencies are able to reach approximately 100% and tend to decrease with increasing cell sizes (at least more than 3.1 μm in mean diameter). A wide range of biologically important elements, such as C, Mg, Al, P, S, K, Ca, Cr, Mn, Fe, and Zn, were tested for reasonable detection using the ICP-MS system. Results likely corresponding to separate cell events were obtained for some elements present in each microbe.

Multielement analysis of micro-volume biological samples by ICP-MS with highly efficient sample introduction system

A method for multielement analysis of micro-volume biological sample by inductively coupled plasma mass spectrometry (ICP-MS) with a highly efficient sample introduction system was presented. The sample introduction system was the combination of (1) an inert loop injection unit and (2) a high performance concentric nebulizer (HPCN) coupled with a temperature controllable cyclone chamber. The loop injection unit could introduce 20 μL samples into the carrier liquid flow of 10 μL min(-1) producing a stable signal for 100s without any dilution. The injection loop is continuously washed with 0.1M HNO(3) carrier solution during the measurement, thereby much improving sample throughput. The HPCN is a triple tube concentric nebulizer, which can generate fine aerosols and provide a stable and highly measurement sensitivity in ICP-MS at a liquid flow rate less than 10 μL min(-1). With the combination of the chamber heating at 60°C, the sensitivity obtained with the proposed sample introduction system at the liquid flow rate of 10 μL min(-1) was almost the same as that with a common concentric nebulizer and cyclone chamber system at the liquid flow rate of 1 mL min(-1), though the sample consumption rate of the HPCN was two orders of the magnitude lower than that of the common nebulizer. The validation of the proposed system was performed by analyzing the NIST SRM 1577b Bovine Liver. The observed values for 12 elements such as Na, P, S, K, Ca, Mn, Fe, Co, Cu, Zn, Mo, Cd were in good agreement with their certified values and information value. Satisfactory analytical results for 14 elements such as Na, Mg, P, S, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Y, Ba in Escherichia coli sample were also obtained. The proposed sample introduction system was quite effective in the cases when only micro-volume of biological sample is available.

Effective and selective recovery of gold and palladium ions from metal wastewater using a sulfothermophilic red alga, Galdieria sulphuraria.

The demand for precious metals has increased in recent years. However, low concentrations of precious metals dissolved in wastewater are yet to be recovered because of high operation costs and technical problems. The unicellular red alga, Galdieria sulphuraria, efficiently absorbs precious metals through biosorption. In this study, over 90% of gold and palladium could be selectively recovered from aqua regia-based metal wastewater by using G. sulphuraria. These metals were eluted from the cells into ammonium solutions containing 0.2M ammonium salts without other contaminating metals. The use of G. sulphuraria is an eco-friendly and cost-effective way of recovering low concentrations of gold and palladium discarded in metal wastewater.

Development of salt-tolerance interface for an high performance liquid chromatography/inductively coupled plasma mass spectrometry system and its application to accurate quantification of DNA samples

Accurate quantification of DNA is highly important in various fields. Determination of phosphorus by ICP-MS is one of the most effective methods for accurate quantification of DNA due to the fixed stoichiometry of phosphate to this molecule. In this paper, a smart and reliable method for accurate quantification of DNA fragments and oligodeoxythymidilic acids by hyphenated HPLC/ICP-MS equipped with a highly efficient interface device is presented. The interface was constructed of a home-made capillary-attached micronebulizer and temperature-controllable cyclonic spray chamber (IsoMist). As a separation column for DNA samples, home-made methacrylate-based weak anion-exchange monolith was employed. Some parameters, which include composition of mobile phase, gradient program, inner and outer diameters of capillary, temperature of spray chamber etc., were optimized to find the best performance for separation and accurate quantification of DNA samples. The proposed system could achieve many advantages, such as total consumption for small amount sample analysis, salt-tolerance for hyphenated analysis, high accuracy and precision for quantitative analysis. Using this proposed system, the samples of 20 bp DNA ladder (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 base pairs) and oligodeoxythymidilic acids (dT(12-18)) were rapidly separated and accurately quantified.

A coupling system of capillary gel electrophoresis with inductively coupled plasma-mass spectrometry for the determination of double stranded DNA fragments

The coupling system of capillary gel electrophoresis (CGE) and inductively coupled plasma-mass spectrometry (ICP-MS) was newly developed and successfully applied to the double-stranded (ds) DNA quantification. The developed system combines the separation technique for large biomolecules and element selective detection of ICP-MS. This coupling was achieved by using the modified high performance concentric nebulizer (HPCN) with the PTFE tube (HPCN-PT), which can produce the liquid jet by the flow focusing effect. The HPCN-PT effectively nebulizes the highly viscous solution containing gel buffer even at a low flow rate. At a liquid flow rate of 0.010 mL min(-1) and a nebulizer gas flow rate of 1 L min(-1), the Sauter mean diameter (D3,2) of primary aerosols generated by the HPCN-PT was 3.4 μm, and over 90% (v/v) of the aerosol droplets were less than 10 μm in diameter. The electrophoresis capillary filled with gel buffer was connected to the HPCN-PT via the interface. This interface has two connectors and an electrode that can connect CE and ICP-MS. After the electrophoretic separation at atmospheric pressure, the samples were transferred to the ICP-MS through the interface by applying additional pressure. Fragments of dsDNA, which were commercially available as a ladder marker solution, were successfully separated and analyzed by measuring (31)P(+) with CGE-ICP-MS, and a linear calibration curve of the phosphorus standard solution (R(2) = 0.999) was obtained from 2.7 to 27 mg kg(-1). The detection limit (LOD) and absolute detection limit of P were 3.7 μg kg(-1) and 0.6 pg (equivalent to 6 pg of DNA), respectively. This absolute detection limit value was equal to the conventional fluorescence determination of DNA.

Modified high performance concentric nebulizer for inductively coupled plasma optical emission spectrometry

The high performance triple-tube concentric nebulizer (HPCN) was modified and evaluated for sample introduction into inductively coupled plasma optical emission spectrometry (ICP-OES) at liquid flow rates of 0.25 to 0.8 mL min−1. The acceptable liquid flow rate for the use of HPCN was extended by replacing the tapered center capillary tube of the nebulizer (i.d./o.d.: 50 μm/150 μm) with a large bore tube (i.d./o.d.: 110 μm/170 μm). The nebulization efficiency was much improved by reducing the inner diameter of the nebulizer nozzle from 250 μm to 200 μm. At a liquid flow rate of 0.8 mL min−1 and a nebulizer gas flow rate of 1 L min−1, the Sauter mean diameter (D3,2) of the primary aerosol generated by the modified HPCN was 3.4 μm, and over 90% (v/v) of the aerosol droplets were below 10 μm in diameter. The D3,2 value was smaller than those generated by conventional nebulizers, Meinhard nebulizer type C (8.2 μm), Glass Expansion Conikal nebulizer (15.8 μm), SeaSpray (14.8 μm), and MiraMist (5.6 μm). The amount of the tertiary aerosol of the modified HPCN generated through a non-baffled cyclone chamber was approximately 1.8 to 3.1 times higher than those of the other nebulizers, with similar size distributions. The sensitivity in ICP-OES with the modified HPCN was 1.5- to 3.2-fold higher than those with the other nebulizers when the liquid flow rates were the same. The plasma robustness estimated from the commonly used ratio Mg(II)/Mg(I) was the same or slightly better than that of the conventional nebulizers. The HPCN also showed a good tolerance to high total dissolved solids (TDS) using 20% NaCl solution. Validation of the modified HPCN was performed by a recovery test of spiked seawater and analyzing the NMIJ CRM 7502-a white rice flour. The observed values for the ten elements Na, Mg, P, K, Ca, Mn, Fe, Cu, Zn and Cd were in good agreement with their certified values. We concluded the modified HPCN is a very useful nebulizer for ICP-OES with good performance at a large range of sample flow rates.

Development of vial wall sorptive extraction and its application to determination of progesterone in human serum

A novel sample preparation method, vial wall sorptive extraction (VWSE), which uses a vial whose internal wall is coated with polydimethylsiloxane (PDMS), was developed. The method was applied to the determination of progesterone in human serum sample. Human serum sample (0.5 mL) spiked with progesterone-13C2 was pipetted into the VWSE device and vortex mixing was performed for 30 min. Then, the serum sample was removed and the vial rinsed with purified water. Fifty microliter of methanol as liquid desorption (LD) solvent was pipetted into the VWSE device and vortex mixing was performed for 10 min. Then, the extract was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The correlation coefficient (r) of the calibration curve over the concentration range of 0.5-200 ng mL(-1) was 0.999. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 and 0.5 ng mL(-1), respectively. The relative recoveries were 97.9% (RSD: 4.4%, n=6) and 102.8% (RSD: 1.1%, n=6) for progesterone spiked at 5 and 50 ng mL(-1), respectively. This simple, accurate, sensitive, and selective analytical method is applicable to the trace analysis of a minute amount of sample.

Permeation regulation of charged species by the component change of polyion complex membranes

Three kinds of polyion complex membranes were prepared on a glassy carbon electrode: polycation (poly-L-lysine)-rich membrane, polyanion (DNA)-rich membrane, and equivalent membrane. The permeation of electroactive species (e.g., hydrogen peroxide, L-ascorbate, urate, dopamine) through the membrane was measured by the oxidation current of species at base electrode. Permeation of the anionic species can be depressed through the anion-rich membrane, and permeation of the cation can also be regulated through the cation-rich membrane. It is obvious that the charge exclusion can be controlled by changing the component ratio of polycation and polyanion during preparation.