SE153:/S1/M1/D1

Sample Set Information

ID	TSE1309
Title	Solid-phase extraction for metabolomic analysis of high-salinity samples by capillary electrophoresis-mass spectrometry.
Description	Environmental samples such as soil solutions contain inorganic ions such as NH4(+), K(+), Na(+), NO3(-), and PO4(3-) in high concentrations, which must be removed prior to capillary electrophoresis-mass spectrometry analysis to obtain accurate results. However, the separation of these inorganic ions from ionic metabolites, which are the target compounds in capillary electrophoresis-mass spectrometry analysis, is difficult because the physicochemical properties of the inorganic ions are similar to those of the ionic metabolites. In this study, we used various solid-phase extraction (SPE) columns for the purification of the samples containing inorganic ions in high concentrations. We found that cation-exchange SPE columns successfully filtered out the inorganic ions while retaining most of the organic compounds, which were easily collected with high recovery rates. In addition, 17 cationic metabolites in the soil solution were quantified by CE-MS analysis following the SPE purification process. The results suggest that our method can be used to analyze other environmental samples containing inorganic ions in high concentrations.
Authors	Oikawa A, Fujita N, Horie R, Saito K, Tawaraya K.
Reference	J Sep Sci. 2011 May;34(9):1063-8. doi: 10.1002/jssc.201000890. Epub 2011 Mar 18.
Comment

PubMed

Sample Information

ID	S1
Title	Andisol soil
Organism - Scientific Name
Organism - ID
Compound - ID
Compound - Source
Preparation	Andisol soil was collected from a native pasture in Tsuruoka, Yamagata Prefecture, Japan. Ammonium sulfate, superphosphate, and potassium sulfate were added to the air-dried Andisol soil at rates of 4.76, 5.88, and 1.85 g/kg of the soil, respectively. The pH (H2O) of the soil was adjusted to 5.80 by adding lime at the rate of 8.93 g/kg. Two kilograms of the fertilized Andisol soil were transferred to a 1:5000-A Wagner pot. Six mullite ceramic tubes (50mm x 0.6 mm) and a Teflon tube (200mm x 0.7 mm) were embedded into the fertilized soil. Rice (Oryza sativa L.) seeds were sown around the tubes and three seedlings were grown around each tube. The plants were grown in a glasshouse at the Yamagata University, Tsuruoka, Japan, from August 17 to August 28, 2009. Pots without rice plants were also prepared. Following the apparatus setup used by Tawaraya et al., a plastic syringe (5 mL) was connected to a Teflon tube 11 days after the rice seeds were sown, and 1mL of the soil solution was collected in the syringe. Of the collected soil solutions, 96 mL was mixed with 4 mL of 5-mM internal standard (200 mM at final concentration) and then purified on the SPE columns.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	CE-MS analysis
Method Details ID	MS1
Sample Amount	5 µL
Comment

Analytical Method Details Information

ID	MS1
Title	CE-MS analysis
Instrument	CE:Agilent CE capillary electrophoresis system (Agilent Technologies) TOF-MS:Agilent G3250AA LC/MSD TOF system (Agilent Technologies) CE-MS:Agilent G1603A
Instrument Type
Ionization	ESI
Ion Mode	positive and negative
Description	SPE purification SPE columns were conditioned with adequate amounts of methanol and then water. A standard mixture of organic and inorganic compounds was dissolved in 1% formic acid (FA); thereafter, 500 mL of the mixture was loaded on to each of the columns. The columns were then washed and the organic metabolites were eluted with the solvents listed in Table 1. The solvent volume used for washing and elution was 20 times the volume of the solid-phase packing. The eluted solution was dried by centrifugal concentration and then resuspended in 20 mL of water. Five microliters of the solution was used for CE-MS analysis. The conditions used in CE-MS analysis have been described in a previous report. CE-MS analysis All CE-TOFMS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA, USA), an Agilent 1100 series binary HPLC pump, a G1603A Agilent CE-MS adapter, and a G1607A Agilent CE-ESI-MS sprayer kit. G2201AA Agilent ChemStation and Analyst QS software were used for CE and TOFMS, respectively. Separations were carried out using a fused silica capillary (50 mm id x 100 cm total length) filled with 1M FA or 20mM ammonium formate (pH 10.0) as the electrolyte for cation and anion analyses, respectively. The capillary temperature was maintained at 201C. Fifteen nanoliters of the sample solutions was injected at 50 mbar for 15 s. The sample tray was cooled below 41C. Prior to each run, the capillary was flushed with the electrolyte for 5 min. The voltage used for the separation was set at 30 kV. Methanol/water (50%, v/v) containing 0.5 mM reserpine was delivered as the sheath liquid at 10 mL/min. ESI-TOFMS was conducted in the positive-ion mode for cation analyses and in the negative-ion mode for anion analyses, and the capillary voltage was set at 4 kV. The flow rate of heated dry nitrogen gas (heating temperature 3001C) was maintained at 10 psig. The fragmentor, skimmer, and Oct RFV voltage were set at 110, 50, and 160 V, respectively, for cation analysis and at 120, 60, and 220 V, respectively, for anion analysis. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The methanol dimer ion ([2M+H]+, m/z=65.0597) and reserpine ([M+H]+, m/z=609.2806) for cation analyses or the FA dimer ion ([2M–H]-,m/z=91.0037) and reserpine ([M–H]-, m/z=607.2661) for anion analyses provided the lock mass for exact mass measurements. Exact mass data were acquired at a rate of 1.5 cycles/s over a 50–1000 m/z range.
Comment_of_details

PubMed

Table 1. SPME cartridges and solvents used in this study

Sorbent category	Column name	Solvents
		Wash	Elution 1	Elution 2
Non-polar	BE C18	Water	MeOH
	BE C8
	BE C2
	BE CH
	BE PH
	A PLS-3
	IS Pharma
Mixed mode	BE Plexa
	BE AccuCAT
Cation exchange (polymer)	Oasis MCX	2% FA/MeOH	MeOH (MCX1)	6% NH4OH/MeOH (MCX2)
	BE Plexa PCX	2% FA/MeOH	MeOH (PCX1)	6% NH4OH/MeOH (PCX2)
		2% FA/MeOH	6% NH4OH/MeOH (PCX3)
		MeOH/MeCN (1:1)	MeOH/MeCN (1:1)+6% NH4OH (PCX4)
Cation exchange (silica)	BE SCX	2% FA/MeOH	MeOH (SCX1)	6% NH4OH/MeOH (SCX2)
		2% FA/MeOH	6% NH4OH/MeOH (SCX3)
		MeOH/MeCN (1:1)	MeOH/MeCN (1:1)+6% NH4OH (SCX4)

Before loading of samples, columns were conditioned by loading a sufficient volume of methanol and water. Mixture of standard organic and inorganic compounds was solved in 1% (v/v) aqueous formic acid (FA), and then loaded onto each of the columns.
a) BE (Bond Elut) columns were purchased from Varian (Lake Forest, CA), A (Aqusis) and IS (InertSep) columns from GL Science (Tokyo,Japan), and Oasis column was provided by Waters (Milford, MA, USA).

b) All ‘%’ in this table means ‘% (v/v)’.

Data Analysis Information

ID	D1
Title	Quantification
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS1
Title	Quantification
Description	For normalization, the area of the detected peak was divided by the area of the internal standard peak. Based on the calibration curves for standard compounds, metabolite amounts were quantified. Standard compounds were prepared using the SPE columns as with the soil solutions.
Comment_of_details