Lc 30ad hplc system

Manufactured by Shimadzu

Sourced in Japan, United States

The LC-30AD HPLC system is a high-performance liquid chromatography system manufactured by Shimadzu. It is designed to perform efficient and reliable liquid chromatography analysis. The LC-30AD system features a dual-plunger parallel design and provides accurate and precise solvent delivery.

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Lab products found in correlation

Qtrap 6500, by AB Sciex (6 mentions) Zorbax eclipse plus c18 column, by Agilent Technologies (5 mentions) Triple tof 5600 1, by AB Sciex (1 mentions) Agilent zorbax sb c18 column, by Agilent Technologies (1 mentions) Tripletof 6600, by AB Sciex (1 mentions) Jupiter c4 column, by Phenomenex (1 mentions) Analyst 1, by AB Sciex (1 mentions) 4500 triple quadrupole mass spectrometer, by Shimadzu (1 mentions) Sil 30ac autosampler, by Shimadzu (1 mentions) Intrada amino acid column, by Imtakt (1 mentions) Intrada organic acid column, by Imtakt (1 mentions)

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HPLC system (1016 citations) LC-30AD (81 citations) LC-30AD system (12 citations) Nexera X2 LC-30AD HPLC system (7 citations) HPLC 30AD (3 citations)

9 protocols using lc 30ad hplc system

HPLC-QTOF/MS Protocol for Compound Analysis

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A Shimadzu LC30-AD HPLC system (Shimadzu, Kyoto, Japan) combined with a quadrupole time-of-flight mass spectrometer (Triple TOF 5600-1, AB SCIEX, Redwood City, CA, USA) was used. An Agilent Zorbax SB-C18 column (4.6 mm × 50 mm, 1.8 μm, Agilent Technologies, USA) with a column temperature maintained at 30 °C was employed. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B), using a gradient elution of 10–20% B at 0–2 min, 20–22% B at 2–9 min, 22–40% B at 9–16 min, 40–70% B at 16–17 min, 70–100% B at 17–18 min, and 100% B at 18–19 min with an equilibrium for 4 min. The flow rate was set at 0.4 mL min⁻¹ with an injection volume of 2 μL.
MS detection was performed using QTOF/MS in negative ionization mode with a DuoSpray ion source. The QTOF/MS was calibrated in high sensitivity mode and the automated calibration device system (CDS) was set to perform an external calibration every four samples using a calibration solution. The source parameters were optimized: collision voltage (CE), 50 eV; ion spray voltage floating (ISVF), 4500 V; temperature, 500 °C; nebulizing gas (GS1), 60 psi; heater gas (GS2), 60 psi; curtain gas, 35 psi. The MS was operated in full-scan TOF/MS (100–2000 amu) and MS/MS mode (100–2000 amu) through data-independent acquisition (DIA) in a single-run analysis.^{16 (link)}

Zhang L.N., Wang L., Shi Z.Q., Li P, & Li H.J. (2018). A metabolomic strategy based on integrating headspace gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry to differentiate the five cultivars of Chrysanthemum flower. RSC Advances, 8(17), 9074-9082.

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Intact Mass Determination of Proteins

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Proteins were prepared in RutR storage buffer at concentrations of 1 μg/μL for intact mass determination. All samples were analyzed on a Triple TOF 6600 (Sciex) mass spectrometer equipped with a DuoSpray Ion Source that was coupled to a Shimadzu LC30AD HPLC System (Shimadzu). Approximately 100 ng of intact proteins (in 0.1% formic acid in water) were loaded onto a Jupiter C4 column (1 mm × 150 mm, 5 μm, 300 A, Phenomenex) and chromatographically separated at a constant flow rate of 100 μL/min using the following gradient: 20-60% solvent B (0.1% formic acid in acetonitrile) within 6 minutes followed by 85% solvent B for 1 minute and 20% B for 3 minutes. MS1 scans were acquired from 600-1600 m/z with 250 msec accumulation time. MS1 scans were summed across the chromatographic protein peak and the in summary spectra were exported in simple txt format for further analysis in the MagTran 1.02 deconvolution software^{138 (link)}.

Kremer M., Schulze S., Eisenbruch N., Nagel F., Vogt R., Berndt L., Dörre B., Palm G.J., Hoppen J., Girbardt B., Albrecht D., Sievers S., Delcea M., Baumann U., Schnetz K, & Lammers M. (2024). Bacteria employ lysine acetylation of transcriptional regulators to adapt gene expression to cellular metabolism. Nature Communications, 15, 1674.

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Phytohormone Analysis by UPLC-MS/MS

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Analysis of phytohormones was performed on a UPLC–ESI (+/−)–MS/MS system consisting of a AB SCIEX 4500 triple quadrupole mass spectrometer (Foster City, CA, USA) with an electrospray ionization source (Turbo Ionspray), a Shimadzu LC-30AD.
HPLC system (Tokyo, Japan) with two 30AD pumps, a SIL-30AC auto sampler, a CTO-30A thermostat column compartment, and a DGU-20A5R degasser. Data acquisition and processing were performed using AB SCIEX Analyst 1.6 software (Foster City, CA, USA).
The HPLC separation was performed on a on a Shim-pack XR-ODS Ш column (75 mm × 2.0 mm i.d., 1.6 μm) purchased from Shimadzu (Tokyo, Japan) at 40 °C. A 52-min gradient of 0.1% FA (A) and ACN (B) was employed for the separation with a flow rate of 0.4 mL/min. A gradient programme of 4 min 5–5% B, 6 min 5–7% B, 10 min 7–20% B, 20 min 20–80% B, 2 min 80–5% and 5 min 5% B was used.
Multiple reaction monitoring (MRM) and the appropriate product ions were chosen to quantify phytohormones (Additional file 1: Table S2). The optimized conditions of MRM experiments were as follows: curtain gas, 40 psi; ion spray voltage, 5000 V for positive ion mode and −4500 V for negative ion mode; turbo heater temperature (TEM), 500 °C; nebulizing gas (Gas 1), 55 psi; heated gas (Gas 2), 40 psi. Data acquisition, peak integration, and the calculations were performed using Analyst 1.6.1 software (AB Sciex).

Cai W.J., Ye T.T., Wang Q., Cai B.D, & Feng Y.Q. (2016). A rapid approach to investigate spatiotemporal distribution of phytohormones in rice. Plant Methods, 12, 47.

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Quantifying Lipid Mediators by LC-MS/MS

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Deuterated internal standards d₄-LTB₄, d₈-5-HETE, d₄-PGE₂, and d₅-RvD2, representing each chromatographic region of identified LMs, were added to the samples (500 pg each) to facilitate quantification. The samples were extracted by SPE on C18 columns as previously described^{45 (link)} and were subjected to LC-MS/MS. The system consisted of a Q-Trap 6500 (Sciex) equipped with a Shimadzu LC-30AD HPLC system. A ZORBAX Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 µm, Agilent Technologies) was used with a methanol/water/acetic acid gradient of 55:45:0.01 to 98:2:0.01 (v/v/v) at a 0.4 ml/min flow rate. For monitoring and quantifying the levels of targeted LMs, the multiple reaction monitoring (MRM) method was developed with signature ion pairs Q1 (parent ion)/Q3 (characteristic fragment ion) for each molecule. Identification was conducted with published criteria using the LC retention time, specific fragmentation patterns, and at least six diagnostic fragmentation ions. Quantification was carried out on the basis of the peak area of the MRM chromatograph, and the linear calibration curves were obtained with authentic standards for each compound.

Tsuda S., Shinohara M., Oshita T., Nagao M., Tanaka N., Mori T., Hara T., Irino Y., Toh R., Ishida T, & Hirata K.I. (2017). Novel mechanism of regulation of the 5-lipoxygenase/leukotriene B4 pathway by high-density lipoprotein in macrophages. Scientific Reports, 7, 12989.

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Quantitative Lipid Mediator Analysis

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Deuterated internal standards d₄-LTB₄, d₈-5-HETE, d₄-PGE₂, and d₅-RvD2 representing each chromatographic region of identified LMs were added to the samples (500 pg each) to facilitate quantification. Samples were extracted by SPE on C18 columns as previously described^{33 (link)}. They were then subjected to LC/MS/MS using a Q-Trap 6500 (Sciex) equipped with a Shimadzu LC-30AD HPLC system. A ZORBAX Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 μm, Agilent Technologies) was used with a methanol/water/acetic acid gradient of 55:45:0.01 to 98:2:0.01 (v/v/v) at a 0.4 ml/min flow rate. For monitoring and quantifying the levels of targeted LMs, a multiple reaction monitoring (MRM) method was developed with signature ion pairs Q1 (parent ion)/Q3 (characteristic fragment ion) for each molecule. Identification was based on published criteria using the LC retention time, specific fragmentation patterns, and at least six diagnostic fragmentation ions. Quantification was carried out on the basis of peak area of the MRM chromatograph, and linear calibration curves were obtained with authentic standards for each compound.

Yamada H., Saegusa J., Sendo S., Ueda Y., Okano T., Shinohara M, & Morinobu A. (2021). Effect of resolvin D5 on T cell differentiation and osteoclastogenesis analyzed by lipid mediator profiling in the experimental arthritis. Scientific Reports, 11, 17312.

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Lipidomic Analysis of Skin Mediators

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Various lipid mediators in the skin were analyzed by liquid chromatography/mass spectrometry (MS)/MS‐based lipidomics as previously described.
²¹ (link) Deuterated internal standards d₄‐leukotriene B₄, d₈‐5‐HETE, d₄‐prostaglandin E₂, and d₅‐resolvin D2, representing each chromatographic region of identified lipid mediators, were added to the samples (500 pg each) to facilitate quantification. The samples were extracted by solid‐phase extraction on C18 columns and were subjected to liquid chromatography–MS/MS. The system consisted of a Q‐Trap 6500 (Sciex) equipped with a Shimadzu LC‐30AD HPLC system. A ZORBAX Eclipse Plus C18 column (100 mm×4.6 mm, 3.5 μm; Agilent Technologies) was used with a methanol/water/acetic acid gradient of 55:45:0.01 to 98:2:0.01 (v/v/v) at a 0.4 mL/min flow rate. For monitoring and quantifying the levels of targeted lipid mediators, the multiple reaction monitoring method was developed with signature ion pairs Q1 (parent ion)/Q3 (characteristic fragment ion) for each molecule. Identification was conducted with published criteria using the liquid chromatography retention time, specific fragmentation patterns, and at least 6 diagnostic fragmentation ions. Quantification was carried out on the basis of the peak area of the multiple reaction monitoring chromatograph, and the linear calibration curves were obtained with authentic standards for each compound.

Tanaka T., Sasaki N., Krisnanda A., Shinohara M., Amin H.Z., Horibe S., Ito K., Iwaya M., Fukunaga A., Hirata K, & Rikitake Y. (2024). Novel UV‐B Phototherapy With a Light‐Emitting Diode Device Prevents Atherosclerosis by Augmenting Regulatory T‐Cell Responses in Mice. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 13(2), e031639.

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Simultaneous BCAA and BCKA Quantification

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The LC-MS system consisted of a Q-Trap 6500 (Sciex, Framingham, MA, USA) equipped with a Shimadzu LC-30AD HPLC system (Shimadzu Corporation, Kyoto, Japan). The stable isotope-labeled internal standards APDSTAG® Amino Acids Internal Standard Mixture Solution (FujiFilm-Wako, Osaka, Japan) and KIV-¹³C₅ (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) were added to the samples to facilitate quantification. For BCAA analysis, an Intrada Amino Acid column (100 × 3 mm, 3.0 μm; Imtakt, Kyoto, Japan) was used with an acetonitrile/100 mM ammonium formate/formic acid gradient of 85:15:0.1 to 0:100:0 (v/v/v) and a flow rate of 0.6 mL/min. For BCKA analysis, an Intrada Organic Acid column (150 mm × 2 mm, 3.0 μm; Imtakt) was used with an acetonitrile/water/100 mM ammonium formate/formic acid gradient of 10:90:0:0.1 to 10:0:90:0 (v/v/v/v) and a flow rate of 0.2 mL/min. For monitoring and quantifying BCAA and BCKA levels, a multiple reaction monitoring (MRM) method was developed with signature ion pairs Q1 (parent ion)/Q3 (characteristic fragment ion).

Yoshida N., Yamashita T., Osone T., Hosooka T., Shinohara M., Kitahama S., Sasaki K., Sasaki D., Yoneshiro T., Suzuki T., Emoto T., Saito Y., Ozawa G., Hirota Y., Kitaura Y., Shimomura Y., Okamatsu-Ogura Y., Saito M., Kondo A., Kajimura S., Inagaki T., Ogawa W., Yamada T, & Hirata K.I. (2021). Bacteroides spp. promotes branched-chain amino acid catabolism in brown fat and inhibits obesity. iScience, 24(11), 103342.

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Quantification of Plasma S1P by LC-MS/MS

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Measurement of S1P in plasma by liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed as in a previous study
³⁴⁾with modifications. Plasma (10 µl) was mixed with 10 µl of S1P (d17:1, 1 µM, an internal standard), and 100 µl of ice-cold methanol was then added for extraction. The extract was centrifuged at 15,000 rpm for 10 min and transferred to an auto-injector vial for analysis. The system consisted of a Q-Trap 6500 (SCIEX) equipped with a Shimadzu LC-30AD HPLC system. A ZORBAX Eclipse Plus C18 column (100 mm×4.6 mm, 3.5 µm, Agilent Technologies) was used for sample separation. The mobile phase consists of (A) methanol/acetonitrile/water (1:1:3) and (B) isopropanol both containing 5 mM ammonium acetate, 500 nM EDTA, and 0.025% NH
₃water with gradient of 0% of B to 95% of B at a 0.4 ml/min flow rate. For monitoring and quantifying the level of S1P, the multiple reaction monitoring method was developed with signature ion pairs Q1 (parent ion)/Q3 (characteristic fragment ion), 366.1/250.1 for S1P (d17:1, internal standard) and 380.3/264.2 for S1P (d18:1).

Ganbaatar B., Fukuda D., Shinohara M., Yagi S., Kusunose K., Yamada H., Soeki T., Hirata K.I, & Sata M. (2020). Inhibition of S1P Receptor 2 Attenuates Endothelial Dysfunction and Inhibits Atherogenesis in Apolipoprotein E-Deficient Mice. Journal of Atherosclerosis and Thrombosis, 28(6), 630-642.

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Quantitative Profiling of Lipid Mediators

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Incubations were terminated by adding a 2× volume of ice-cold methanol. Then, the deuterated internal standards, leukotriene B4-d4 (d4-LTB4), d8-5-hydroxyeicosatetraenoic acid (d8-5-HETE), d4-prostaglandin E2 (d4-PGE2), and d5-resolvin D2 (d5-RvD2), which represented each chromatographic region of the identified LMs, were added to the samples (500 pg each) to facilitate quantification. Samples underwent solid phase extraction (SPE) on C18 columns and were subjected to liquid chromatographymass spectrometry (LC-MS/MS). The system consisted of a Qtrap 6500 (Sciex, Framingham, MA, USA), equipped with a Shimadzu LC-30AD HPLC system. A ZORBAX Eclipse Plus C18 column (100 mm×4.6 mm, 3.5 µm; Agilent Technologies, Santa Clara, CA, USA) was used with a gradient of methanol/water/acetic acid from 55:45:0.01 (v/v/v) to 98:2:0.01 at a flow rate of 0.4 mL/min. A multiple reaction monitoring (MRM) method was developed for the signature ion pairs Q1 (parent ion)/Q3 (characteristic fragment ion) of each molecule to monitor and quantify the levels of targeted LMs. The LMs were identified using published criteria, such as LC retention time, specific fragmentation patterns, and diagnostic fragmentation ions. The samples were quantified based on the peak area of the MRM chromatograph, and linear calibration curves were obtained with authentic standards for each compound. 19

Tanaka N., Irino Y., Shinohara M., Tsuda S., Mori T., Nagao M., Oshita T., Mori K., Hara T., Toh R., Ishida T, & Hirata K.I. (2018). Eicosapentaenoic Acid-Enriched High-Density Lipoproteins Exhibit Anti-Atherogenic Properties. Circulation journal : official journal of the Japanese Circulation Society, 82(2).

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