An Agilent HPLC 1100 series was used with a Waters Acquity CSH™ Phenyl-Hexyl 1.7μM 2.1 x 50mm column. A Sciex API 4000 triple quadrupole mass spectrometer with an ESI source was used in positive mode with first scan event a full MS scan at 55.0-1000m/z. For positive polarity detection of targeted tryptophan/kynurenine pathway metabolites, as well as other metabolites a gradient of 95% buffer A was set at 0.00–1.00min, buffer B increased to 15% at 4.00min, then buffer B increased to 95% to 7.00, maintained to 8.00 min then decreased to 5% buffer B at 8.50min through 10.0min. For negative polarity detection of metabolites a gradient of 95% buffer A (H2O and 0.1% formic acid) and 5% buffer B (methanol) at 0–0.5min, increased buffer B to 95% at 5min, increased buffer B to 98% at 8.5min and then decreased buffer B to 5% to 9.0–10.0min at a flow rate of 0.15ml/min. In negative polarity mode for analysis similar parameters were set at 55.0–1000 m/z.
Hplc 1100 series
Manufactured by Waters Corporation
The HPLC 1100 series is a high-performance liquid chromatography system designed for analytical and preparative chromatography applications. It provides precise control of mobile phase flow, solvent delivery, and sample injection, allowing for accurate and reproducible separation of complex mixtures.
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5 protocols using hplc 1100 series
1
Targeted Metabolite Analysis by HPLC-MS
2
Quantification of DEX Release from Nanoparticles
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DEX release was measured by dispersing the DEX-coated nanoparticles (10 mg) into 1 mL of a buffer media (acetate buffer pH=5, and phosphate buffer pH=7.3) and incubated at 37°C in Eppendorf.
Daily, the suspension was centrifuged at 4000 g for 10 mins at room temperature; the supernatant was withdrawn for quantification and the medium was replaced with an equal volume of fresh buffer and the nanoparticles resuspended.
The amount of DEX/DEX-P released from the coating layer was determined using reverse-phase High Performance Liquid using an Agilent Technologies® HPLC (1100 series) equipped with a Waters-Spherisorb ODS2 column (Pore size – 80 Å, 5 µm, and packing dimension of 4.6 mm×150 mm). The injection volume was 10 µL. The mobile phase was a mixture of PBS-acetonitrile-glacial acetic acid 70:26:4 at a flow rate of 1 mL/min; a UV detector at 244 nm was employed.
Stock solutions of DEX-P and DEX with a concentration of 1 mg/mL were prepared separately and a series of standards ranging from 0.4 to 250 µg/mL were prepared for calibration.
Daily, the suspension was centrifuged at 4000 g for 10 mins at room temperature; the supernatant was withdrawn for quantification and the medium was replaced with an equal volume of fresh buffer and the nanoparticles resuspended.
The amount of DEX/DEX-P released from the coating layer was determined using reverse-phase High Performance Liquid using an Agilent Technologies® HPLC (1100 series) equipped with a Waters-Spherisorb ODS2 column (Pore size – 80 Å, 5 µm, and packing dimension of 4.6 mm×150 mm). The injection volume was 10 µL. The mobile phase was a mixture of PBS-acetonitrile-glacial acetic acid 70:26:4 at a flow rate of 1 mL/min; a UV detector at 244 nm was employed.
Stock solutions of DEX-P and DEX with a concentration of 1 mg/mL were prepared separately and a series of standards ranging from 0.4 to 250 µg/mL were prepared for calibration.
3
Targeted Metabolomics Analysis using HPLC-MS/MS
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An Agilent HPLC 1100 series was used with a Waters Acquity CSHTM Phenyl-Hexyl 1.7 μM 2.1 × 50-mm column. A Sciex API 4000 triple quadrupole mass spectrometer with an ESI source was used in positive mode with first scan event a full mass spectrometry scan at 55.0 to 1000 m/z. For positive polarity detection of 230 targeted metabolites with a gradient of 98% buffer A was set at 0.00 to 1.00 minute, buffer B increased to 15% at 4.00 minutes, then buffer B increased to 95% to 7.00 minutes, maintained to 8.00 minutes, then decreased to 2% buffer B at 8.50 minutes through 10.0 minutes. For negative polarity detection of metabolites, a gradient of 98% buffer A (H2O and 0.1% formic acid) and 2% buffer B (methanol) at 0 to 0.5 minutes, increased buffer B to 95% at 5 minutes, increased buffer B to 98% at 8.5 minutes, and then decreased buffer B to 2% to 9.0 to 10.0 minutes at a flow rate of 0.15 mL/min. In negative polarity mode for analysis, similar parameters were set at 55.0 to 1000 m/z.
4
Chlorophyll Pigment Analysis Protocol
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Leaf samples (~200mg) were ground to a fine powder with liquid nitrogen, and pigments were extracted with 80% acetone. The samples were centrifuged at 15 000 g for 10min and the supernatant was used for Chl estimation and HPLC analysis (Roca et al., 2004 (link)). Chls were determined according to Arnon (1949) (link) using a Shimadzu UV-VIS spectrophotometer (model UV-2600, Japan). HPLC analysis of Chl pigments was done according to Jiang et al. (2007) (link) by using an Agilent HPLC 1100 series equipped with a C-18 column (Waters Nova-Pak, 3.9×150mm) and a dual absorbance detector. Separation was carried out on an elution gradient with the mobile phases (A) ion pair reagent (1M ammonium acetate in water)/methanol (1:4, v/v) and (B) acetone/methanol (1:4, v/v), at a flow rate of 1.2ml min−1. The gradient was isocratic A for 4min, isocratic B for 20min, and a return to A for 6min, and detection was at 660nm. The absorption spectrum of each peak was obtained from their respective chromatograms in the HPLC profile. The eluted Chl pigment samples were collected in special eppendorf tubes. The HPLC-purified samples were vacuum centrifuged for 2h to a powdered form for further analysis.
5
Chlorhexidine Release from Coated Nanoparticles
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Coated-particles were dispersed (10 mg/mL) in either acetate buffer pH = 5 or phosphate buffer pH = 7.3, and incubated statically at 37 °C. The medium was withdrawn daily for quantification and the replaced with an equal volume of fresh buffer. All experiments were performed on 3 independent nanoparticles batches.
The amount of chlorhexidine released from the coated nanoparticles was determined using reverse-phase High Performance Liquid Chromatography with a Agilent Technologies ® HPLC (1100 series) equipped with a Waters-Spherisorb ODS2 column (Pore size-80Å 5 µm and packing dimension of 4.6 mm X 150 mm) and a UV detector at 239 nm. The injection volume was 10 L while the mobile phase was 0.1 M acetate buffer pH = 5 acetonitrile 58:42 at a flow rate of 1 mL/min. Stock solutions of chlorhexidine (ranging from 0.4-25 µg/mL) were prepared separately for calibration.
The amount of chlorhexidine released from the coated nanoparticles was determined using reverse-phase High Performance Liquid Chromatography with a Agilent Technologies ® HPLC (1100 series) equipped with a Waters-Spherisorb ODS2 column (Pore size-80Å 5 µm and packing dimension of 4.6 mm X 150 mm) and a UV detector at 239 nm. The injection volume was 10 L while the mobile phase was 0.1 M acetate buffer pH = 5 acetonitrile 58:42 at a flow rate of 1 mL/min. Stock solutions of chlorhexidine (ranging from 0.4-25 µg/mL) were prepared separately for calibration.
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