Pharmacokinetic experiments for ND-2110 were performed by dosing DBA1 mice either i.v. at 3 mg/kg in 10% 2-hydroxypropyl-β-cyclodextrin, or by oral gavage at 10 mg/kg in 0.5% methylcellulose. Blood was collected into sodium heparin tubes before dose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, and 8 h after dosing for the i.v. administration arm or 0.25, 0.5, 1, 2, 4, 6, and 8 h after dosing for the oral gavage administration arm. Plasma was prepared and ND-2110 level was quantified by LC/MS/MS with API 5000 Triple Quadropole System (Applied Biosystems). Pharmacokinetic experiments for ND-2158 were performed by dosing male C57BL/6 mice either i.v. or i.p. at 3 mg/kg in 10% 2-hydroxypropyl-β-cyclodextrin. Blood was collected into sodium heparin tubes before dose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after dosing. Plasma was prepared and ND-2158 level was quantified by LC/MS/MS using the API 4000 Q TRAP system (Applied Biosystems). Pharmacokinetic parameters were calculated using Phoenix WinNonlin.
Api 4000 q trap system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The API 4000 Q TRAP system is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument designed for qualitative and quantitative analysis. It features a triple quadrupole design that provides enhanced sensitivity and selectivity for a wide range of applications.
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Lab products found in correlation
Agilent 1200 hplc system, by Agilent Technologies (2 mentions)
Absoluteidq p180 kit, by Biocrates (2 mentions)
Agilent 1200 hplc, by Agilent Technologies (1 mentions)
Multiquant software, by Thermo Fisher Scientific (1 mentions)
Analyst 1, by Thermo Fisher Scientific (1 mentions)
Agilent 1200 series hplc system, by Agilent Technologies (1 mentions)
5 protocols using api 4000 q trap system
1
Pharmacokinetic Evaluations of ND-2110 and ND-2158
2
Serum Metabolite Quantification Protocol
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Serum metabolite quantification for 2579 participants was carried out using an AbsoluteIDQ p180 kit (BIOCRATES Life Science, Innsbruck, Austria) following the manufacturer’s instructions. An API 4000 QTRAP system (Applied Biosystems, Foster City, CA, USA) equipped with an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) was used to perform liquid chromatography and flow injection analysis-mass spectrometry. Concentrations of kynurenine and tryptophan used in this study were measured in μM units with a MetVal software package (BIOCRATES Life Sciences). Serum metabolites that passed the following quality control (QC) criteria were included in this study: (i) the coefficient of variance for each metabolite in the reference standards < 25%, (ii) half of the analyzed metabolite concentrations in the reference standards > limit of detection (LOD), and (iii) half of the analyzed metabolite concentrations in the experimental samples > LOD. Detailed information for the QC procedure has been described in a previous study [23 (link)]. The ratio of kynurenine to tryptophan (K/T) was calculated to estimate the IDO activity.
3
HPLC-MS/MS Analysis of AAI and ALI
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Agilent 1200 HPLC (Agilent Technologies, Santa Clara, CA, USA) was used. Kinetex-C 18 110A column (3 × 30 mm, 2.6 μm, Phenomenex) was operated at 30 °C and the flow rate was set at 0.8 mL/min for separation of AAΙ and ALΙ. The mobile phase consisted of (A) water containing of 10 mmol/L ammonium acetate and (B) acetonitrile. Gradient conditions were as follows: 0–0.5 min, 10% B; 0.5–0.8 min, 10–95% B; 0.8–2.5 min, 95% B. A 10 μL aliquot of each sample was injected. All samples were kept at 4 °C throughout the analysis.
MS was performed on an API 4000 Qtrap system (Applied Biosystems, Foster City, CA, USA). Electrospray ionization (ESI) was performed in the positive ion mode. Curtain gas (CUR), nebulizer gas (GS1), and turbo-gas (GS2) were set at 15 psi, 60 psi, and 60 psi, respectively. The ionspray voltage was 5.0 kV, and the temperature was 550 °C. Nitrogen was employed as the collision gas. AAΙ and ALΙ were analyzed using the scheduled MRM. Data acquisitions were performed using the Analyst 1.5.2 software (Applied Biosystems). Multiquant software (Applied Biosystems) was used to quantify AAΙ and ALΙ.
MS was performed on an API 4000 Qtrap system (Applied Biosystems, Foster City, CA, USA). Electrospray ionization (ESI) was performed in the positive ion mode. Curtain gas (CUR), nebulizer gas (GS1), and turbo-gas (GS2) were set at 15 psi, 60 psi, and 60 psi, respectively. The ionspray voltage was 5.0 kV, and the temperature was 550 °C. Nitrogen was employed as the collision gas. AAΙ and ALΙ were analyzed using the scheduled MRM. Data acquisitions were performed using the Analyst 1.5.2 software (Applied Biosystems). Multiquant software (Applied Biosystems) was used to quantify AAΙ and ALΙ.
4
Quantifying Kynurenine and Tryptophan Levels
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To quantify kynurenine and tryptophan, serum samples collected from 2579 participants were analyzed using an AbsoluteIDQ p180 kit (BIOCRATES Life Science, Innsbruck, Austria) according to the manufacturer’s instructions. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) was conducted using an API 4000 QTRAP system (Applied Biosystems, Foster City, CA, USA) equipped with an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) to measure metabolites. The quality control (QC) process for analyzed metabolites has been described in detail elsewhere [29 (link)]. Briefly, both kynurenine and tryptophan used in this study met the following criteria: the coefficient of variance for each metabolite in the reference standards < 25%, 50% of the analyzed metabolite concentrations in the reference standards > limit of detection, and 50% of the analyzed metabolite concentrations in the experimental samples > limit of detection. Pooled human normal serums were used as reference standards. IDO activity was estimated as the ratio of kynurenine to tryptophan.
5
Mass Spectrometry Protocol for Sample Analysis
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The MS data were acquired by electrospray ionization (ESI) mass spectrometry with an API 4000 Q TRAP system (Applied Biosystems, Foster City, CA, USA) in positive ion mode ([M+H]+) that was equipped with an Agilent 1200 series HPLC system. The MS operating conditions were as follows: ion spray voltage (5.5 kV), curtain gas (20 psi), nebulizing gas (50 psi), heating gas (50 psi), high purity nitrogen (N2), heating gas temperature (550°C), declustering potential (100 V), entrance potential (10 V), and spectra scanning range (m/z 100–1000) (scan time 4.8 sec).
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