Vitamins and several reaction products in P‐media samples were measured with a LC‐MS/MS method. A complete list of the reported components is presented in the Supporting Information Table 1 . P‐media samples of the DoE study (Table 1 ) were frozen directly after media preparation and stored at −70˚C. Samples were thawed at room temperature and under constant protection from light exposure. All samples were measured in triplicates using an InfinityLab Poroshell Hydrophilic interaction chromatography‐Z column (Agilent Technologies, USA). MS/MS was performed on a QTRAP® 6500+ triple quadrupole mass spectrometer (Sciex, USA) equipped with an ESI source and coupled to an Agilent 1260 series binary HPLC system (Agilent Technologies, USA). Nitrogen was used as curtain and collision gas. Data was acquired using MultiQuant 3.0.3 software (Sciex, USA).
Agilent 1260 series binary hplc system
Manufactured by Agilent Technologies
Sourced in Germany
The Agilent 1260 series binary HPLC system is a high-performance liquid chromatography instrument designed for analytical applications. It features a binary pump, autosampler, column compartment, and detector, providing the core functionality for liquid chromatography separations and analysis.
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Lab products found in correlation
Qtrap 6500 triple quadrupole mass spectrometer, by AB Sciex (2 mentions)
Multiquant 3, by AB Sciex (2 mentions)
Konelab prime60i, by Thermo Fisher Scientific (1 mentions)
7890b gc system, by Agilent Technologies (1 mentions)
Cedex hires analyzer, by Roche (1 mentions)
Diode array detector, by Agilent Technologies (1 mentions)
Synergi hydro rp c18, by Phenomenex (1 mentions)
4 protocols using agilent 1260 series binary hplc system
1
Quantitative Analysis of Vitamins and Metabolites in P-Media
2
Comprehensive Analytical Workflow for Cell Culture Monitoring
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Cultivation samples were taken every 24 h and cell counting and viability determination was performed using a Cedex HiRes analyzer (Roche). The metabolites glucose, lactate, and ammonia were measured with a Konelab™ Prime60i (Thermo Fisher Scientific) device. Antibody concentration was determined with a Protein‐A HPLC method (Thermo Fisher Scientific). Amino acid analytics were performed with a 7890B GC system (Agilent Technologies). Cysteine was excluded from the amino acid method due to the known stability issues of this component (Prade et al., 2020 ; Wang et al., 2017 ). LC‐MS/MS was applied to determine vitamin concentrations and additional metabolites such as 2‐hydroxybutyrate, indole‐3‐lactic acid, indole‐3‐carboxylic acid, and 2‐deoxycitidine. LC‐MS/MS samples were measured using an InfinityLab Poroshell Hydrophilic interaction chromatography (HILIC)‐Z column (Agilent Technologies) as described previously (Krattenmacher et al., 2018 ). Tandem mass spectrometry was performed on a QTRAP® 6500 + triple quadrupole mass spectrometer (Sciex) equipped with an ESI source and coupled to an Agilent 1260 series binary HPLC system (Agilent Technologies). Nitrogen was used as curtain and collision gas. Data were acquired using MultiQuant 3.0.3 software (Sciex).
3
Quantification of Cellular Carotenoids by HPLC
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The cellular carotenoid content was determined according to a previously described method with minor modifications (Diao et al., 2020 (link)). Briefly, freeze-dried cells (50 mg) were mixed with 500 µL acetone and 300 mg zirconia beads and then homogenized. After centrifuging at 12,000 × g for 5 min, the upper phase containing the carotenoid was collected for high-performance liquid chromatography (HPLC) analysis.
HPLC analysis was conducted following the previous method (Diao et al., 2020 (link)). Briefly, Agilent 1,260 series binary HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a Symmetry C18 column (5 μm × 4.6/250 mm) (Waters, Milford, MA, United States) was used. Pigments were eluted at a flow rate of 0.7 mL/min with a 25-min gradient of ethyl acetate (0%–100%) in acetonitrile-water-triethylamine (9:1:0.01, vol/vol/vol) and detected using the Agilent diode array detector at 470 nm. Individual carotenoids were identified by their absorption spectra, and their typical retention times were compared with standard samples of pure carotenoids. β-carotene was identified via comparison to commercially available authentic standards β-carotene (SC8140, Solarbio, China).
HPLC analysis was conducted following the previous method (Diao et al., 2020 (link)). Briefly, Agilent 1,260 series binary HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a Symmetry C18 column (5 μm × 4.6/250 mm) (Waters, Milford, MA, United States) was used. Pigments were eluted at a flow rate of 0.7 mL/min with a 25-min gradient of ethyl acetate (0%–100%) in acetonitrile-water-triethylamine (9:1:0.01, vol/vol/vol) and detected using the Agilent diode array detector at 470 nm. Individual carotenoids were identified by their absorption spectra, and their typical retention times were compared with standard samples of pure carotenoids. β-carotene was identified via comparison to commercially available authentic standards β-carotene (SC8140, Solarbio, China).
4
Metabolomic Sample Preparation for LC-MS
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Samples preparation for LC-MS metabolomics analysis followed the previous study (Li et al., 2015 (link)). Briefly, cells grown at the 24 or 48 h were centrifuged at 8,000 × g for 5 min at 25°C (Eppendorf 5430R, Hamburg, Germany). The supernatant was discard. The sediment was resolved in 900 µL of solution 1 (80:20 MeOH/H2O, stored at −80°C), quickly frozen in liquid nitrogen, and thawed on dry ice to release metabolites (Park et al., 2011 (link)). The cells were frozen-thrawed for three times to release the whole metabolites, and the supernatants were collected by centrifugation at 150,00 × g for 5 min at 4°C (Park et al., 2011 (link)). The left cell debris were then re-suspended in solution 1 and the above extraction process was repeated. The twice supernatants were mixed and stored at −80°C until LC-MS analysis. An Agilent 1,260 series binary HPLC system (Agilent Technologies, Waldbronn, Germany) using a SYnergi Hydro-RP (C18) 150 mm × 2.0 mm I.D., 4 μm 80 Å 549 particles column (Phenomenex, Torrance, CA, United States), coupled to an Agilent 6410 550 triple quadrupole mass analyser equipped with an electrospray ionization source was used for LC-MS analysis. Data processing and statistical analysis were conducted according to the method described previously (Wang et al., 2019 (link)).
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