Triple quadrupole lcms 8050

For the quantification of 54 metabolic compounds in MEFs, 100 mg cell pellets (1 × 10⁷ cells per line, for 8 WT versus 8 ClpP^−/−) were snap frozen in liquid nitrogen, stored at −80 °C, and shipped on dry ice to the company Metabolomic Discoveries (Potsdam, Germany). Targeted profiling with unambiguous characterization and relative quantification by LC-tandem mass spectrometry was performed on a Shimadzu (Kyoto, Japan) triple quadrupole LCMS-8050 equipped with an electrospray ionization (ESI) source and operated in multiple reaction mode (MRM). For statistical evaluation, raw data as peak abundances normalized to internal standards and, if necessary, protein content were detailed, complemented by means and standard deviation values, as well as a differential analysis sheet with ANOVA across all groups as a global p-value, and an adjusted global p-value, followed by a local p-value from pairwise t-tests and ratios of the group means as a log₂ value, as well as absolute fold changes. Absolute numbers were normalized against the mean of WT samples. Bar graphs were created with GraphPad Prism (GraphPad Software, San Diego, CA, USA) version 9.

All solvents and additives were LCMS grade and purchased from Sigma-Aldrich. Internal standard of sphingosine-1-phosphate, ceramides C16, C18, and C24:1, and deuterated ceramide mix were purchased from Avanti Polar Lipids (Alabaster). For the extraction of sphingolipids, human plasma (20 μl) was diluted to 400 μL with Milli-Q water (Sigma-Aldrich), and 3 μl of the internal standard was added, after which samples were extracted by the addition of 400 μL of isopropanol/ethyl acetate 18/85 (v/v); the solution was vortexed for 20 seconds and centrifuged at 16,110 x g for 5 minutes. The upper organic phase was collected, while the aqueous phase was acidified with 20 μL of formic acid and reextracted with 400 μL of fresh isopropanol/ethyl acetate 18/85 (v/v%). The obtained solution was vortexed and centrifuged. The supernatants were pooled and dried under a gentle stream of nitrogen. For sphingolipid extraction, cell media (500 μL) was diluted with 1 mL isopropanol/ethyl acetate 18/85 (v/v%) and processed as described above. Samples were resolubilized with 150 μL of methanol and 1 mM HCOONH₄ plus 0.2% HCOOH (v/v). UHPLC-MS/MS analysis was carried out with a Shimadzu Nexera coupled online to a triple-quadrupole LCMS 8050 (Shimadzu) by an ESI source.

For the quantification of metabolic compounds, 8 WT and 8 CLPP-null sex-matched (50% males, 50% females) 12-month-old mouse cerebella were snap-frozen in liquid nitrogen, stored at −80 °C, and shipped on dry ice to the company Metabolomic Discoveries (Potsdam, Germany). Targeted profiling with unambiguous characterization and relative quantification by means of LC–tandem mass spectrometry was performed on a Shimadzu (Kyoto, Japan) triple quadrupole LCMS-8050 equipped with an electrospray ionization (ESI) source and operated in multiple reaction mode (MRM). For statistical evaluation, the raw data as peak abundances normalized to internal standards and, if necessary, protein content were detailed, complemented by means and standard deviation values, as well as a differential analysis sheet with ANOVA across all groups as a global p-value, an adjusted global p-value, a local p-value from pairwise t-tests, ratios of the group means as a log₂ value, and absolute fold changes.

Bisphenol A (BPA) and various phthalate concentrations, including monomethyl phthalate (MEP), mono isobutyl-phthalate (MIbP), mono (2-ethyl-5-hydroxylhexyl) phthalate (MEHHP), mono benzyl phthalate (MBzP), and mono (2-ethylhexyl) phthalate (MEHP), were measured in clean, mid-stream urine samples, as per the method described by Lee et al. [23 (link)]. Concurrently, creatinine concentrations were determined. The urine samples were collected in polypropylene cups intended for urine culture, and participants were instructed to store the cups in a cool environment until they could be transported. These urine samples were then transported under refrigeration, divided into tubes made from phthalate-free materials, and stored at −80 °C until the time of analysis.
BPA and phthalate concentrations were quantified using RP-UHPLC-MS/MS (Shimadzu, Milan, Italy) on a Nexera UHPLC coupled online to a triple quadrupole LCMS8050 (Shimadzu, Kyoto, Japan). Detailed extraction and UHPLC-MS/MS conditions for the BPA and phthalate metabolites can be found elsewhere [2 (link)].
Creatinine levels were measured using routine methods on an Atellica^® CH Analyzer (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) and expressed as _mol/L. All urinary samples were considered acceptable because the creatinine concentrations were within the range of 0.3–3.0 g/L [24 ].