Api 4000 qtrap

Samples were prepared by adding internal standards (Additional file 3: Table S3) and 20 µl (Ryu and Wang and single-extraction) or 40 µl (single-extraction with repeat extraction) of sample corresponding to 0.2 mg extracted leaf dry mass. Samples were brought to 1.4 ml by adding chloroform: methanol: 300 mM ammonium acetate in water (30/66.5/3.5, v/v/v) for mass spectrometric analysis. Analysis was performed on a triple quadrupole mass spectrometer with an electrospray ionization source (API 4000 QTRAP, Applied Biosystems, Foster City, CA) in direct infusion mode. Samples were introduced using an autosampler (LC Mini PAL, CTC Analytics AG, Zwingen, Switzerland) fitted with the required injection loop for the acquisition time and presented to the electrospray ionization needle at 30 µl/min. Samples were analyzed with neutral loss and precursor scans. Most instrument settings were as indicated by Xiao et al. [19 (link)], but scan-specific analytical parameters are listed in Additional file 4: Table S4.

Quantitative analyses of polar lipids, (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), LPC, DGDG or MGDG) lipids were carried out using electrospray ionization tandem triple quadrupole mass spectrometry (API 4000 QTRAP; Applied Biosystems; ESI-MS/MS) as described previously by Gonzalez-Thuillier et al. (2015) (link). The internal standards for polar lipids were supplied by Avanti (Alabama, USA), incorporated as; 8 pmol 13:0-LPC, 0.086 nmol di24:1-PC, 0.080 nmol di14:0-PE, 0.05 nmol di18:0-PI, 0.080 di14:0-PG and 0.03 nmol di18:0-PS. The standards dissolved in chloroform and different conditions were used for the aqueous samples, 100 μL foam or 25 μL un-foamed DL were combined with chloroform/methanol/300 mM ammonium acetate (300:665:3.5 v/v) to make a final volume of 1 mL. The lipid extracts were infused at 15 μL/min with an autosampler (HTS-xt PAL, CTC-PAL Analytics AG, Switzerland). Data acquisition and acyl group identification were as described by Gonzalez-Thuillier et al. (2015) (link). The data were processed using the LipidView software (SCIEX, Framingham, MA, U.S.A.), where isotope corrections were applied. The peak area for each lipid was normalized to the internal standard and further normalized to the weight of the initial sample.

Plasma concentrations of vitamins B9 and B12 were determined using a radioisotope dilution assay (simulTRACSNB; ICN Pharmaceuticals, Versailles, France) as previously reported [45 (link)]. Concentrations of homocysteine were measured by high-performance liquid chromatography (Waters, St. Quentin, France) coupled with mass spectrometry (Api 4000 Qtrap; Applied Biosystems, Courtaboeuf, France) [46 (link)].
Plasma levels of total ghrelin was measured in duplicate after appropriate dilution by specific radioimmunoassays using commercial kits (RK-031-31; Phoenix Europe GmbH, Karlsruhe, Germany,), as previously described [13 (link)]. Leptin, peptide YY and insulin were measured using the MILLIPLEX Rat Metabolic Hormone Panel (RMHMAG-84K, Millipore, Fontenay-sous-Bois, France).

We analyzed the serum and tissue concentrations of doxorubicin by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity (Agilent, Santa Clara, CA, USA), followed by tandem mass spectrometry (MS/MS) using API4000QTRAP (Applied Biosystems, Waltham, MA, USA). For the HPLC analysis, a Gemini 5 μm C18, 50 × 2.0 mm analytical column (Phenomenex, Torrance, CA, USA) was used. The mobile phase consisted of 5 mM ammonium acetate and 0.1% acetic acid acetonitrile with a flow rate of 0.3 ml/min and a 25 °C column temperature over 7.5 min.
The MS/MS was equipped with a positive ionization mode with Turbo Spray, and multiple reaction monitoring was used for quantification. The nebulizer and desolvation gas pressure was 50 psi, both using nitrogen. MS/MS was conducted under a needle voltage of 5000 V and a set temperature of 400 °C. The acquisition delay was 0 s with a pause time of 5 ms.

Plasma concentrations of vitamin B12 and folate were determined by radio-dilution isotope assay (simulTRAC-SNB, ICN, Costa Mesa, USA). Homocysteine, Methylmalonic acid, Succinic acid, SAM and SAH concentrations were measured in plasma by High Performance Liquid Chromatography (Waters, St Quentin, France) coupled to mass spectrometry (Api 4000 Qtrap Applied Biosystems, Courtabœuf, France), as described previously34 (link). Insulin was assayed by radioimmunoassay (MP, Biomedicals, Solon, OH, USA). HOMA-IR was calculated using the formula [fasting plasma glucose (mmol/L) × fasting plasma insulin (μIU/mL)]/22.5. Lipids, glycemia, ASAT, ALAT and other routine biochemical parameters were determined in plasma as described previously10 (link).