Ez faast kit

Plasma amino acid and acylcarnitine concentrations were determined on a Waters Acquity UPLC‐coupled Xevo TQD mass spectrometer using non‐derivatized a semi‐quantitative kit for dried blood spots from Chromsystems (Gräfeling, Germany), which was modified by directly pipetting 1.3 μL plasma, sampled and stored as described above, into extraction buffer. For confirmation, amino acids were additionally quantified from fresh plasma of 30 of the patients using the EZ:faast kit from Phenomenex (Torrance, CA) on a Waters Q‐Micro HPLC‐coupled mass spectrometer with essentially same results (not shown). BCAA concentrations were calculated by addition of valine, leucine and isoleucine values.

For tissue AA concentration, 60.9 ± 27.4 mg of mammary tissue was mixed with PBS and a known amount of universally labeled ¹³C AA mix as an internal standard. The tissue was bead-homogenized, centrifuged at 12× g for 10 min, and the supernatant was deproteinized with 0.5 M perchloric acid. Amino acids were derivatized following EZ-Faast kit instructions (CN KH0-7338, Phenomenex, Torrance, CA, USA). The tissue concentration of the derivatized AA was measured in a Shimadzu 2020 liquid chromatography mass spectrometer (Shimadzu, Kyoto, Japan) as previously reported [36 (link)].

Free AAs were extracted and derivatised as previously described [38 (link)]. Briefly, fresh plant material (1.0 ± 0.05 g) was extracted with 15 mL of a 7:3 (V/V) mixture of methanol and twice distilled H₂O. Extracts were derivatised using an EZ:faast kit (Phenomenex, USA). The prepared samples were analysed on a Hewlett Packard 6890N/5975 MSD gas chromatography-mass spectrometry system (GC-MS; Agilent Technologies, Santa Clara, CA, USA), as previously described [75 (link)].

Plasma amino acids were extracted and derivatized using the EZ: FAAST kit (Phenomenex, USA) as specified by the manufacturer. Along with other internal standards, deuterated citrulline (100 μM) was included in the assay to optimize citrulline quantification in duplicate. Amino acid profiles were achieved using UHPLC coupled to Tandem Quadrupole Mass spectrometry (Acquity H-Class, Xevo TQD, Waters corp., Mildford, MA). Intra- and inter-assay coefficients were lower than 15% for all amino acids and were respectively of 2.6% (n = 244) and 3.7% (n = 18) for citrulline. Citrulline levels were above the LOQ (0.1 µmol/L) for all samples.

Cecal contents from hamsters were weighed at necropsy and flash frozen in liquid nitrogen. Samples were sent to the University of Michigan, Metabolomics Resource Core for amino acid analysis. Amino acids were analyzed using the EZ-faast kit from Phenomenex–Torrance, CA following the instructions provided by the manufacturer. Samples were extracted, semi-purified, derivatized by a proprietary method, and analyzed by EI-GCMS using internal standards for normalization. Analytes were reported as nM/mg of cecal content. Fold changes between clindamycin treated vs. non-antibiotic treated controls, and clindamycin treated vs. C. difficile infected colon samples, were evaluated by Welch's t-test; p<0.05 was considered to be significant.