Open Data LMU (Ludwig-Maximilians-Universität München)

Marchioro, Linda

Supplemental material: Methods for metabolomics analysis of amino acids, 
tricarboxylic acid cycle (TCA) intermediates, phospholipids and acylcarnitines. 
 
Amino acids. Aliquots of the supernatant were prepared using derivatization as previously 
reported [1]. For internal standardization, a labeled amino acid standards set (set A, 
Cambridge Isotope Laboratories) was mixed with L-Asparagine (15N2, 98%, Cambridge 
Isotope Laboratories) and L-Tryptophan (Indole-D5, 98%, Cambridge Isotope Laboratories) 
and added to the precipitation reagent. AA butylester were determined by ion-pair liquid 
chromatography coupled to mass spectrometry detection (LC-MS/MS). 10 μL of the prepared 
sample were injected into the HPLC system (HPLC 1100, Agilent, Waldbronn, Germany) 
and chromatographic separation was performed with a XBridge C18 column (Waters GmbH, 
Eschborn, Germany). MS detection was performed with an API 2000 triple quadrupole 
instrument (Sciex, Darmstadt, Germany) with an APCI source operating in positive ion 
ionization mode. Data acquisition on the mass spectrometer was controlled by Analyst 1.6.2 
software (AB Sciex, Darmstadt, Germany). Data handling and quantification were also 
performed with Analyst 1.6.2 software (AB Sciex, Darmstadt, Germany). 
 
TCA intermediates. Metabolites of the TCA cycle and keto-acids were measured by a 
modified LC-MS/MS method based on previously published methods [2, 3]. D3-
methylmalonic acid was used as internal standard. 100 μL of the supernatant were evaporated 
to dryness and re-suspended in 50 μL water. 5 μL of the extracted samples were injected to 
an Agilent 1200 HPLC and molecular species were separated on a Kinetex F5 core-shell 
HPLC column, 150 x 2.1 mm, 2.6 μm particle size (Phenomenex, Aschaffenburg, Germany). 
The mobile phase A was water with 1% formic acid and mobile phase B was composed of 
methanol/ isopropanol (50:50) with 1% formic acid. The gradient elution at a flow rate of 250 
μL/min was held constant for 1 minute with 1% B, raised to 65% B within 6 minutes, and 
turned back to initial conditions of 1%B within 0.5 minutes. Re-equilibration was held for 5 
minutes at 1% B. The triple quadrupole mass spectrometer (AB Sciex API4000; Applied 
Biosystems, Darmstadt, Germany) was operated in negative scheduled multiple reaction 
monitoring mode using electrospray ionization (ESI). 
 
Phospholipids. Phospholipids were analyzed via flow-injection mass spectrometry (FIA-
MS/MS). 30 μL of the centrifuged supernatant were mixed for 20 min at 600 rpm with 500 µl 
methanol containing 1.2 mM ammonium acetate and then used for FIA-MS/MS analysis. 
Samples were analyzed with a triple quadrupole mass spectrometer (QTRAP4000, Sciex, 
Darmstadt, Germany) with an electrospray ionization (ESI) source which was used in 
positive ionization mode. The MS was coupled to a LC system (Agilent, Waldbronn, 
Germany). MS/MS analysis was run in MRM with 184 Da (choline head group) as product 
ion for the PL. Analyst 1.6.2 software, followed by in-house processing with the statistical 
program R (R Project for Statistical Computing, http://www.r-project.org/), was used to post-
process the entire analytical process. 
 
Acylcarnitines. The supernatant was injected by flow-injection analysis into a LC-MS/MS 
system with isocratic elution with 76% isopropanol, 19% methanol and 5% water. The mass 
spectrometer was equipped with electrospray ionization and operated in positive ionization 
mode. 
 
 
 
  
References 
[1] Harder U, Koletzko B, Peissner W. Quantification of 22 plasma amino acids combining 
derivatization and ion-pair LC-MS/MS. Journal of chromatography B, Analytical 
technologies in the biomedical and life sciences. 2011;879:495-504. 
[2] Luo B, Groenke K, Takors R, Wandrey C, Oldiges M. Simultaneous determination of 
multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic 
acid cycle by liquid chromatography-mass spectrometry. Journal of chromatography A. 
2007;1147:153-64. 
[3] Birkler RI, Stottrup NB, Hermannson S, Nielsen TT, Gregersen N, Botker HE, et al. A 
UPLC-MS/MS application for profiling of intermediary energy metabolites in microdialysis 
samples--a method for high-throughput. Journal of pharmaceutical and biomedical analysis. 
2010;53:983-90. 
 


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