Fig 1.
Biochemical pathway of propionate metabolism (simplified).
3OHPA: 3-hydroxypropionic acid; MMA: methylmalonic acid; MCA: methylcitric acid; Cbl: cobalamin. Drawn using Inkscape 0.91pre4 (Open Source Software licensed under the GNU General Public License).
Table 1.
Optimized MS/MS settings in the second-tier LC-MS/MS assay (negative electrospray ion mode).
Table 2.
Assay validation in calibrator dried blood spots (n = 10).
Fig 2.
LC-MS/MS chromatogram of DBS from a healthy newborn.
Peak 1: 3-hydroxypropionic acid (3OHPA); peak 2: lactic acid; peak 3: succinic acid; peak 4: methylmalonic acid (MMA); peaks 5a and 5b: methylcitric acid (MCA); peak 0: interfering peak; DBS: dried blood spot. Numbered black peaks: unlabelled analytes; numbered unfilled peaks: deuterated internal standards. All intensities (Y-axes) have been normalized to 4*105 cps for better comparability (not on the inserted zoom-in figures) and have been plotted against retention time (X-axes). Chromatograms were generated by MassLynx 4.1 (Waters, Milford, MA, USA) and modified using Inkscape 0.91pre4 (Open Source Software licensed under the GNU General Public License).
Fig 3.
LC-MS/MS chromatogram of DBS from a PA patient.
Peak 1: 3-hydroxypropionic acid (3OHPA); peak 2: lactic acid; peak 3: succinic acid; peak 4: methylmalonic acid (MMA); peaks 5a and 5b: methylcitric acid (MCA); peak 0: interfering peak; DBS: dried blood spot; PA: propionic acidemia. Numbered black peaks: unlabelled analytes; numbered unfilled peaks: deuterated internal standards. All intensities (Y-axes) have been normalized to 4*105 cps for better comparability (not on the inserted zoom-in figures) and have been plotted against retention time (X-axes). Chromatograms were generated by MassLynx 4.1 (Waters, Milford, MA, USA) and modified using Inkscape 0.91pre4 (Open Source Software licensed under the GNU General Public License).
Fig 4.
LC-MS/MS chromatogram of DBS from a MMAmut(0) patient.
Peak 1: 3-hydroxypropionic acid (3OHPA); peak 2: lactic acid; peak 3: succinic acid; peak 4: methylmalonic acid (MMA); peaks 5a and 5b: methylcitric acid (MCA); peak 0: interfering peak; DBS: dried blood spot; MMAmut(0): methylmalonyl-CoA mutase deficiency (complete). Numbered black peaks: unlabelled analytes; numbered unfilled peaks: deuterated internal standards. All intensities (Y-axes) have been normalized to 4*105 cps for better comparability (not on the inserted zoom-in figures) and have been plotted against retention time (X-axes). Chromatograms were generated by MassLynx 4.1 (Waters, Milford, MA, USA) and modified using Inkscape 0.91pre4 (Open Source Software licensed under the GNU General Public License).
Table 3.
Assay validation in dried blood spots using quality control samples at three concentrations (n = 10).
Table 4.
Results of dried blood spot samples from patients with known diagnosis: Retrospective analysis of NBS specimens (untreated) and samples during therapy.
Data are provided as medians (ranges).
Table 5.
Results of External Quality Assurance dried blood spot samples (n = 7).