Fig 1.
Chemical structures of four cobalamins.
R group may be: OH (hydroxycobalamin, OHCbl), CN (cyanocobalamin, CNCbl), 5’-deoxyadenosyl (adenosylcobalamin, AdoCbl) and CH3 (methylcobalamin, MeCbl).
Fig 2.
Schematic diagram of the online SPE and chromatographic separation system: (A) valve position I at loading samples, (B) valve position II at separating samples.
Table 1.
The online SPE-HPLC-MS/MS gradient elution and valve switching program.
Table 2.
Specific MRM parameters and retention times (RT) for each analyte and internal standard (IS).
Fig 3.
MS/MS spectra of (A) OHCbl and (B) CNCbl. Representative MRM chromatograms of (C) OHCbl and (D) CNCbl in standards. Representative MRM chromatograms of (E) OHCbl and (F) CNCbl in plasma.
Table 3.
Linear equation, correlation coefficient (r) and LOD of the four cobalamins.
Table 4.
Method validation for SPE-HPLC-MS/MS analysis of four cobalamins: Precision and recovery values at different concentrations (0 ng/ml, 0.2 ng/ml, 1.0 ng/ml and 4.0 ng/ml) in quality control samples.
Table 5.
Stability of four cobalamins in rat plasma in various stresses determined by SPE-HPLC-MS/MS (n = 3).
Fig 4.
Histograms showing concentrations of OHCbl and CNCbl in PTZ-induced kindling rat model and controls (n = 8).
Each bar represents the mean value; variability is shown by standard error of mean (SEM). Asterisks represent significant difference.
Table 6.
Plasma OHCbl, CNCbl and AdoCbl concentrations (ng/ml) and scores in rat model (8 PTZ-induced kindling rats and 8 controls).