Sensitive quantitation of vitamin B1 and B6 in human whole blood
Abstract
In this technical note, the combination of a rapid sample preparation procedure consisting of a protein precipitation with a robust and sensitive LC-MS/MS method using the SCIEX QTRAP 6500+ enabled accurate quantitation of vitamin B1 and B6 extracted from human whole blood. Excellent linearity was observed across clinically relevant concentrations. Low-nmol/L level sensitivity was achieved at the lowest calibrator with signal-to-noise ratios (S/N) of 769:1 for vitamin B1 at 39 nmol/L and 499:1 for vitamin B6 at 21 nmol/L. In addition, the method showed excellent precision and accuracy at low-level concentrations, demonstrating the quantitative performance of the assay.
B vitamins play an important role in energy production, brain function, and red blood cell formation. More specifically, neurotropic vitamins such as vitamin B1 (thiamine-diphosphate) and vitamin B6 (pyridoxine-5’ phosphate) are micronutrients that are crucial for energy production to support nerve function and overall brain health. As a result, accurate quantitation of these vitamin complexes is important.
Key benefits of vitamin B1 and B6 analysis from human whole blood using the QTRAP 6500+ system
Low-nmol/L level sensitivity and excellent quantitative performance: Sensitive quantitation of vitamin B1 and vitamin B6 was performed with excellent precision (5.2% for vitamin B1 and 1.8% for vitamin B6) and accuracy 99.6% for vitamin B1 and 100.8% for vitamin B6 at the lowest calibrator levels (39 nmol/L for vitamin B1 and 21 nmol/L for vitamin B6)
Rapid sample preparation: Vitamin B complexes were extracted from human whole blood samples using a protein precipitation procedure
Chromatographic separation: Optimized LC conditions enabled fast (4 minutes) chromatographic separation of vitamin B1 and vitamin B6
Excellent linearity: Calibration curves for vitamin B1 and vitamin B6 showed r2 values above 0.99 across the calibration range
Methods
Sample preparation: Vitamin B1 and vitamin B6 were extracted from human whole blood using a protein precipitation procedure. In short, 50 µL of blank human whole blood spiked with vitamin B1 and vitamin B6 at six concentration levels were added to a centrifuge tube to which 50 µL deuterated vitamin B1 and vitamin B6 internal standards were added 400 µL of a 12% (v/v) trichloroacetic acid deproteinization solution was added to the tube and vortexed for 30 seconds. The tube was immediately shaken for 30 minutes and centrifuged for 5 minutes at 10,000 x g. 200 µL of the supernatant was transferred to a vial for analysis.
Liquid chromatography: Chromatographic separation was achieved using a Phenomenex Kinetex Luna Omega Polar C18 column (100 x 2.1 mm, 1.6 µm, 00D-4748-AN). Mobile phase A was water and mobile phase B was methanol. The LC flow rate was 600 μL/min and the total run time was 5 minutes. The injection volume was 20 μL. The LC gradient program is presented in Table 1.
Results and discussion
Figure 1 shows the chromatographic separation of vitamin B1 and vitamin B6 into a control human whole blood sample at final concentration of 39.0 nmol/L for vitamin B1 and 21.0 nmol/L for vitamin B6, respectively. The 4 min gradient, in combination with the column selection and mobile phase composition, resulted in baseline separation of vitamin B1 and vitamin B6. The extracted ion chromatograms showed a S/N of 769:1 for vitamin B1 and 499:1 for vitamin B6, at the lowest matrix calibrator measured (39.0 nmol/L for vitamin B1 and 21.0 nmol/L for vitamin B6), calculated using a peak-to-peak algorithm.
Figure 2 shows the representative extracted ion chromatograms (XICs) for A) vitamin B1 and B) vitamin B6 across their respective concentration ranges (39.0-657.0 nmol/L for vitamin B1 and 21.0-361.0 nmol/L for vitamin B6). The quantitative performance of the method was investigated by injecting a series of calibrator samples spiked at concentrations ranging from 39.0 to 657.0 nmol/L for vitamin B1 and 21.0 to 361.0 nmol/L for vitamin B6, respectively.
Linearity, accuracy and precision were assessed across the calibration ranges for each of the two analytes. Figure 3 shows the calibration curves for vitamin B1 (left) and vitamin B6 (right) over the analytes’ respective calibration ranges. The plots show excellent linear responses across the calibration series, with r2 values greater than 0.99 for both analytes.
The accuracy and precision values were calculated by 3 replicates of the lowest matrix calibrators analyzed (39.0 nmol/L for vitamin B1 and 21.0 nmol/L for vitamin B6). The accuracy was 99.6% for vitamin B1 and 100.8% for vitamin B6, respectively. The precision (%CV) was 5.2% for vitamin B1 and 1.8% for vitamin B6, respectively.
Conclusion
A fast and sensitive LC-MS/MS method for the detection of vitamin B1 and B6 extracted from human whole blood samples was developed. The method demonstrated:
- Fast sample preparation which consisted of a simple protein deproteination, requiring 50 μL of human whole blood sample
- Chromatographic separation of vitamin B1 and vitamin B6
- Excellent linear responses across the calibration series consisting of 6 calibrators, with r2 values greater than 0.990 for both analytes
- High quantitation performance of the method, resulting in excellent precision (5.2% for vitamin B1 and 1.8% for vitamin B6) and accuracy 99.6% for vitamin B1 and 100.8% for vitamin B6 at the lowest calibrator levels (39 nmol/L for vitamin B1 and 21 nmol/L for vitamin B6)