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Abstract Introduction
Vitamin C (Ascorbic acid, AA) is an important vitamin involved in many physiological activities in living organisms such as the synthesis of collagen, acts as antioxidant, metabolism and synthesis of adrenaline, and prevention and treatment of scurvy and cold. In the presence of moisture, air, heat, light or oxygen, ascorbic acid transforms into its oxidative form, dehydroascorbic acid (DHAA). Total Vitamin C concentration in a plasma sample is defined as the sum of AA and DHAA.
Objectives
Our goal for this presentation is to develop a method for the quantification of total vitamin C in plasma in a single operation, using the LDTD-MS/MS Technology at 8 seconds per samples, instead of HPLC method at 7 minutes per sample using reduction into the sample preparation.
Methods
To stabilize ascorbic acid, plasma samples were spiked with a H2SO4 solution (0.2 N) at a ratio of 1:1. The samples were transferred into barcoded tubes, then read, and scanned by a liquid handler where an automatic batch file was created. The liquid handler mixed the stabilized plasma, the internal standard solution, the extraction buffer, and acetonitrile. After centrifugation, 4 µL of the upper layer phase were spotted onto a LazWell96 plate and evaporated to dryness before analysis by LDTD-MS/MS.
LDTD was used with a power ramp of 6 seconds to 45% followed by a 2-second hold. Air is used as carrier gas at 3 L/min. The mass spectrometer was operated in negative MRM mode.
Result
Total vitamin C is calculated as the sum of DHAA and AA. Traditional methods use a reduction agent (DTT) for the transformation of DHAA to AA during the sample preparation before HPLC analysis. The LDTD-MS/MS technology uses the APCI ionisation mode. Under this condition, a complete transformation of ascorbic acid (AA) into its oxidative form (DHAA) is observed. Without any additional reduction or oxidation step during the sample preparation, the total vitamin C content in plasma samples was quantified using DHAA’s MRM transition (173->113).
Calibration curves ranging from 1 to 100 µg/mL and QCs were prepared in an AA-depleted plasma (plasma kept at room temperature/exposed to light for 4 days). Replicate extraction samples were deposited onto a LazWell plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.
The calibration curves were plotted using the peak area ratio and the nominal concentration of the standards. The inter-day correlation coefficients for vitamin C show values greater than 0.999. For the inter-run precision and accuracy experiment, each QCs are analyzed in sextuplicate, on five different days. The inter-run precision and accuracy results obtained for vitamin C ranged between 3.4% and 6.0%, and between 96.2% and 101.6%, respectively.
Following the extraction, sample extracts are kept at 4°C in closed containers to evaluate the wet stability of sample extracts. After 1 day, sample extracts were spotted onto a LazWell plate, dried and analyzed. For the dry stability, extracted samples are spotted onto a LazWell plate, dried and kept at room temperature for 1 hour before analysis. Precision and accuracy of QC samples are within the acceptance criteria (Precision ≤15% CV and accuracy 100±15% of nominal value) for 1 day at 4°C (wet stability) and 1 hour at room temperature (dry stability).
Conclusion
Total Vitamin C quantification in Plasma using LDTD-MS/MS without reduction step in sample preparation at 8 seconds per analysis.
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= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
