TP-076

Fast Analysis of Total Vitamin C in Plasma Using the LDTD-MS/MS Technique

Sylvain Letarte, Jonathan Rochon, Serge Auger, Jean Lacoursière and Pierre Picard
Phytronix Technologies, Québec, CANADA;

OVERVIEW METHOD RESULTS

Purpose Generic SALLE extraction Validation Test
• Total Vitamin C quantification in plasmas with LDTD-MS/MS using optimization of
To stabilize ascorbic acid, plasma samples Calibration curves ranging from 3 to 100 µg/mL (Spike with AA), QC-AA (QCL, QCM and QCH with AA) and QCM-DHAA were prepared in an AA-depleted plasma
an extraction process (plasma kept at room temperature/exposed to light for 4 days). Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The
were spiked with a H2SO4 solution (0.2 N) at a
Method peak area against the internal standard (IS) ratio was used to normalize the signal.
ratio of 1:1. The samples were transferred into
• Plasma stabilization with H2SO4 Linearity Table 2
barcoded tubes, then read, and scanned by a
• Salt Assisted Liquid-Liquid Extraction (SALLE) The calibration curves were plotted using the peak area ratio and Inter-run calibration curve correlation
liquid handler (Figure 3) where an automatic
• Samples dried and analyzed by LDTD-MS/MS the nominal concentration of standards. For the linearity test, the coefficients
batch file was created. The liquid handler following acceptance criteria was used:
Quantification Vitamin C
mixed 40 µL stabilized plasma and 20 µL Curve 1 0.99986
• Linearity: r2> 0.99 over the calibration range Curve 2 0.99981
internal standard solution (Ascorbic acid-13C6, • Linear regression (r) must be ≥ 0.995
• The inter-run precision and accuracy result ranged between 3.4 and 6.0% and Curve 3 0.99885
50 µg/mL in H2SO4 (0.2N)). Then 4.5 µL of Curve 4 0.99972
between 96.2% and 101.6%, respectively. Table 2 shows the inter-run correlation coefficients for Vitamin C.
plasma/IS, 30 µL extraction buffer (KH2PO4, Curve 5 0.99785
• Samples analyzed with a runtime of 9 seconds using LDTD-MS/MS system Figure 3 Azeo: Automated extraction
Values greater than 0.999 are obtained. Figure 6 shows a typical
1mM) and 300 µL acetonitrile are mixed. After calibration curve result for Vitamin C. Figure 6 Vitamin C calibration curve
system
centrifugation, 4 µL of the upper layer phase
QCM-DHAA Precision and Accuracy Table 4
were spotted onto a LazWell96 plate and Inter-run Precision and Accuracy of
DHAA was spiked at 50 Table 3 For the inter-run precision and
evaporated to dryness before analysis by
INTRODUCTION LDTD-MS/MS.
µg/mL and analyzed as a
QC. Table 3 shows the
Inter-run Precision and Accuracy of
Vitamin C (DHAA)
accuracy experiment, each QC was
analyzed in sextuplicate, on five
Vitamin C (AA)
Vitamin C QC-L QC-M QC-H
inter-run precision and different days. Table 4 shows the Conc (µg/mL) 3.0 50.0 75.0
Vitamin C (Ascorbic acid, AA) is an important vitamin involved in many physiological accuracy results for
Vitamin C QC-M (DHAA)
inter-run precision and accuracy N 30 30 30
Conc (µg/mL) 50.0
activities in living organisms such as the synthesis of collagen, acts as antioxidant, Vitamin C. The obtained N 6 results for Vitamin C (AA). The
Mean (µg/mL) 2.9 49.7 76.2
metabolism and synthesis of adrenaline, and prevention and treatment of scurvy and Instrumentation %CV was below 15% and obtained %CV was below 15% and
%CV 6.0 3.6 3.4
Mean (µg/mL) 42.8 %Nom 96.2 99.3 101.6
cold. In the presence of moisture, air, heat, light or oxygen, ascorbic acid transforms into • Ion source: Phytronix Luxon S-960 Ion Source the accuracy was within %CV 2.8 the accuracy was within 15% of the
its oxidative form, dehydroascorbic acid (DHAA). Total Vitamin C concentration in a • Mass spectrometer: Sciex, Q-Trap System 5500 15% of the nominal value. %Nom 85.6 nominal value.
plasma sample is defined as the sum of AA and DHAA.
Stability
Luxon Parameters MS Parameters Table 1 MRM transitions parameters Wet stability Table 5 Wet and Dry stability of Vitamin C
Our goal for this presentation is to develop a method for the quantification of total • Laser power pattern: • APCI (-) Following the extraction, sample extracts are kept at 4°C in closed containers. After 1
vitamin C in plasma in a single operation using the LDTD-MS/MS Technology. - Increase laser power to • Curtain: 10 Q1 Q3 CE day, sample extracts are spotted on a LazWell™ plate, dried and analyzed. Precision Dry stability (1 hour / RT) Wet stability (1 day / 4°C)
Compound QC QC-L QC-M QC-H QC-L QC-M QC-H
45% in 6 sec • CAD: 8 (Da) (Da) (V) and accuracy of QC samples are reported in Table 5. All the results are within the Conc (µg/mL) 3 50 75 3 50 75
- Hold 2 seconds •Time: 20 msec Vitamin C (Qual) 173 99 -10 acceptable criteria range for 1 day at 4°C. N 6 6 6 6 6 6
- Decrease laser power to • MRM mode Vitamin C (Quan) 173 113 -15 Dry stability Mean (µg/mL) 3.1 49.4 76.6 3.2 49.8 80.6
LUXON Ionization Source: 0% IS 179 103 -10 Extracted samples are spotted onto a LazWell™ plate, dried and kept at room %CV 3.4 2.5 1.2 6.3 3.2 2.9
• Carrier gas flow: 3 L/min (Air) temperature for 1 hour before analysis. The precision and accuracy results of QC %Nom 101.7 98.7 102.1 105.0 99.6 107.5
The Luxon Ion Source (Figure 1) is the second-generation
samples are reported in Table 5. All the results are within the acceptable criteria
sample introduction and ionization source based on the range for 1 hour at room temperature.
LDTD technology for mass spectrometry. The Luxon Ion Total Vitamin C Table 6 Matrix effect evaluation for Vitamin C
Source uses a Fiber-Coupled Laser Diode (Figure 2) to Total vitamin C is calculated as the sum of DHAA (Figure 4) and AA (Figure 5). Matrix effect Vitamin C M1 M2 M3 M4 M5 M6
Traditional methods use a reduction agent (DTT) for the transformation of DHAA to Ascorbic acids are spiked in six (6) different AA-depleted plasma samples at QC-M Conc (µg/mL) 50.0 50.0 50.0 50.0 50.0 50.0
obtain unmatchable thermal uniformity giving more N 3 3 3 3 3 3
level and stabilized. After extraction, concentrations are evaluated against a
precision, accuracy and speed. The process begins with AA during the sample preparation before analysis. The LDTD-MS/MS technology uses Mean (µg/mL) 50.3 50.7 49.4 50.6 57.1 49.9
calibration curve. Replicate extractions are deposited onto a LazWell™ plate and
dry samples which are rapidly evaporated using indirect the APCI ionization mode. Under this condition, a complete transformation of ascorbic dried before analysis. The precision and accuracy criteria are used. Results are %CV 1.1 0.7 2.0 1.8 9.1 1.2
acid (AA) into its oxidative form (DHAA) is observed. Without any additional reduction %Nom 100.5 100.9 98.7 101.3 114.3 99.8
heat. The thermally desorbed neutral molecules are Figure 1 Luxon Ion Source shown in Table 6.
carried into a corona discharge region. High-efficiency or oxidation step during the sample preparation, the total vitamin C content in plasma
protonation and strong resistance to ionic suppression samples was quantified using DHAA’s transition (173->113). CONCLUSION
characterize this type of ionization and is the result of
the absence of solvent and mobile phase. This thermal • Efficient Protein precipitation is used to extract the Vitamin C
desorption process yields high-intensity molecular ion • No additional reduction step needed.
signal in less than 1 second sample-to-sample and • High-throughput analysis using Luxon-MS/MS
allows working with very small volumes. Figure 2 Schematic of the • Linearity, precision, accuracy, stability and matrix effect are within the acceptance criteria for all compounds
Luxon Ion Source • Sample-to-sample analysis of 9 seconds
Figure 4 Dehydroascorbic acid (DHAA) Figure 5 Ascorbic acid (AA)
For Research Use Only. Not for use in diagnostic procedures.