Introduction:
The TUP WCX 96-well plate utilizes patented loading technology and high-quality packing materials, providing lower background interference and higher reproducibility for experiments. It is easy to operate and minimizes the risk of cross-contamination.
Sample preparation method:
Preparation of serum samples
Mix 375μL of serum with 10μL of 0.1% formic acid in acetonitrile, 10μL of internal standard, and 375μL of 25mM ammonium acetate solution. Vortex before injection.
Materials
WCX 96-well plate (TUP Catalog No.: H07-00502, 5mg/2ml).
Serum processing procedure
- Activation:Activate with 200 μL methanol first, then activate with 200 μL 25 mM ammonium acetate, controlling the flow rate at 3-5 s/drop.
- Loading:Load all pre-treated samples, apply positive pressure, control the flow rate at 3-5 s/drop, and discard.
- Washing 1:Add 400 μL of 25 mM ammonium acetate to each well, apply positive pressure, control the flow rate at 3-5 s/drop, and discard.
- Washing 2:Add 400 μL of acetonitrile to each well, apply positive pressure, control the flow rate at 3-5 s/drop, and discard.
- Elution:Add 200 μL of 5% FA acetonitrile to each well, press down with positive pressure, control the flow rate at 3-5 s/drop, and collect.
- Nitrogen blowing and reconstitution:The collected eluate was dried under nitrogen at 35 °C, and then reconstituted with 100 μL mobile phase (initial ratio), vortexed for 2 min, transferred to a sample vial, and detected by LC-MS.
LC-MS conditions
HPLC conditions
- Specialized column, 2.1 x 100 mm, 3 μm particle size, 120 Å pore size.
- Column temperature: Set at 40℃.
- Injection volume: Set at 10 μL.
- Mobile phase conditions
A: 10 mM ammonium formate with 0.1% formic acid
B: 0.1% Formic acid, 10 mM Ammonium Formate in 90% Acetonitrile
| Total time (min) | Flow rate (μL/min) | %A | %B |
| 0 | 500 | 0 | 100 |
| 0.1 | 500 | 10 | 90 |
| 0.6 | 500 | 10 | 90 |
| 1.2 | 500 | 40 | 60 |
| 3 | 500 | 40 | 60 |
| 3.01 | 500 | 0 | 100 |
| 6.5 | 500 | 0 | 100 |
MS conditions
- Ionization mode: Electrospray ionization positive mode (ESI+)
- Detection mode: Multiple Reaction Monitoring (MRM)
- Electrospray voltage: 5500V
- Ion source temperature: 500℃
| ID | Q1 | Q2 | DP | CE |
| 3-MT 1 | 168.1 | 119.1 | 50 | 26 |
| 3-MT 2 | 168.1 | 91.1 | 50 | 33 |
| MN 1 | 180.1 | 148.1 | 90 | 25 |
| MN 2 | 180.1 | 120.1 | 90 | 25 |
| MN-d3 1 | 183.1 | 151.1 | 80 | 25 |
| MN-d3 2 | 183.1 | 123.1 | 80 | 24 |
| NMN 1 | 166.1 | 134.1 | 85 | 23 |
| NMN 2 | 166.1 | 121.1 | 85 | 24 |
| NMN-d3 1 | 169.1 | 137.1 | 80 | 25 |
| NMN-d3 2 | 169.1 | 109.1 | 45 | 19 |
| 3-MT-d4 1 | 172.1 | 123.1 | 55 | 26 |
| 3-MT-d4 2 | 172.1 | 95.1 | 55 | 35 |
| E1 | 184.1 | 166.1 | 60 | 12 |
| E2 | 184.1 | 107.1 | 80 | 26 |
| E-d6 | 190.1 | 172.1 | 60 | 14 |
| NE 1 | 152 | 107 | 60 | 24 |
| NE 2 | 152 | 135 | 60 | 17 |
| NE-d6 1 | 158.2 | 111.1 | 76 | 25 |
| NE-d6 2 | 158.2 | 140 | 76 | 20 |
| DA 1 | 154.2 | 137.1 | 40 | 15 |
| DA 2 | 154.2 | 97.1 | 90 | 26 |
| DA-d4 | 158.2 | 141.1 | 40 | 16 |
Results and Discussion
Linear Range and Sensitivity
A calibration curve was prepared using 2% BSA as the matrix with six different concentrations (10, 20, 50, 100, 200, and 400 pg/ml). The samples were processed using the aforementioned pre-treatment method and subjected to analysis. The results obtained are presented in the table below.
| ID | Regression equation | Correlation coefficient |
| MN | y=3.52x+0.167 | y=3.52x+0.167 |
| NMN | y=8.77x+0.552 | y=8.77x+0.552 |
| 3MT | y=18.13x+0.902 | y=18.13x+0.902 |
| NE | y=0.00612x+0.00721 | y=0.00612x+0.00721 |
| E | y=0.00316x+0.00164 | y=0.00316x+0.00164 |
| DA | y=0.00543x+0.00211 | y=0.00543x+0.00211 |
Recovery results
| ID | Spiked concentration
pg/ml |
Average recovery rate
（n=3） |
RSD
（n=3） |
| MN | 20 | 20 | 20 |
| 99.1% | 99.1% | 99.1% | |
| NMN | 20 | 20 | 20 |
| 98.7% | 98.7% | 98.7% | |
| 3MT | 20 | 20 | 20 |
| 99.9% | 99.9% | 99.9% | |
| NE | 20 | 20 | 20 |
| 99.3% | 99.3% | 99.3% | |
| E | 20 | 20 | 20 |
| 101.8% | 101.8% | 101.8% | |
| DA | 20 | 20 | 20 |
| 99.2% | 99.2% | 99.2% |