3-Methyl-5-Isopropylhydantoin (MVH), a metabolite of DMF was selected as the biomarker to monitor occupational exposure to DMF. A rapid sample preparation method to extract MVH from hemoglobin was established utilizing Cleanert MAS-B 96-well Plate (25mg/well). 3-Methy-5-isobutylhydantion (MIH) was used as internal standard which had similar structure with MVH.
Table 1 Information of MIH and MVH
Diluted the stock solution of MVH and MIH with methanol/water(1:1,v/v) to required concentrations.
0.1g hemoglobin was weighed accurately and placed in 15mL tube. 0.25 mL of 20 μg/mL MIH and 4.75 mL of
hydrochloric acid/ acetic acid (1:1,v/v) was added into the tube and vortexed for 3 min. Then the tube was heated in
boiling water to degrade hemoglobin for 1 h. There might be bubbles emerged during the degradation process which was a spontaneous phenomenon. The tube was cooled to room temperature and brought the total volume of the sample to 5 mL by adding methanol/water (1:1,v/v). Then 200 μL of the treated sample was transferred into Cleanert® MAS-B 96-well Plate and mixed with 600 μL of 1% formic acid in acetonitrile. Finally, the treated sample was passed through the plate under vacuum for 2 min to 4 min. The fractions were collected and analyzed by LC-MS/MS.
Instrumentation: LC-MS/MS, API 4000+
LC Instrument: Shimadzu LC-20A
HPLC Column: Venusil ASB C18, 150mm×2.1 mm, 5 μm, 100 Å
Mobile Phase: Acetonitrile/0.1 % formic acid-Water, 35:65(v/v)
Flow rate: 0.2 mL/min
Injection volume: 2 μL
Ion source: ESI Positive
Scan mode: MRM
Table 2 Precursor/Product Ions of MVH and MIH
Retention time (min)
Table 3 LOD of MVH and MIH standard
(2) Recoveries and Precision
Standard solutions of MVH and MIH were added in to hemoglobin samples accurately. The recoveries of the spiked
samples were listed in Table 4.
Figure 1 Chromatogram of MVH and MIH (250ng/mL)
Figure 2 Chromatogram of hemoglobin spiked with 50mg/kg MVH
Figure 3 Chromatogram of actual hemoglobin
Table 4 Recoveries and Precisions
Mean recoveries (n=5)
Cleanert MAS-B 96-well Plate is suitable to remove the matrix of hemoglobin which might interfere the analysis of MVH by LC-MS/MS. The usage of the 96-well plate is sufficient for high throughput sample clean-up procedure prior to LC-MS/MS. This experiment provided a solution for monitoring cumulative exposure to DMF.
Cleanert MAS-B Plate
Venusil AQ C18
2.1 mm×150 mm, 5 μm, 100 Å
96-well collection plate
2.2 mL Squaral well
96-well vacuum manifold
adapt to 96well plate
HPLC, 4 L
1.5 mL vials
1.5 mL short thread vial, amber glass, label and filling lines
1.5 mL vials caps
9 mm screw neck cap, center hole; red silicone/ white PTFE septa
φ13; 0.22 μm