In DMPK research, there is a universal problem to develop a treatment of plasma samples with high efficiency and satisfying purification. We develop a method named solid supported liquid-liquid extraction (SLE) by taking advantage of diatomite which has a porous structure. The aqueous sample such as plasma will spread as a thin film on the hydrophilic surface of diatomite’s pore, then when a water immisicible organic solvent elutes the diatomite, the target compound will be extracted by the organic solvent while protein and phospholipids will be adsorbed by the hydrophilic diatomite’s surface.
A plasma sample containing Dexamethasone was treated by SLE. By contrast, we compared SLE with SPE and found SLE method has a better recovery for its better ability of impurities removal.
Instrument: Agilent 1200 HPLC system with API 4000+ MS detector
Column：Venusil® ASB C18 (2.1×150 mm, 3μm)
Mobile phase：38% ACN: 62% 0.01 mol/L ammonium acetate
Flow rate：0.25 mL/min
Injection volume： 5 µL
ESI(+),MRM: m/z 496/184 (phospholipids), m/z 393.4/373.3（Dexamethasone）
Table 1 the parameter of API4000+ MS detector
5 500 V
5 500 V
Sample treatment with Oasis HLB SPE column ( 30mg/1mL)
1). Activated HLB column with 1 mL MeOH, then 1 mL Water;
2). Loaded 50 µL sample diluted by 50 µL water;
3). Washed columns with 1mL Water-Methanol (8:2);
4). Elute sample with 1 mL ACN-Methanol (9:1).
Sample treatment with Cleanert® SLE column (200 mg/3 mL)
1). 50 µL plasma diluted by 50 µL water;
2). Load sample solution, stands for 10min;
3). 1.5 mL MTBE elution in 2 times.
All the elution were dried by Nitrogen blowing and reconstituted with 1mL mobile phase of LC/MS.
Results and Discussion
Figure 3 the chromatogram of Dexamethasone in the sample treated with HLB
Figure 4 the chromatogram of Dexamethasone in the sample treated with SLE
Table 2 Comparison of response of Dexamethasone between SPE and SLE
Figure 5 Chromatogram of phospholipids in the sample treated by SPE
Figure 6 Chromatogram of phospholipids in the sample treated by SLE
The SLE can remove phospholipids better than SPE with Oasis HLB, which lead to ion suppression of Dexamethasone in LC/MS analysis. So the response of Dexamethasone in the sample from SLE is higher than that from SPE.
When Cleanert® SLE applies Diatomite to achieve solid supported liquid-liquid extraction, the hydroxyl on the surface of Diatomite provides an adsorption of phospholipids so that the matrix effect is reduced remarkably. Also, the SLE is a liquid-liquid extraction actually, so the feasibility of operation is predicted to transfer traditional LLE method to SLE method in DMPK sample treatment.