Comprehensive Study of SLE as a Sample. Preparation Tool for Bioanalysis

Transcription

1 Comprehensive Study of SLE as a Sample Preparation Tool for Bioanalysis Wan Wang, Warren Chen, Jerry Wang Bonna-Agela Technologies 179 Southern Street, West TEDA, Tianjin, China Abstract A simple, fast, efficient and high through-put sample preparation method is necessary to remove the interferences from plasma samples prior to LC-MS/MS in bioanalysis. This paper describes a comparison study of four sample preparation methods, LLE, PPT, RP-SPE and SLE, with spiked plasma. Dexamethasone is used in the study. The results showed that the responses of SLE are about five times higher than that of others. Keywords: SLE plate, sample preparation, plasma Introduction A vast number and increased diversity of molecular entities of drug candidates arise new challenges to bioanalysis particularly in the field of drug metabolism and pharmacokinetic (DMPK) studies. It becomes a criteria in the development of sample pretreatment method that the method should be simple, fast, and efficient with high throughput. LC MS/MS has well been wide accepted in DMPK studies. One of the main challenges is to remove interferences from bio samples such as plasma. Protein precipitation (PPT) with organic solvents such as acetonitrile (ACN), liquid liquid extraction (LLE) and SPE are commonly used techniques to prepare plasma samples However, It is clear that those traditional techniques are insufficient to remove proteins and phospholipids in plasma. Those methods share some common disadvantages when filtering out phospholipids from plasma. The residues of phospholipids in plasma will significantly affect the reliability of results of LC MS /MS analysis, which is most commonly used today for profiling a drug and its metabolites. Removing both phospholipids and proteins is the key issue to the effectiveness of a clean-up procedure for plasma samples. Recently, diatomaceous earth or diatomite-like substrate have draw many attentions of scientists for bio sample preparation. The porous and channel structures of diatomite offer excellent dispersive properties to trap large molecules such as phospholipids. When a plasma sample passes the treated diatomite material, it will spread and penetrate into the pores, to

2 form an aqueous film on the surface of the packing material. When a water-immiscible liquid is passing through the diatomite, LLE occurs on the solid support. The procedure is called supported liquid extraction (SLE). Thus, analytes will be extracted while lipids are retained. It is believed that the acidic property of the hydroxyl groups and metal centers on the substrate contribute to the retention of phospholipids via the interaction with its chorine and phosphate groups. In this study, we have compared LLE, PPT, RP-SPE and SLE methods to treat dexamethasone in plasma prior to LC-MS/MS. The results showed that the responses of SLE are about five times higher than rest methods. Materials and Methods Materials Cleanert protein precipitation plates (2 ml/well) were used for protein precipitayion. Oasis HLB plates (30mg/well) were used for solid phase extraction and Cleanert SLE plates (200 mg/well) were utilized for solid supported extraction. All chemicals were in analytical grade. Instrumentation Agilent 1200 HPLC system coupled with API MS detector. Column:Venusil ASB C18 ( mm, 5μm) from Bonna-Agela Column Temperature:25 Mobile phase:38% acetonitrile, 62% 0.01 mol/l ammonium acetate solution (containing 0.1% formic acid) Flow rate:0.2 ml/min Injection Volume:5 μl ESI(+),MRM: m/z 496/184(phospholipids), m/z 393.4/373.3(Dexamethasone) Table1. The parameter of API4000+ MS detector analytes DP CE CXP EP IS TEM GS1 GS2 CUR phospholipids 63 V 20 V 25 V 10 V V Pa 50 Pa 13 Pa Dexamethasone 50 V 14 V 9 V 10 V V Pa 60 Pa 12 Pa Sample preparation Spik the plasma samples with proper concentration of dexamethasone standard solution and then vortex them. The concentration of dexamethasone in plasma were 10 ng/ml, 100 ng/ml, 1000 ng/ml respectively. LLE method: Add 50 μl plasma and 250 μl acetonitrile to a tube, vortex and centrifuge for 10 min, and then take the supernatant and dry with nitrogen, re-dissolved the sample with 200μL mobile phase.

3 Protein precipitation method: Put the protein precipitation plate and collection plate into the vacuum device. Add 50 μl plasma and 250 μl acetonitrile to each well rapidly. Then hold for 3 minutes and blow the liquid in to the collection plate and further to dryness. The residues are re-dissolved with 200 μl mobile phase. SPE method 1). Activated HLB plate with 1 ml MeOH followed by 1 ml Water; 2). Loaded 50 µl sample diluted with 50 µl water; 3). Washed columns with 1mL Water-Methanol (8:2); 4). Elute sample with 1mL ACN-Methanol (9:1). SLE method 1). Add 50 µl plasma diluted by 50 µl water to Cleanert SLE plate 2). Load sample solution, stands for 10min; 3). 1.5 ml MTBE elution in 2 times. Blow the eluate to dryness with nitrogen, re-dissolved the sample with 200 μl mobile phase. Result and Discussions The comparison results of various sample preparation methods are summarized in Table 2. It is clear that SLE method enhances the response of the analytes by factors of four- to six-times significantly. Furthermore, the amount of the residues of phospholipids after SLE is only 1/100 to 1/50 less than that of remained in the sample with PPT, LLE and SPE methods (Fig 3 to Fig 4). Table 2. Comparison results of LLE,PPT, RP-SPE and SLE Level PPT LLE RP-SPE SLE (ng/ml) E E E E E E E E+04

4 Fig.1 the chromatogram of Dexamethasone in the sample treated with HLB Fig.2 the chromatogram of Dexamethasone in the sample treated with SLE Fig.3 The Chromatogram of phospholipids in the sample treated by SPE Fig.4 The Chromatogram of phospholipids in the sample treated by SLE It is clear that the method of protein precipitation and RP-SPE are not able to remove phospholipids effectively, causing serious matrix interference while the samples are treated by SLE contain much less phospholipids, avoiding matrix effect significantly. Conclusions SLE methods described provide a sufficient pretreatment solution for plasma samples in DMPK studies. The results demonstrated in this paper shown that diatomaceous earth or diatomite-like substrate applied for SLE maybe a suitable material for

5 removing interferences from plasma samples efficiently.