MSACL 2016 US Abstract

Cleanert HFMF¡ªA New Sample Clean-up Technique for Plasma Sample Prior to LC-MS/MS in Bio-analysis

Suzi Qin (Presenter)
Bonna-Agela Technologies

Authorship: Wan Wang(1), Suzi Qin(2), Warren Chen(3)
Bonna-Agela Technologies, Tianjin 300462, China

Short Abstract

Plasma represents a major sample form in bio-analysis. The major challenge in the preparation of plasma samples prior to LC-MS/MS is to remove both proteins and phospholipids. This article introduces a new sample pretreatment technique by utilizing Cleanert HFMF£¨hollow fiber membrane filtration£©, a hollow fiber membrane grafted with cyclic acid to filtrate proteins and phospholipids by centrifugation. A comparison study between Cleanert HFMF, PPT, LLE and SPE was carried out in terms of the efficiency to eliminate the matrix effects of proteins and phospholipids. The results showed HFMF enjoys the best capacity for removing the major impurities in plasma.

Long Abstract

Introduction

Plasma represents a major sample form in bio-analysis, particularly in the area of drug metabolism and pharmacokinetic studies, and clinical diagnosis. LC-MS/MS is mostly used technique today in profiling a drug and its metabolites[1]. Although LC-MS/MS has been accepted widely in bio-analysis it is a challenge to efficiently remove proteins and phospholipids, the major interferences to successive LC-MS/MS determination.

Current techniques involving in the preparation of blood prior to LC-MS/MS can summarized as protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE). Partial removal of proteins can be achieved with simple and fast operation when using PPT and LLE techniques. SPE method shows better results comparing to LLE and PPT, but its operation involved a multi-step procedure. These three methods mentioned above, however, share some common disadvantages to filter out phospholipids from plasma. In some cases the residues of phospholipids in pretreated plasma will affect the reliability of results of LC-MS analysis significantly.

The article introduces a new sample pretreatment technique by utilizing Cleanert HFMF (hollow fiber membrane filtration), a hollow fiber membrane grafted with cyclic acid to remove proteins and phospholipids by centrifugation. Cyclic acid grafted to polysulfone hollow fiber ultra filtration membrane by radiation to introduce carboxyl functional group into the pore in the membrane. Then the grafted hollow fiber ultra filtration membrane was packed into a centrifuge tube for easy operation.

A comparison study between PPT, LLE, SPE and Cleanert HFMF was carried out to investigate the efficiency of the elimination of proteins and phospholipids. The results indicate Cleaner HFMF emerges the best capacity in terms of removing the major impurities from plasma.

Materials and Methods

Materials and instrumentation

Formic acid and ammonia were HPLC-grade obtained from Mreda. Arachidonic acid (purity of 99%) was pursed from Sigma. Methanol, acetonitrile and ethyl acetate were HPLC-grade obtained from Honeywell. Water was LC-MS grade obtained from Honeywell. Human plasma was obtained from local hospital. Stock solution was prepared by dissolving arachidonic acid withmethanol. The stock solution was diluted with mobile phase to obtain work solution. Vortex mixer and centrifuge purchased from Bonna-Agela Technologies. Cleanert PPT, Cleanert PEP, Cleanert HFMF plates were products of Bonna-Agela Technologies.

The LC-MS/MS system was consisted of Shimadzu LC-20A HPLC with a binary pump and an auto-injector coupled with a triple quadrupole tandem mass spectrometer API4000+ (SCIEX).

Chromatographic separation was achieved on Venusil ASB C18 column (3 ¦Ìm, 150 Å, 2.1 mm¡Á50 mm, Bonna-Agela Technologies) at 30¡æ under an isocratic condition with mobile phase of acetonitrile/water (75:25, v/v) at a flow rate of 0.2 mL/min. The injection volume was 5 ¦ÌL. The target compounds were detected by the electrospray ionization interface with negative ion mode for arachidonic acid and phospholipids with positive ion mode, respectively. These MS/MS transitions utilized for arachidonic acid were m/z 303/259.1 and 303/205.1, and 496.3/184.3 for phospholipids. An UV detector at 254 nm wavelength was applied for the detection of proteins.

Sample preparation Methods

Method A- Cleanert HFMF

Arachidonic acid was used as target analyte spiked in plasma to investigate the effectiveness of Cleanert HFMF. 100¦ÌL plasma was mixed with 100¦ÌL of 3% ammonia in methanol and loaded into each well of 96-well Cleanert HFMF plate. After vortex and centrifugation at 6000 rpm, the collected fraction was analyzed by LC-MS/MS.

Method B- PPT

100 ¦ÌL plasma diluted with 100 ¦ÌL 1% formic acid was loaded into each well of Cleanert PPT plate, then precipitated protein by 400¦ÌL acetonitrile. The eluates were concentrated and reconstituted with 200 ¦ÌL mobile phase for further analysis.

Method C - LLE

100 ¦ÌL plasma diluted with 100 ¦ÌL 1% formic acid and 5 ¦ÌL methanol. LLE was achieved by 500¦ÌL ethyl acetate. The supernatants were transferred and concentrated, then reconstituted with 200 ¦ÌL mobile phase.

Method D - SPE

100 ¦ÌL plasma diluted with 100 ¦ÌL 1% formic acid and loaded into each well of Cleanert PEP plate which was pre-conditioned with 1mL methanol and 1mL water sequentially. The SPE plate was washed by methanol:water (5:95,v/v). The target compounds were eluted with 2mL of 5% ammonia in acetonitrile. The eluates were concentrated and reconstituted with 200 ¦ÌL mobile phase.

Results and Discussions

Comparison of sample pretreatment methods for removing proteins and phospholipids

The results demonstrate that in Method B proteins were not eliminated efficiently which have similar retention behaviors as arachidonic acid on reversed phase HPLC column while Method A, Method C and Method D shown good ability for removing proteins. The present of the residues of proteins in Method B may cause matrix effect on the determination of arachidonic acid. Therefore, in terms of removing proteins from plasma all the methods except Method B are applicable.

The response of phospholipids in plasma samples treated by PPT, LLE, SPE and HFMF were 1.58 e+06, 1.601e+6, 1.462e+6, 7.53e+04, respectively. PPT, LLE and SPE showed poor ability to remove phospholipids from plasma. HFMF, which grafted with cyclic acid, could provide weak cation exchange to adsorb phospholipid effectively. The results showed HFMF enjoys the best capacity for removing the major impurities in plasma.

Liner Range, LOD and recovery data

The calibration curve of arachidonic acid ranged from 10 to 2500 ng/mL with a sufficient linearity (r2=0.9999). The recoveries were in the range of 98.13% to 101.34% with RSD less than 8%. The limit of detection is 3ng/mL.

Conclusions

A novel sample treatment, hollow fiber membrane filtration, can be used for eliminating matrix effect of phospholipids and proteins prior to the analysis of arachidonic acid in plasma by LC-MS/MS. A sufficient recovery with acceptable precision is obtained. The HFMF treatment method has potential to spread in other target compounds with quick and simple feature.


References & Acknowledgements:

1. Chambers E, Wagrowski-Dieh DM, and Z. Lu, et al, ¡°Systematic and comprehensive strategy for reducing matrix effects in LC¨CMS/MS analyses,¡± J. Chromatogr. B Anal. Technol. Biomed. Life Sci, vol. 852, no. 1¨C2, pp. 22¨C34, 2007.


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