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An Optimized High Throughput Clean-Up Method Using Mixed-Mode SPE Plate for the Analysis of Free Arachidonic Acid in Plasma by LC-MS/MS.

Wang W, Qin S, Li L, Chen X, Wang Q, Wei J - Int J Anal Chem (2015)

Bottom Line: The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r (2) = 0.9999).The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%.The limit of detection is 3 ng/mL.

View Article: PubMed Central - PubMed

Affiliation: School of Material Science and Engineering, Tianjin Polytechnic University, Tianjin 300387, China.

ABSTRACT
A high throughput sample preparation method was developed utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. Plasma was mixed with 3% aqueous ammonia and loaded into each well of 96-well plate. After washing with water and methanol sequentially, 3% of formic acid in acetonitrile was used to elute arachidonic acid. The collected fraction was injected onto a reversed phase column at 30°C with mobile phase of acetonitrile/water (70 : 30, v/v) and detected by LC-MS/MS coupled with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r (2) = 0.9999). The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%. The limit of detection is 3 ng/mL.

No MeSH data available.


Related in: MedlinePlus

Chromatograms and peak areas of phospholipids in plasma treated with various clean-up methods, where “A” = peak area of phospholipids.
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Related In: Results  -  Collection


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fig3: Chromatograms and peak areas of phospholipids in plasma treated with various clean-up methods, where “A” = peak area of phospholipids.

Mentions: It is well known that phospholipids in plasma will result in matrix effect on mass spectrometry [19, 20]. Therefore, it is necessary to remove phospholipids from the samples before injection. Although PPT is a common method for biosample preparation [22], the method is unable to remove the phospholipids from plasma efficiently. As shown in Figure 3 (PPT), the eluate obtained from Method A results in a big phospholipids peak that may influence the analysis of arachidonic acid. Although LLE is a widely used sample preparation method to extract the target compounds from aquatic samples [23], the result of Method B (Figure 3 LLE) indicates that a wide peak of phospholipids appeared. Comparing with LLE, SPE has become a popular sample preparation technique in terms of reproducibility, less usage of organic solvents, and ease of use. Moreover, SPE is very compatible with an automatic system for high throughput analysis. Arachidonic acid is a hydrophobic compound with Log Kow 7.27; in Method C, therefore, a Cleanert PEP plate packed with nonpolar polymer material was used. However, as shown in Figure 3 (SPE), a broad high peak of phospholipids still remains after SPE clean-up. It is apparent to see from Figure 3 (MAS-M) that Method D is the best one for removing phospholipids from plasma. Cleanert MAS-M plate is packed with mixed resins with nonpolar, anion exchange, and cation exchange interactions. Since the pKa of arachidonic acid is 4.77, it is retained on the plate by both anion exchange and nonpolar interactions while the phospholipids and some proteins are retained on the plate by cation exchange and nonpolar interactions under experiment conditions during sample loading. Water soluble interferences are washed out with water and the nonpolar interferences are removed by methanol. Since the arachidonic acid is adsorbed on the plate by anion interaction, there is no loss when the plate is washed by methanol. Finally, after optimizing, 600 μL of 3% formic acid in acetonitrile is applied to release arachidonic acid from the plate while phospholipids with choline groups and proteins with poly amino-groups are retained by the Cleanert MAS plate.


An Optimized High Throughput Clean-Up Method Using Mixed-Mode SPE Plate for the Analysis of Free Arachidonic Acid in Plasma by LC-MS/MS.

Wang W, Qin S, Li L, Chen X, Wang Q, Wei J - Int J Anal Chem (2015)

Chromatograms and peak areas of phospholipids in plasma treated with various clean-up methods, where “A” = peak area of phospholipids.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4383463&req=5

fig3: Chromatograms and peak areas of phospholipids in plasma treated with various clean-up methods, where “A” = peak area of phospholipids.
Mentions: It is well known that phospholipids in plasma will result in matrix effect on mass spectrometry [19, 20]. Therefore, it is necessary to remove phospholipids from the samples before injection. Although PPT is a common method for biosample preparation [22], the method is unable to remove the phospholipids from plasma efficiently. As shown in Figure 3 (PPT), the eluate obtained from Method A results in a big phospholipids peak that may influence the analysis of arachidonic acid. Although LLE is a widely used sample preparation method to extract the target compounds from aquatic samples [23], the result of Method B (Figure 3 LLE) indicates that a wide peak of phospholipids appeared. Comparing with LLE, SPE has become a popular sample preparation technique in terms of reproducibility, less usage of organic solvents, and ease of use. Moreover, SPE is very compatible with an automatic system for high throughput analysis. Arachidonic acid is a hydrophobic compound with Log Kow 7.27; in Method C, therefore, a Cleanert PEP plate packed with nonpolar polymer material was used. However, as shown in Figure 3 (SPE), a broad high peak of phospholipids still remains after SPE clean-up. It is apparent to see from Figure 3 (MAS-M) that Method D is the best one for removing phospholipids from plasma. Cleanert MAS-M plate is packed with mixed resins with nonpolar, anion exchange, and cation exchange interactions. Since the pKa of arachidonic acid is 4.77, it is retained on the plate by both anion exchange and nonpolar interactions while the phospholipids and some proteins are retained on the plate by cation exchange and nonpolar interactions under experiment conditions during sample loading. Water soluble interferences are washed out with water and the nonpolar interferences are removed by methanol. Since the arachidonic acid is adsorbed on the plate by anion interaction, there is no loss when the plate is washed by methanol. Finally, after optimizing, 600 μL of 3% formic acid in acetonitrile is applied to release arachidonic acid from the plate while phospholipids with choline groups and proteins with poly amino-groups are retained by the Cleanert MAS plate.

Bottom Line: The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r (2) = 0.9999).The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%.The limit of detection is 3 ng/mL.

View Article: PubMed Central - PubMed

Affiliation: School of Material Science and Engineering, Tianjin Polytechnic University, Tianjin 300387, China.

ABSTRACT
A high throughput sample preparation method was developed utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. Plasma was mixed with 3% aqueous ammonia and loaded into each well of 96-well plate. After washing with water and methanol sequentially, 3% of formic acid in acetonitrile was used to elute arachidonic acid. The collected fraction was injected onto a reversed phase column at 30°C with mobile phase of acetonitrile/water (70 : 30, v/v) and detected by LC-MS/MS coupled with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r (2) = 0.9999). The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%. The limit of detection is 3 ng/mL.

No MeSH data available.


Related in: MedlinePlus