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.. Table 2 Recoveries of arachidonic acid in spiked samples treated by four sample pretreatment methods. The recoveries of arachidonic acid by various sample pretreatment methods in two concentration levels are listed in Table 2. As discussed in Sections 3.1 and 3.2, the elimination of phospholipids and proteins is not satisfactory by Method A (PPT), Method B (LLE), and Method C (single-mode SPE with Cleanert PEP). The matrix effect caused by phospholipids and proteins is considered as the main factor to cause the variable recoveries of arachidonic JQ1 chemical structure acid. In contrast, Method D with Cleanert MAS-M results in efficient removing of impurities and obtains a sufficient, robust recovery of arachidonic acid. For quantitation, in order to compare the absolute recoveries of various sample preparation methods, the external standard method is used so that the matrix effect on the absolute recoveries can be observed clearly by contrast with Method D and others. 3.4. Method Validation 3.4.1. Linearity, Limits of Detection, and Quantitation The calibration curve range of 10~2500?ng/mL was calculated by a regression analysis of the data to a linear fit with a weighting factor of 1/x2 E-64 for the ratio of the peak area of arachidonic acid against the nominal concentration. In this range, a linear curve was obtained with correlation coefficients (r2) better than 0.9999; the result of the linear regression analysis for arachidonic acid is y = 5.18e + 3x + 9.45e + 4 (r2 = 0.9999). The limit of R428 mw detection, defined as the concentration giving the signal to noise ratio of 3, was estimated to be 3?ng/mL for arachidonic acid. 3.4.2. Precision and Accuracy The recoveries and precision of the proposed method with Method D are summarized in Table 2. Two concentration levels at 500?ng/mL and 2?��g/mL were measured. The average recoveries are in the range of 99.38%~103.21% with RSD ranged from 5.17% to 5.34%. It is found that adding 100??L of 3% ammonium hydroxide to dilute plasma is critical to improve the recovery of arachidonic acid, because it will damage the binding of analyte and proteins in plasma by ionizing arachidonic acid. Also, the ionized arachidonic acid will be adsorbed strongly in the loading process by the resin with the combination of anion exchange and nonpolar interaction. By contrast, the recovery of arachidonic acid was insufficient when 100?��L of water was used to dilute plasma. 3.5. Applications of the Proposed Methods The optimized clean-up method using Cleanert MAS-M plate coupled with LC-MS/MS was applied to analyze free arachidonic acid in human plasma samples obtained from local hospital. The results are listed in Table 3. Table 3 Free arachidonic acid in some human plasma samples. The method was also applied in medical research center of local hospital to determine more than 100 actual plasma samples from patients of coronary heart disease and healthy people.
