42 February / March 2016
Figure 9: ACN:MeOH precipitates pre and post dilution [1].
sample resulting in the cloudy sample and/ or the water addition caused many of the particulates (and possibly analytes) to fall out of the previously all organic solution.
Figure 10. ZnSO4 ACN precipitate post dilution [1].
Based on the lack of turbidity observed with the acidic precipitated samples post dilution (Figure 11), it could suggest that formic acid is at least partially responsible for the cloudiness. However, it is also possible that the
same matrix components that caused the turbidity in the organic precipitates were not present in the acidified precipitation since they may not have been miscible in the acidic solution and thus were never there to be crashed out in the first place.
Solid phase extraction
Table 1. Compounds in pain panel [1].
Figure 11: 6% Perchloric acid precipitate post dilution (left) and 10% Trichloroacetic acid precipitate post dilution (right) [1].
Table 1 summarises the suite of compounds employed in this study that range from polar bases (opiates) to hydrophobic neutrals (benzodiazepines). Because of the neutral and basic drugs in this panel, a polymeric mixed mode cation-exchange SPE cartridge (Strata-X-C, Phenomenex) was chosen to utilise both its hydrophobic retention as well as its
cation-exchange capability. The SPE protocol and pretreatment use a 0.1% formic acid wash followed by a 30% organic wash. The formic acid wash keeps analytes protonated and helps remove any lightly bound polar and acid interferences while the 30% methanol wash is strong enough to remove any lightly bound moderately hydrophobic interferences, but not too strong such that it would compromise the recovery of non-ionic bases such as the benzodiazepines. The elution scheme of Ethyl Acetate:IPA:Ammonium hydroxide (7:2:1) v/v work together to disrupt hydrophobic, polar and ionic interactions ensuring complete recovery is achieved by the elution solvent. These are optimised elution conditions as this percentage of ethyl acetate is strong enough to dislodge hydrophobic analytes, but is not strong enough to elute things like phospholipids and fatty acids present from the matrix. The final elution solvents for each pretreatment step are visually compared in Figure 12.
SPE Procedure:
Pretreatment: Add 100 µL 5% (w/v) ZnSO4 to 0.5 mL whole blood (with EDTA preservative) in a glass tube and vortex for 3-5 seconds. Add 1.5 mL chilled (~0o
C) 90:10 ACN/
MeOH while vortexing. Centrifuge the samples at 6,000 rpm for 10 minutes and transfer the supernatant to a new glass tube. Add 4 mL of aqueous 0.1% formic acid to the supernatant to acidify and dilute the mixture. The sample is now ready for SPE.
Condition: 1 mL Methanol Equilibrate: 1 mL Water Wash 1: 1 mL 0.1% Formic acid in water Wash 2: 1 mL 30% Methanol in water Dry: 3 to 4 minutes at high vacuum (~10” Hg) Elute: 2x 500 µL (2 aliquots) of 500 µL) Ethyl acetate:Isopropanol:Ammonium hydroxide (7:2:1)
Dry Down: Evaporate to dryness under nitrogen at 40-45o
C
Reconstitute: With 500 µL of 85:15 (A:B) of LC mobile phase
Figure 12: Final Elution for each pretreatment option [1].
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