43 LC/MS/MS


This method was run using a Kinetex 2.6 µm core-shell Biphenyl HPLC/UHPLC column (Phenomenex). This superficially porous column offers improved efficiency in comparison to fully porous columns and the selectivity of the biphenyl ring structure is a great choice for pain panel methods that include opiates, benzos and other drugs containing aromatic moieties. The resulting LC/MS/MS method is outlined below and the resulting chromatogram is depicted in Figure 13.


LC/MS/MS Method Dimensions: 50 x 3.0 mm


Mobile Phase: A: 0.1% Formic acid in water B: 0.1% Formic acid in methanol


Flow Rate: 0.7 mL/min Gradient: Time (min)


0.00 2.50 3.50 3.51 5.00


Temperature: Ambient Detection: MS/MS, ESI+ (4000 QTRAP®


Injection: 10 µL


Comparison of Pretreatments: Acidic Pretreatments While the acidic pretreatments of HClO4


% B 100


100 100 10 10


Figure 13: Representative Chromatogram of Basic Compounds [1].


, SCIEX)


and


TCA produced the clearest supernatants upon aqueous dilution (Figure 11), they also produced the poorest recovery (Figures 14, 15 and 16). This could possibly be explained by solubility or stability issues. Since the majority of the compounds in this suite are hydrophobic and basic it is plausible to suspect that the analytes themselves were not miscible in the very polar acidic solution which forced co-precipitation along with the proteins.


While acidic precipitation yielded generally the lowest recoveries, it is important to note that the recovery of some compounds were close to the optimal precipitating solvent. The compounds


Figure 15: Comparison of the effects of various pretreatment options on Codeine (peak 1) and Hydrocodone (peak 2) Chromatograms are overlaid with time shift to provide clarity [1].


Figure 14: Comparison of the effects of various pretreatment options on amphetamine. Chromatograms are overlaid with time shift to provide clarity [1].


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