45 ZnSO4 Pretreatment + ACN The ZnSO4 and acetonitrile combination
yielded the best results for Benzoylecognine and Amphetamines (Figures 14 and 16). While the strictly organic pretreatment yielded slightly better recoveries for the hydrocodone and codeine, they also performed adequately with ZnSO4
in place
of the osmotic breakdown. A poor performer under these conditions was EDDP. A Log D value of 2.15 at pH7 implies that it was too hydrophobic to be efficiently extracted by the ACN even in the presence of ZnSO4
,
and required the addition of 10% methanol (v/v) to achieve acceptable recovery.
ZnSO4 MeOH
Pretreatment + 90:10 (v/v) ACN/
Based on what was learned in the above organic pretreatments, this method was further optimised to improve the recovery of the opiates by using ZnSO4
in combination
with 90:10 (v/v) ACN/MeOH. Overall this pretreatment produced the most consistent results for many of the compounds in the suite (Table 3). This solvent cocktail proves to be most effective as it provides a diverse set of modifications. The ZnSO4
helps lyse
the cells, lowering pH and decreasing Log D values of ionisable compounds. The use of a protic solvent like methanol helps to solubilise stubborn hydrophobic compounds in the presence of the more polar protein precipitating ACN.
Other Analytes of Note:
MDMA (Figure 19) and Tramadol (Figure 20) display relatively similar responses in all of the precipitating solvents. Their polar nature at acidic and neutral pH (Table 2) support the claim that these compounds are well solubilised by any of these precipitating reagents and maybe offer a benchmark for compounds of similar chemical structure.
Conclusion:
While there is no one pretreatment option that yields the best results for each class of compounds, in this study it is shown that a very effective pretreatment using ZnSO4
Figure 19: Comparison of the effects of various pretreatment options on MDMA, Chromatograms are overlaid with time shift to provide clarity [1].
and
90:10 (v/v) ACN/MeOH has been developed for most compounds that are often found in a large pain panel suite representative of a typical application in forensic toxicology. By successfully preparing samples for a mixed mode cation-exchange SPE cartridge prior to LC/MS/MS analysis, column lifetime is
preserved and system maintenance is abated while also eliminating other downstream chromatographic difficulties.
One of the pretreatment options not investigated in this study is a technique that uses dilution in buffer followed by physically denaturing sample via sonication. The
promises of such a method are elaborated on in (Chen et al., 1992) [3], where it is demonstrated that using this pretreatment for a range of acidic, basic and neutral drugs extracted on a similar mixed mode cation- exchange resin yielded the best recoveries in comparison to the other techniques
Figure 17: Comparison of the effects of various pretreatment options on Nordiazepam. Chromatograms are overlaid with time shift to provide clarity [1].
Figure 18: Comparison of the effects of various pretreatment options on EDDP, Chromatograms are overlaid with time shift to provide clarity [1].
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