Page 17 - Advances in Forensic Applications of Mass Spectrometry - Jehuda Yinon

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full-scan GC/MS detection. Negative-ion chemical ionization (NICI) allows
markedly lowering the detection limits, 20,24,44 but this technique is not suitable
for comprehensive screening because the analytes must contain an electrone-
gative moiety and the NICI mass spectra are less informative and reproduc-
ible than EI spectra.
In conclusion, urine is still the sample of choice for comprehensive
screening for, and identification of, unknown drugs or poisons, mainly
because concentrations of drugs are relatively high in urine and the samples
can be taken noninvasively. 1,2,4,16,18,32 However, the metabolites of these
unknowns must be identified, additionally or even exclusively. In (horse)
doping control, urine is also the common sample for screening. 55

1.1.2 Sample Preparation

Suitable sample preparation is an important prerequisite for GC/MS analysis
in biosamples. It may involve cleavage of conjugates, isolation, and derivati-
zation, preceded or followed by cleanup steps. Cleavage of conjugates can be
performed by fast acid hydrolysis or by gentle but time-consuming enzymatic
hydrolysis. However, the enzymatic hydrolysis of acyl glucuronides (ester
2
glucuronides of carboxy derivatives like NSAIDs) may be hindered due to
acyl migration, an intramolecular transesterification at the hydroxy groups
56
of the glucuronic acid, which leads to b-glucuronidase-resistant derivatives.
If the analysis must be finished within a rather short time as in emergency
toxicology, it is preferable to cleave the conjugates by rapid acid hydroly-
sis. 57–62 Alkaline hydrolysis is only suitable for cleavage of ester conjugates.
However, the formation of artifacts during chemical hydrolysis must be con-
63
sidered. A compromise over both cleavage techniques is the use of a column
packed with immobilized glucuronidase/arylsulfatase. It combines the advan-
tages of both methods — the speed of acid hydrolysis and the gentle cleavage
64
of enzymatic hydrolysis. Acyl glucuronides, e.g., of acidic drugs are readily
cleaved under the conditions of extractive alkylation (alkaline pH, elevated
temperature) and need no extra cleavage step. 65–69
Isolation can be performed by liquid–liquid extraction (LLE) at a pH at
which the analyte is nonionized or by solid-phase extraction (SPE) preceded
or followed by cleanup steps. Sample pretreatment for SPE depends on the
sample type: whole blood and tissue (homogenates) need deproteinization
and filtration/centrifugation steps before application to the SPE columns,
whereas for urine usually a simple dilution step and/or centrifugation is
satisfactory. Whatever SPE column is used, the analyst should keep in mind
that there are large differences from batch to batch, and that comparable
sorbents from different manufacturers may also lead to different results. 70
Therefore, use of a suitable internal standard (e.g., deuterated analytes) is
recommended.

© 2004 by CRC Press LLC