Page 109 - Advances in Forensic Applications of Mass Spectrometry - Jehuda Yinon

1522_C02.fm Page 94 Wednesday, November 12, 2003 9:36 AM

trazepam in blood or plasma. The comparison of ionization response showed
that APCI gave a signal 7 to 30 times stronger than ESI for all compounds
except 7-aminoflunitrazepam. After solid-phase extraction on C18 car-
tridges, the drugs were separated on a C18 column in ACN–ammonium
formate buffer (pH 3.0) and detected with APCI/MS. For each compound,
a protonated quasi-molecular ion was monitored. The LODs ranged from
0.2 to 1 mg/l. This method has been applied in routine toxicological case-
7
work. LeBeau et al. determined flunitrazepam, 7-aminoflunitrazepam, and
N-desmethylflunitrazepam in blood and urine after SPE in a mixed-phase
129
cartridge. A LC/ESI/MS/MS (ion trap) procedure was applied. Protonated
quasi-molecular ions of drugs involved were monitored and the identity was
confirmed by fragmentation. The LODs in blood were from 0.5 to 1 mg/l. An
ion trap LC/ESI/MS/MS was also used for determination of flunitrazepam
130
in serum by Darius et al. The drug was extracted with tertiary butylmethyl
ether and separated on C18 column in ACN–water mobile phase. Product
ions of flunitrazepam and internal standard (clonazepam) were monitored.
The LOD was 0.2 mg/l. Unfortunately, the metabolites, particularly 7-ami-
noflunitrazepam, were not determined. Yuan et al. described an automated
in-tube solid-phase microextraction combined with LC/ESI/MS for determi-
nation of diazepam, nordiazepam, temazepam, oxazepam, 7-aminofluni-
trazepam, and N-desmethylflunitrazepam in serum and urine. 131
Flunitrazepam itself was not included in this study. The isolation technique
used allowed solvent-free, automatic extraction in 15 min for each consecu-
tive sample. The extraction procedure was optimized, using six various
extraction capillaries and various extraction conditions. The drugs were
detected with LC/ESI/MS (full scan m/z 100 to 400) and SIM. The LODs of
0.02 to 2 mg/l were achieved. Two drawbacks of the procedure are relatively
low recovery in serum (below 50%) and peak broadening caused by auto-
matic desorption. A LC/APCI/MS/MS (ion trap) procedure was applied for
determination of flunitrazepam, 7-aminoflunitrazepam and N-desmeth-
ylflunitrazepam in plasma after SPE on Oasis MCX cartridges by Kollroser
132
et al. The analytical recoveries were above 90% for all compounds; the
limits of detection were 0.25 to 2 mg/l.
Midazolam is a short-acting benzodiazepine used for induction of anes-
thesia that may also be abused as an incapacitating agent. This drug and its
active hydroxylated metabolite were extracted from serum with ether–isopro-
panol (98:2) at alkaline pH and separated on ODS column (Nucleosil C18,
150 ¥ 1 mm). The drugs were determined with ESI/LC/MS in SIM mode.
Protonated quasi-molecular ions and fragments of both compounds were
133
monitored. The LOQ for both compounds was 0.5 mg/l. In another study,
midazolam and hydroxymidazolam, as well as triazolam and hydroxytriaz-
olam used as internal standards, were extracted from plasma with Oasis HLB

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