Biomedical Engineering Reference
In-Depth Information
Among these, both LC-MS and LC-MS/MS approaches have been described
using different mass analyzers operating in the positive ion mode scan such as triple
stage quadrupole (TSQ) mass spectrometers [
145,
194,
226,
227,
235,
239,
240,
242-
247
] and hybrid quadrupole-linear ion trap (LTQ) [
173,
193,
218,
238,
241
]
mass spectrometers working in SRM mode as well as time-of-flight (TOF) [
195
]
and hybrid quadrupole-TOF (Q-TOF) [
236,
237
] mass spectrometers working in the
MS mode.
3.3
Chromatographic Conditions and Tamoxifen
Metabolites Separation
Since the introduction of ionization sources working at atmospheric pressure such
as ESI interface, LC-MS has become the gold standard in the field of quantitative
bioanalysis due mainly to the selectivity, sensitivity, and high-throughput detection
in LC-MS systems. However, LC-MS features depend not only on the ionization
technique and mass spectrometer unrivaled inherent selectivity, sensitivity, and
speed acquisition but are also challenged, notably in drug metabolism studies, by
the availability of stable isotope labeled (SIL) version of metabolites (see below)
and the need of efficient and adequate chromatographic resolution of multiple ana-
lytes from interfering metabolites or endogenous biological components in a mini-
mum time frame.
Reversed-phase LC (RPLC) methods using conventional, microbore [
218
] , nar-
row-bore [
194,
242
] , and short [
241
] HPLC columns have been used for the separa-
tion of tamoxifen/metabolites either under isocratic or gradient elution conditions.
Narrow-bore columns present the advantages of being solvent saving and by the
need of low sample injection (or loading) volumes. These advantages were illus-
trated by Beer et al. [
218
], who developed an analytical method for the separation
of tamoxifen, anastrozole, and letrozole under gradient of 30 ml/min of acetone in
aqueous heptafluorobutyric acid solution and volumes as low as 2 ml, from the pro-
cessed samples, were injected into the system. Furlanut et al. [
241
] used a short
analytical column for the separation of tamoxifen and two of its metabolites within
almost 8 min under isocratic conditions at flow rate of 1 mL/min. Although, the use
of conventional short columns is a simple method for shortening analytical run
times, these columns suffer from a loss in efficiency and resolution.
For enhanced throughput, fast RPLC methods using monolithic silica columns
[
238
], small size particles (3 mm) packed columns [
173,
195,
226,
235-
237,
239,
240
], ultra high pressure liquid chromatography (UHPLC) columns packed with
sub-2 m m particles [
145,
196,
227,
245,
247
] and 2.6 mm core-shell particles HPLC
columns [
246
] have been proposed for the high-throughput separation and
quanti fi cation of tamoxifen/metabolites.
Five UHPLC methods have already been described to improve speed, resolution,
and sensitivity of HPLC assays for the quantification of tamoxifen phase I as well
as phase II metabolites. These methods exclusively enabled, within run times of
