essential. The typical instrumental settings and operational conditions for LC/
MS/MS systems are described below.
An instrument comprises a HPLC, often consisting of an autosampler, a
column compartment, and a binary pumping unit, and a triple quadrupole mass
spectrometer (MS/MS). The column effluents are directly diverted into the MS/
MS, serving as the detector for the HPLC. The quasi-molecular ions (e.g., a
protonated molecules), formed after the molecules in the effluents are ionized in
the ion source (Q0), are selected based on the mass-to-charge (m/z) ratios at the
first quadrupole (Q1), and are fragmented upon the collisions with molecules of
the inert gases, for example, nitrogen, helium, or argon in the second quadrupole
(Q2; also called the collision cell). The fragmented product ions are detected at
the third quadrupole (Q3). Although a tandem MS can serve as a single
quadrupole MS when the function of either Q1 or Q3 is used, sensitive and
specific quantitative methods require the use of MS/MS functions.
13.4.2.2 General Operations
HPLC Conditions Columns: Reverse phase, and particularly C18 columns
such as Waters Symmetry Shield RP18, are most frequently used (Li et al.,
2003; Soars et al., 2002; Zhang et al., 2002).
Mobile phases: There are several frequently used combinations of aqueous
and organic solvents. The options for the organic mobile phases are rather
limited, usually either acetonitrile (AcCN) or methanol (MeOH). The aqueous
mobile phases are often lightly acidified and/or buffered (Li et al., 2003; Zhang
et al., 2002). To limit the potential for ion suppression, the salt concentrations
in aqueous mobile phases are usually in the low mM range, much lower than
what typically applied in the traditional HPLC/UV methods (Fasco et al.,
1977).
Flow rate: 250 100 mL/min for a narrow bore column (with 2.0–3.0 mm
diameter).
Gradient (B%): Depending largely on the physicochemical properties of the
analytes, the following gradients, assuming a 10-min run, can be used to
initiate the method development.
General: 10% (0–1 min), 90% (4–9.5 min), and 10% (10 min).
Hydrophobic compounds: 20% (0–0.5 min), 95% (2–9.5), and 20% (10 min).
Hydrophilic compounds: 5% (0–2 min), 90% (6–9.5 min), and 5% (10 min).
The HPLC run time dictates the gradient program. If the run time is
required to be very short, a mobile phase in the isocratic mode should be
considered, with 30–50% organic solvents.
Internal standard (IS): Commercially available small molecules (MW: 200–
800), exhibiting the appropriate chromatographic properties and the potential
to be ionized in the MS, besides the structural analogs of the analytes, are in
principle applicable as internal standards.
QUANTITATIVE ANALYTICAL METHODS 433