Biomedical Engineering Reference
In-Depth Information
3
Chromatographic Separation of Antidepressants
by LC-MS(/MS)
LC is a very versatile technique that allows separation of nonvolatile, thermolabile
and high polar analytes, without the need to perform derivatization processes. LC
coupled to traditional detectors such as UV requires separation of interferences
from the analyte of interest, and between them when several analytes are included
in the analytical method; this is usually traduced in long chromatographic run times.
The hyphenation of the LC system to the MS significantly reduced the chromato-
graphic run, as there is no need for complete chromatographic resolution of the
analytes included in the method. However, as previously mentioned, although
endogenous interferences are not observed when analyte specific masses ( m/z ) or
transitions are monitored, they can cause ion suppression or enhancement if they
coelute with the analyte(s) of interest. Therefore, efficient chromatographic separa-
tion can minimize matrix effects and increase precision and accuracy of the assay.
In LC, several parameters such as the mobile phase, stationary phase, and
column temperature can be optimized for a specific analytical application.
3.1
Mobile Phase
Mobile phase options are quite restricted, as only volatile buffers are suitable for
LC-MS. In addition, ion-pairing agents traditionally used in LC to improve peak
shape and retention time such as trifluoroacetic acid (TFA), have shown to produce
ion suppression, and they are not recommended for LC-MS analysis [ 27 ] . Therefore,
although few applications for specific antidepressants employ TFA or its ammo-
nium salt due to sensitivity enhancement [ 48, 81- 85 ], aqueous phases in most
LC-MS analytical methods are composed by formic or acetic acid in water, or its
ammonium buffers. Although acid mobile phases are by far the most common, basic
aqueous mobile phases (pH ranging from 8 to 10) have been used for specific appli-
cations in order to increase antidepressant retention time or to couple the online SPE
elution to chromatographic analysis [ 57, 72, 86, 87 ] . Organic phase composition
was typically ACN and/or MeOH.
3.2
Stationary Phase
Antidepressant separation was usually performed by reversed-phase chromatog-
raphy with typical C8 or C18 alkyl chain columns, although phenyl [ 30, 59 ] or
cyano [ 48, 64, 84 ] stationary phases were also employed. As an exception, hydro-
philic interaction liquid chromatography (HILIC), a variation of normal phase
chromatography, was employed in two analytical methods for duloxetine [ 38 ] and
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