Biomedical Engineering Reference
In-Depth Information
Table 1
Comparison of offline and online SPE for the quantitation of raltegravir in human plasma
using LC-MS/MS
Of fl ine SPE
Online SPE
LC-MS
Mass Spec.
Sciex API 5000™ ESI (-)
Sciex API 4000™ ESI (-)
Source temperature
500 °C
500 °C
Column
Waters Xbridge
®
Phenyl 2 × 50 mm
None
Flowrate
0.600 mL/min
0.600 mL/min
Mobile phase
A: 0.1 % Formic acid in water
A: 0.1 % formic acid in water
B: MeCN
B: MeCN
Needle Wash
1: 0.1 % Formic acid in water/MeCN
1: TEA and EDTA in water/
DMF
2: 0.1 % Formic acid in water
2: 0.1 % formic acid in water
LC Program
Isocratic
gradient
Cycle time
3 min
100 s
SPE
Automated SPE
N/A
Symbiosis™ Pharma
Cartridge/plate
Phenomenex Strata
®
X 33 m m
Spark-Holland HySphere
®
C18 HD 7 m m
Conditioning
MeCN
MeOH
Equilibration
5 % Formic acid in water
5 % Formic acid in water
Wash
5 % Formic acid in water
5 % Formic acid in water
Elution
5 % Formic acid in water/MeCN
Mobile phase from gradient
pumps
Cartridge fl ush
N/A
MeCN/MeOH
elution solvent does not match the pH of the mobile phase or the composition of the
elution solvent is stronger than that of mobile phases, an addition step called “peak
focusing” need to be considered. Additionally, because samples injected are plasma
diluted with internal standard and certain buffer, analytes that are unstable in plasma
may not be suitable. Finally, it may have greater potential for carryover and is
difficult to troubleshoot due to the complexity of the system. In the case of raltegra-
vir, we found that the combination of triethylamine (TEA) and ethylenediaminetet-
raacetic acid (EDTA) mixture solution is the key to eliminate system carryover and
SPE cartridge carryover.
Based on the newly gained knowledge of phospholipids as interferences in bio-
analytical LC-MS/MS analysis, several specialty PPE stationary phases such as
HybridSPE
®
(Sigma-Aldrich) and Ostro™ 96-well plate (Waters), Isolute
®
PPT
+
plate (Biotage AB Corporation) have merged specifically designed for the depletion
of phospholipid interferences during sample preparation. Per manufacturer, this
type of specialty stationary phases can reduce ion-suppression through the complete
removal of phospholipids and precipitated proteins concurrently. It can serve as
generic sample cleanup procedure and minimal method development for bioanalyti-
cal applications.
