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LC/MS/MS Analysis of Diuretics in Urine:
Biphenyl Column Takes Matrix
Out of the Equation
By Amanda Rigdon, Clinical/Forensic Innovations Chemist, Takeo Sakuma, AB Sciex, and Becky Wittrig, Ph.D., AB Sciex
Diuretics can mask the presence of performance enhancing
drugs since they act to dilute the urine. Because of this, the
use of diuretics has been banned by the World Anti-Doping
Agency (WADA) and diuretic compounds are included in drug
testing of athletes. Most common diuretics are highly func-
tionalized compounds, making them hydrophilic and difficult
to retain using C18 columns. Phenyl columns are a good alter-
native, as they generally have better hydrophilic retention;
however, not all phenyl columns are retentive enough to
ensure adequate resolution. While chromatographic resolution
is not always required for LC/MS/MS analyses, it is necessary when isobaric interfer-
ences are present, such as when testing for diuretics in urine.
Better Retention Reduces Matrix Interference
Ultra II® Biphenyl columns can retain hydrophilic compounds longer than other phenyl-
based stationary phases, due to the unique selectivity of the Biphenyl ligand for highly-
functionalized aromatic compounds. As shown in Figure 2, using an Ultra II® Biphenyl
column ensures complete separation of the diuretic amiloride from matrix peaks (k’ =
5). In contrast, matrix interference occurs on a Gemini® C6-Phenyl (phenyl hexyl) col-
umn (k’ = 0.6), preventing accurate quantitation. During this experiment, 10 diuretics
from 4 classes were analyzed and excellent retention and resolution were obtained for
all compounds in just 8 minutes, including re-equilibration time, on an Ultra II®
Biphenyl column (see full chromatogram and conditions at ).
Despite the power of modern LC/MS/MS instrumentation, isobaric matrix interferences
often complicate analyses involving biological samples and using a column that produces
adequate retention is critical for accurate quantitation. An Ultra II® Biphenyl column, in com-
bination with LC/MS/MS, provides fast, reliable results when analyzing diuretics in urine.
For the complete version of this condensed article, visit
• Ultra II® Biphenyl columns separate compounds that
coelute on phenyl hexyl columns.
• Improve quantitation through resolution of diuretics
from isobaric matrix interferences.
• Fast analysis time supports high sample throughput.
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