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By Amanda Rigdon
• Quantify 29 drug compounds from four drug classes in a
fast, 5.5-minute analysis.
• Ultra Biphenyl column separates isobaric compounds for
more definitive results.
• Highly reproducible retention times reduce down-
time and reanalysis.
As demand for therapeutic drug monitoring rises, laboratories are
under increased pressure to implement streamlined, cost-effective
testing procedures. As with any high-volume application, the meth-
ods developed for therapeutic drug monitoring must be fast, robust,
and easy to implement. Methods that can be used to quantify a wide
variety of drug chemistries from a single analysis are particularly ben-
eficial, as they reduce costs and save time. The objective of this work
was to develop a fast, robust LC-MS/MS method for the quantification
of 29 therapeutic drugs and metabolites in urine from several drug
classes including opiates, benzodiazepines, tricyclic antidepressants,
and anticonvulsants. Results from this partial validation indicate that
the method used here produces good linearity, accuracy, and preci-
sion for most of the drugs tested in a fast, 5.5-minute analysis.
The method employed here uses a Shimadzu UFLC
XR
HPLC coupled
to an AB SCIEX API 4000 MS/MS and a 5 µm Ultra Biphenyl (100 mm x
2.1 mm, cat.# 9109512) analytical column with a matching guard
column (cat.# 910950212). The Biphenyl column was chosen for this
work because of its versatility; it combines the performance of a
traditional alkyl (e.g., C18) column with that of a phenyl column, and
it offers excellent retention of both polar and nonpolar compounds.
The adaptability of the Biphenyl phase makes it particularly useful for
methods developed to analyze drugs from multiple classes. Matrix
standards and samples were prepared using dilute-and-shoot meth-
odology as described in Figure 1.
Linear Range and Sensitivity
To evaluate linearity and sensitivity, an 11-point calibration curve cover-
ing a concentration range of 1-1,000 ng/mL was prepared in matrix.
Calibration curves for each compound were built from triplicate injec-
tions using either a linear or quadratic equation, depending on the
Fast, Robust LC-MS/MSMethod for Quantification of Multiple Therapeutic
Drug Classes Using an Ultra Biphenyl Column
Compound Name
LOQ
(ng/mL)
Linearity
(r)
%Accuracy at
LOQ
%CV at
LOQ
S/N at
LOQ
Morphine
5.0
0.9995
95
5
20
Oxymorphone
5.0
0.9994
101
2
30
Pregabalin
5.0
0.9994
95
5
40
Hydromorphone
2.5
0.9993
91
1
40
Gabapentin
10.0
0.9994
98
5
10
Codeine
10.0
0.9990
109
18
50
Oxycodone
5.0
0.9989
112
10
40
Hydrocodone
5.0
0.9997
106
2
30
7-Aminoclonazepam 2.5
0.9978
85
14
50
Tapentadol
2.5
0.9993
95
7
30
Zopiclone
10.0
0.9911
102
12
20
Norbuprenorphine
25.0
0.9955
124
19
30
7-Aminoflunitrazepam 5.0
0.9993
91
12
40
Zolpidem
1.0
0.9994
96
11
200
Citalopram
2.5
0.9996
101
7
50
Fentanyl
1.0
0.9996
97
14
70
Buprenorphine
5.0
0.9996
99
2
40
Doxepin
5.0
0.9996
100
9
90
Paroxetine
5.0
0.9994
88
2
100
Promethazine
1.0
0.9997
94
12
30
Nortriptyline
1.0
0.9990
101
8
50
Amitriptyline
5.0
0.9995
92
7
100
EDDP
5.0
0.9997
91
4
200
Lorazepam
5.0
0.9994
99
13
20
Sertraline
10.0
0.9946
113
23
40
Methadone
1.0
0.9998
101
5
3
Clonazepam
2.5
0.9997
104
6
20
Flunitrazepam
1.0
0.9996
90
9
10
Diazepam
2.5
0.9994
84
6
40
Table I:
Partial validation results for 29 therapeutic drugs and drug
metabolites.