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7 authors, including:
Hongyue Guo
Chengdong Xu
13 PUBLICATIONS 62 CITATIONS
SEE PROFILE
SEE PROFILE
Zachary Breitbach
Daniel W. Armstrong
SEE PROFILE
SEE PROFILE
Department of Chemistry & Biochemistry, University of Texas at Arlington, Arlington, TX 76019, USA
Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC, 88040-900, Brazil
c
Cerilliant Corp., 811 Paloma Dr., Suite A, Round Rock, TX 78665, USA
b
a r t i c l e
i n f o
Article history:
Received 4 December 2014
Received in revised form 5 August 2015
Accepted 7 August 2015
Available online 21 August 2015
Keywords:
Performance-enhancing drugs
High performance liquid chromatography
(HPLC)
Paired ion electrospray ionization (PIESI)
Mass spectrometry (MS)
Sensitivity
a b s t r a c t
The detection window for drugs of abuse, including performance-enhancing drugs, is limited by the
sensitivity of analytical methodologies. Herein, paired ion electrospray ionization mass spectrometry
(PIESI-MS) was employed for sensitive analysis of performance-enhancing drugs and drugs of abuse by
detecting their glucuronide and sulfate conjugates. The proposed approach provides enhanced sensitivity
for these drug metabolites, and overcomes the drawbacks of the less sensitive negative ion mode ESI-MS
by detecting the anionic metabolites in the positive ion mode at higher m/z where the background noise
is less. Absolute LODs down to sub-pg levels were obtained with the use of the optimal symmetrical
or unsymmetrical ion pairing reagents. One to three orders of magnitude improvement were obtained
compared to other reported methods performed in the negative ion mode. Structurally similar steroid
conjugates were chromatographically separated and detected by HPLC coupled with PIESI-MS. Finally, an
off-line solid phase extraction (SPE) protocol was successfully developed to eliminate any matrix effects
in the analysis of human urine samples.
2015 Elsevier B.V. All rights reserved.
1. Introduction
Detection times and elimination times of drugs and their
metabolites in humans are of clinical and forensic interest [1,2]. In
the case of performance-enhancing drugs, the detection window
subsequent to the administration or consumption of such drugs is
particularly relevant and can vary considerably. The pharmacokinetics and pharmacodynamics of most drugs are known, but these
are usually for highly controlled single dose experiments [37].
Further, it is well known that different classes of drugs and even different drugs within a class can have unique metabolic pathways and
therefore very different elimination half-lives [711]. In the case
of performance-enhancing drugs, several mitigating factors can
affect individual drug-metabolite elimination/lifetimes/detection
times. These include: an individuals duration of use, the dosage,
the form of the drug (e.g., salt, neutral molecule, crystal structure
if solid, particle size, diluents or excipients present), the nature
Corresponding author.
E-mail address: sec4dwa@uta.edu (D.W. Armstrong).
http://dx.doi.org/10.1016/j.ijms.2015.08.005
1387-3806/ 2015 Elsevier B.V. All rights reserved.
15
Table 1
Structures, abbreviations, and exact masses of the symmetrical and unsymmetrical ion pairing reagents used in this study.
Ion pairing reagent
Abbreviation
1,5-Pentanediyl-bis-(1butypyrrolidinium)
diuoride
C5 (bpyr)2
324.4
1,9-Nonanediyl-bis(3methylimidazolium)
diuoride
C9 (mim)2
290.3
1,3-Propanediylbis(tripropylphosphonium)
diuoride
C3 (triprp)2
362.3
1,5-Pentanediyl-bis(3benzylimidazolium)
diuoride
C5 (benzim)2
386.3
1-Butyl-1-[5-(1-tetradecyl-1pyrrolidiniumyl)pentyl]pyrrolidinium
diuoride
UDC1
464.5
N1-dodecyl-N1,N1,N5,N5,N5pentamethyl-1,5pentanediaminium
diuoride
UDC2
342.4
ionization mass spectrometry (ESI-MS) than with atmosphericpressure chemical ionization (APCI) [16]. In addition to being more
sensitive, the positive ion mode produced more abundant and diagnostic fragment ions than the negative ion [16,27]. In this work we
focus on mass spectrometry (MS) and MS/MS detection methods
that use very small amounts of specically designed and synthesized agents for the ultra-sensitive detection of anionic metabolites
in the positive ion mode. This approach is known as paired ion
electrospray ionization (PIESI) [5160]. PIESI provides a facile
approach for doing ESI-MS and ESI-MS/MS of negatively charged
analytes in the positive ion mode. Further it moves the detection
of analytes away from a higher noise, low m/z region to a relatively lower noise, high m/z region [52,60]. The ionization efciency
also appears to be enhanced in PIESI [56,59]. However, an anions
response can be quite different when using different cationic pairing agents. Thus choosing the optimal agent is important.
The goal of this work was to examine the feasibility of adapting
the PIESI approach for the sensitive detection and quantitation of
drug metabolites, specically, glucuronide and sulfate conjugates.
This is compared to direct MS and MS/MS analysis of these anionic
metabolites in the negative ion mode, as well as the commonly used
HPLCMS methodologies previously reported in the literature. The
optimized PIESI approach was then coupled to HPLC for the analysis
of selected metabolites in urine samples.
Structure
Exact mass of
the dication
2. Experimental
2.1. Reagents and chemicals
Acetonitrile (ACN), methanol and water were of HPLCMS grade
and purchased from Honeywell Burdick and Jackson (Morristown,
NJ, USA). The chemical structures and abbreviations of the symmetrical and unsymmetrical ion pairing reagents used in this study
are shown in Table 1. These ion pairing reagents were originally
synthesized in our laboratories [52,59], and were anion exchanged
from bromide to their uoride salt form prior to analysis to maximize ion pairing reagent/anion complex formation [60]. Notably,
four of our symmetrical ion pairing reagents have become commercially available from SigmaAldrich. The more recently developed
unsymmetrical ion pairing reagents were selected as they provided
enhanced detection sensitivity for some anions [59]. Ethyl--dglucuronide (EtG), ethyl sulfate (ES), morphine-3-d-glucuronide
(M3G), oxazepam glucuronide (OxaG) and 5-androsten-3-ol17-one sulfate (dehydroepiandrosterone sulfate, DHEAS) were
gifts from Cerilliant (Round Rock, TX, USA). 5-Androstan3-ol-17-one sulfate (androsterone sulfate, AS), androstadiene3-one-17-ol (boldenone sulfate, BS), 5-androstane-17-diol
(17-dihydroepiandrosterone sulfate,17-DHEAS), 5-androsten3-ol-17-one glucuronide (dehydroepiandrosterone glucuronide,
16
Table 2
Structures, abbreviations, and exact masses of the drug metabolites used in this study.
Analyte
Abbreviation
Structure
Exact mass
Ethyl--D-glucuronide
EtG
222.1
Morphine-3--D-glucuronide
M3G
461.2
Oxazepam glucuronide
OxaG
462.1
Dehydroepiandros-terone glucuronide
DHEAG
464.2
Testosterone glucuronide
TG
464.2
17
Table 2 (Continued)
Analyte
Abbreviation
Ethyl sulfate
EtS
125.0
Dehydroepiandrosterone sulfate
DHEAS
367.2
Androsteronea sulfate
AS
369.2
Boldenone sulfate
BS
365.1
17-Dihydroepiandros-terone sulfate
17-DHEAS
371.2
Testosterone sulfate
NTS
367.2
Structure
Exact mass
Androsterone is an endogenous steroid hormone with an androgenic potency of 14% that of testosterone.
2.2. Instrumental
All experiments were performed on a Thermo Finnigan HPLC
system coupled with a LXQ linear ion trap mass spectrometer
(Thermo Fishier Scientic, San Jose, CA, USA). A scheme of the
instrumental setup for the PIESI-MS detection study is shown in
Fig. 1. Briey, a carrier ow consisting of methanol and water
(67/33, v/v) was delivered by a binary LC pump at 300 L/min, while
18
Sample
LOD (pg)
C5 (bpyr)2
EtG
M3G
OxaG
DHEAG
TG
EtS
DHEAS
AS
BS
17-DHEAS
NTS
a
b
c
d
e
C3 (triprp)2
C5 (benzim)2
C9 (mim)2
SIM (m/zd )
SRM (m/ze )
SIM (m/z)
SRM (m/z)
SIM (m/z)
SRM (m/z)
SIM (m/z)
SRM (m/z)
SIM (m/z)
SRM (m/z)
500 (545.5)
1500(784.4)
180 (785.4)
400 (787.5)
190 (787.5)
6.0 (449.2)
7.5 (691.5)
8.0 (693.6)
8.0 (689.4)
19 (695.5)
15 (677.5)
37 (418.3)
130(657.4)
28 (658.3)
10 (660.5)
6.0 (660.5)
5.0 (322.2)
4.5 (214.2)
8.5 (294.3)
5.5 (294.3)
19 (294.3)
4.2 (294.3)
150 (607.3)
1100(846.4)
250 (847.3)
60 (849.3)
40 (849.3)
75 (511.3)
7.5 (753.6)
3.0 (755.6)
7.0 (751.5)
3.0 (757.5)
3.8 (739.4)
170 (385.3)
1100(385.3)
100 (385.3)
60 (385.3)
40 (385.3)
18 (227.2)
5.0 (227.2)
5.0 (483.3)
7.0 (227.2)
5.0 (227.2)
9.5 (227.2)
250 (583.4)
10,000(822.5)
1200 (823.4)
120 (825.6)
22 (825.6)
17 (487.3)
1.2 (729.5)
4.0 (731.5)
6.0 (729.5)
4.5 (733.5)
2.5 (715.5)
250 (187.2)
15,000(361.1)
2500 (361.1)
400 (361.1)
190 (361.1)
17.5 (187.2)
7.5 (459.3)
6.0 (459.3)
750 (459.3)
100 (459.3)
1000 (459.3)
400 (511.3)
1500(750.4)
1200(751.3)
600 (753.5)
750 (753.5)
200 (415.3)
5.0 (657.5)
45 (659.4)
25 (655.5)
65 (661.5)
10 (643.4)
500 (289.3)
1500(289.3)
880 (289.3)
100 (289.3)
97 (289.3)
500 (289.3)
1.2 (387.3)
3.7 (387.3)
75 (289.3)
150 (289.3)
60 (289.3)
100(221.1)
800(460.2)
250(461.1)
70 (463.2)
30 (463.2)
100(125.0)
10 (367.2)
12 (369.2)
16 (367.2)
20 (371.2)
10 (353.1)
100(202.9)
NAc
NA
70 (445.3)
30 (445.3)
NA
NA
NA
NA
NA
NA
The best LOD for each drug metabolite obtained using symmetrical ion pairing reagents is in bold type.
Times improvement of best LODs obtained using symmetrical ion pairing reagents vs. LODs obtained in the negative ion mode without using ion pairing reagents.
Not detectable.
The m/z of the analyte/ion pairing reagent complex ion monitored in SIM mode.
The m/z of the most abundant fragment ion generated from the analyte/ion pairing reagent complex in SRM mode; the precursor ion was the same as the m/z of the complex used in SIM mode.
Improvement
factorb
3
6
9
7
5
20
8
4
3
7
4
Table 3
LODs of the drug metabolites detected in the positive ion mode using symmetrical ion pairing reagents and in the negative ion mode without using ion pairing reagents.a
19
Mobile phase
Mixing
Device
Xcalibur 2.0
ESI-MS
40 M aqueous solution of
ion pairing reagent
Fig. 1. The HPLC-PIESI-MS instrumental setup. PIESI-MS detection was performed without using column. For chromatographic separations, an analytical column was placed
between the injection valve and Y-type mixing device.
Table 4
LODs of the drug metabolites detected in the positive ion mode using unsymmetrical ion pairing reagents.a
Sample
EtG
M3G
OxaG
DHEAG
TG
EtS
DHEAS
AS
BS
17-DHEAS
NTS
UDC1
UDC2
SIM (m/ze )
SRM (m/zf )
SIM (m/z)
SRM (m/z)
450 (685.6)
1100(924.7)
250 (925.7)
75 (927.7)
75 (927.7)
9.0 (589.5)
2.5 (831.7)
5.0 (833.6)
3.7 (831.7)
10 (835.7)
5.0 (817.6)
250(418.4)
200(657.5)
90 (798.6)
5.0 (660.5)
8.5 (660.5)
3.0 (462.4)
1.5 (354.5)
3.0 (354.5)
3.7 (434.4)
6.0 (434.4)
2.5 (434.4)
150 (563.5)
1100(803.6)
500 (804.6)
220 (805.5)
30 (805.5)
2.7 (467.4)
3.5 (709.6)
5.0 (711.5)
4.0 (707.6)
7.5 (713.6)
5.0 (695.5)
150 (504.4)
1000(743.7)
250 (744.7)
75 (746.8)
30 (746.8)
2.1 (408.5)
1.2 (380.4)
1.5 (380.4)
0.80 (380.4)
1.5 (380.4)
1.0 (380.4)
Improvement factor
(unsymmetrical vs.
symmetrical ion
pairing reagents)c
Total improvement
factor (PIESI-MS vs.
negative ion
mode)d
37
130
28
10
6.0
5.0
1.2
3.0
5.5
3.0
2.5
0.25
0.65
0.31
2.0
0.70
2.4
1.0
2.0
7.0
2.0
2.5
3
6
9
14
5
48
8
8
20
13
10
The best LOD for each drug metabolite obtained using unsymmetrical ion pairing reagents is in bold type.
Data were obtained from Table 3.
c
Times improvement of best LODs obtained using unsymmetrical ion pairing reagents vs. LODs obtained using symmetrical ion pairing reagents
d
Times improvement of best LODs obtained using both symmetrical and unsymmetrical ion pairing reagents in PIESI-MS vs. LODs obtained in the negative ion mode
without using ion pairing reagents.
e
The m/z of the analyte/ion pairing reagent complex ion monitored in SIM mode.
f
The m/z of the most abundant fragment ion generated from the analyte/ion pairing reagent complex was monitored in SRM mode. The precursor ion in SRM mode was
the same as the m/z used in SIM mode.
b
20
EtG/EtS
10000
Intensity (AU)
Segment 1
Segment 2
Segment 3
BS/NTS
DHEAS
5000
AS
17-DHEAS
M3G
Impurity from
OxaG
OxaG
10
DHEAG
TG
15
20
Time (min)
Fig. 2. Total ion chromatogram of separation of the eleven metabolites of PEDs by HPLC-PIESI-MS. Column: AscentisTM C18 (2.7 m, 2.1 150 mm); mobile phase A: 0.1%
formic acid in water (pH = 2.7), B: 0.1% formic acid in methanol; gradient elution conditions: 05 min, 3% B; 56 min, 330% B; 622 min, 3080% B; primary LC pump ow rate:
300 L/min; injection volume: 5 L. The separation was carried out with the outperformed IPR, C5 (bpyr)2 monitored in SIM mode with three segments (see experimental).
Segment 1
Segment 2
Segment 3
400 ng/mL
50 ng/mL
Urine blank
10
15
20
Time (min)
Fig. 3. Extracted ion chromatogram of OxaG by HPLC-PIESI-MS/MS. Column: AscentisTM C18 (2.7 m, 2.1 150 mm); mobile phase A: 0.1% formic acid in water (pH = 2.7),
B: 0.1% formic acid in methanol; isocratic elution condition: 60% B; primary LC pump ow rate: 300 L/min; injection volume: 5 L; ion pairing reagent: C5 (bpyr)2 . The m/z
transition of OxaG/C5(bpyr)2 was monitored in SRM mode in segment 1 (05 min, 785.4 658.3). The nal concentrations of OxaG in urine samples prior to injection were
40 ng/mL and 500 ng/mL respectively.
21
AS
Segment 1
Segment 2
Segment 3
Internal standard
400 ng/mL
50 ng/mL
Urine blank
10
15
20
Time (min)
Fig. 4. Extracted ion chromatogram of AS and the internal standard by HPLC-PIESI-MS/MS. Column: AscentisTM C18 (2.7 m, 2.1 150 mm); mobile phase A: 0.1% formic
acid in water (pH = 2.7), B: 0.1% formic acid in methanol; isocratic elution condition: 60% B; primary LC pump ow rate: 300 L/min; injection volume: 5 L; ion pairing
reagent: C5 (bpyr)2 . The m/z transition of AS/C5(bpyr)2 and internal standard/C5(bpyr)2 were monitored in segment 2 (512 min, 697.5 294.3) and segment 3 (1222 min,
693.6 294.3) respectively. The nal concentrations of AS in urine samples prior to injection were 50 ng/mL and 400 ng/mL respectively. The nal concentrations of internal
standard were 100 ng/mL in all these samples.
22
Table 5
Recovery results of standard drug metabolites from urine spiked at two concentration levels.
Drug metabolite
OxaGa
ASa
a
Spiked standard
Spiked standard
Concentration (ng/mL)
Recovery (%)
RSD (%)
Concentration (ng/mL)
Recovery (%)
RSD (%)
50
50
90%
107%
15%
7.9%
400
400
89%
98%
4.5%
3.5%
Table 6
Comparison of instrumental LOD (pg) of steroid glucuronides and sulfates measured by PIESI-MS method as to other HPLCMS methodologies performed in the negative ion
mode.a
Ionization mode, MS analyzer used
Scan mode
Reference
ESI, ITb
ESI, QTOFc
ESI, QTOF
API, QqQd
SSIe , IT
PIESI, IT
SRM
SRM
SRM
SRM
SIM
SIM/SRM
15300
2001000
50500
4000050000
40800
0.86.0g
[28]
[30]
[33]
[39]
[50]
Current method
a
LODs represent instrumental detection limits. For the methods using SPE to concentrate samples for analysis, the LODs have been corrected by the SPE concentration
factors.
b
Ion Trap.
c
Quadrupole coupled with Time of Flight.
d
Triple Quadrupole.
e
Sonic Spray Ionization.
f
Only the LODs for steroid glucuronides and sulfates that belong to the same categories as in this study are considered in these references.
g
The LODs for these steroid metabolites were measured in the human urine matrix under the same SPE, LC and MS conditions as the recovery study.
a natural substance in the urine of all normal individuals. For quantitation, the recovery of OxaG, AS, and the internal standard from
the SPE were determined and the relative response factors (HPLCPIESI-MS) between the internal standard and the metabolites were
found.
3. Results and discussion
3.1. Ion pairing reagents and drug metabolites
The ion pairing reagents play an essential role in the detection
limits obtained using the PIESI-MS approach. The structures for all
the tested ion pairing reagents are shown in Table 1. The symmetrical ion pairing reagents tested here (Table 1) were selected because
they gave the best performance for anion detection in previous
studies [51,52,58]. The four symmetrical ion pairing reagents have
diverse structures in their cationic moieties, which includes pyrrolidinium (C5 (bpyr)2 ), imidazolium (C9 (mim)2 and C5 (benzim)2 ),
and phosphonium (C3 (triprp)2 ). The two charged moieties of each
ion pairing reagent are separated by an alkyl chain with different
lengths. This type of bolaform structure makes the ion pairing
reagents somewhat exible, which has proven to be advantageous
in PIESI-MS [51,52,58]. Two unsymmetrical ion pairing reagents,
UDC 1 (unsymmetrical dication 1) and UDC 2 (unsymmetrical dication 2), were designed based on their symmetrical analogs by
introducing a long alkyl chain to one end (see Table 1). These unique
structures give higher surface activity compared to their symmetrical analogs. These surfactant-like ion pairing reagents often show
superior performance for anion detection compared to the symmetrical ion pairing reagents due to enhanced ionization efciency
[59].
Table 2 gives the structures and masses of eleven tested drug
metabolites, along with their abbreviations. Except oxazepam, all
of these drugs are prohibited by World Anti-Doping Agency [61].
These metabolites are glucuronide or sulfate conjugates of the parent drugs. Biologically, they are products of common metabolic
pathways in phase II reactions to form polar water soluble conjugate products, which increases their tendency to be excreted in
urine [23].
23
4. Conclusions
The method developed based on PIESI was shown to be sensitive
and effective for the analysis of performance-enhancing drugs and
drugs of abuse by detecting their glucuronide and sulfate conjugates. LODs in the sub-pg range were obtained for the metabolites,
which had 348 times improvement compared to the negative ion
mode detection. It was also found that the two unsymmetrical
ion pairing reagents provided further sensitivity enhancement and
complimentary performance in detecting these drug metabolites.
For further quantitative analysis, a method based on HPLC-PIESIMS was successfully developed for the simultaneous separation of
these eleven drug metabolites under the optimized conditions. A
SPE protocol can be carried out to eliminate any matrix effects in
the analysis of urine samples, which would provide more precise
and accurate quantication for the analysis of these drug metabolites. Overall, the developed method could be useful for drug testing
in clinical laboratories.
24
Acknowledgement
The authors gratefully thank the Robert A. Welch Foundation
(Y-0026) for its nancial support on this study.
[22]
[23]
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