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Sensitive detection of anionic metabolites of

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Article in International Journal of Mass Spectrometry August 2015
DOI: 10.1016/j.ijms.2015.08.005

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International Journal of Mass Spectrometry 389 (2015) 1425

Contents lists available at ScienceDirect

International Journal of Mass Spectrometry

journal homepage: www.elsevier.com/locate/ijms

Sensitive detection of anionic metabolites of drugs by positive ion

mode HPLC-PIESI-MS
Hongyue Guo a , Maressa D. Dolzan a,b , Daniel A. Spudeit a,b , Chengdong Xu a ,
Zachary S. Breitbach a , Uma Sreenivasan c , Daniel W. Armstrong a,
a

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.

of administration, inter-individual differences and the matrix to

be analyzed (e.g., urine, blood, saliva) [1,2]. The analyst has
no control over any of these factors except perhaps, in limited
cases, the latter. However, the analyst does control one important factor that profoundly affects drug detection, the analytical
methodology.
In general the detection time of a drug is increased if the
analysis is performed on: (a) the most persistent metabolite,
(b) the optimal biological uid and (c) using the most sensitive method of analysis. Therefore, it is not surprising that the
plethora of papers published on drug detection often involve new
and improved instrumental approaches. These are often accompanied by sample pre-concentration and/or pre-treatment steps
such as enzymatic hydrolysis and derivitization [1233]. Indeed the
quest for more sensitive and reliable methods continues unabated,
usually with a focus on hyphenated separations and mass spectrometric approaches [3449]. While many recent papers have
focused on glucuronide and sulfate conjugates of steroids, these
types of metabolites are relevant for most performance-enhancing
drugs, and drugs of abuse, even ethanol [14,20,34,39,50].
A study of anabolic steroid glucuronides concluded that chemical background noise and fragmentation are less with electrospray

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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.

DHEAG), 4-androsten-17-ol-3-one glucosiduronate (testosterone

glucuronide, TG) and 4-androsten-17-ol-3-one sulfate (testosterone sulfate, NTS) were purchased from Steraloids, Inc. (Newport,
RI, USA). Their structures are listed in Table 2. The internal standard,
d6-dehydroepiandrosterone-3-sulphate (d6-DHEAS) was obtained
from Sigma Aldrich (St. Louis, MO, USA). ACS grade formic acid
(88%, w/w, J. T. Baker. Inc., Mallinckrodt Baker, UK) was used as
the additive for the chromatographic separations.

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

Negative ion mode

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

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

19

[Analyte/ion pairing reagent]+

Mobile phase

Mixing
Device

Methanol/water (67/33, v/v)

Xcalibur 2.0

ESI-MS

400 L/min, 10 M ion pairing reagent

in methanol/water
(50/50, v/v) after mixing

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)

Best LODs with

symmetrical ion
pairing reagentsb

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

a 40 M aqueous solution of ion pairing reagent was introduced

by a secondary pump (Shimadzu LC-6A, Shimadzu, Columbia, MD,
USA) at a ow rate of 100 L/min. The two streams were combined
in a low dead volume, Y-type, mixing device and subsequently
a total ow of methanol/water (50/50, v/v) containing 10 M of
ion pairing reagent was introduced into the MS at a ow rate of
400 L/min. Samples were injected into the HPLC system through
a six-port injection valve prior to the mixing device. As a result,
the anionic analyte was associated with the dicationic ion pairing reagent, to form positively charged analyte/ion pairing reagent
complexes, which could be detected by the MS in the positive
ion mode. The MS parameters in the positive ion mode were set
as follows: spray voltage, 3 kV; capillary voltage, 11 V; capillary

temperature, 350 C; sheath gas ow, 37 arbitrary units (AU); and

the auxiliary gas ow, 6 AU. For the chromatographic separations and real sample analysis, an analytical column was inserted
between the mixing device and the injection valve (see Fig. 1). The
injection volume was kept at 5 L for all the experiments. In SRM
mode, the normalized collision energy, Q value and the activation
time were set at 30, 0.25, and 30 ms, respectively.
The detection limits (LODs) obtained in the negative ion mode
was used for comparison to the positive ion mode PIESI results. The
MS parameters in this mode were optimized as follows: spray voltage, 4.5 kV; capillary voltage, 32 V; capillary temperature, 350 C;
sheath gas ow, 50 arbitrary units (AU); and the auxiliary gas
ow, 6 AU. To have equal LC conditions, a carrier ow consisting

20

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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.

of methanol and water (50/50, v/v) at 400 L/min was introduced

into the MS directly without using ion pairing reagent.
2.3. Preparation of standards
Stock solutions used for LOD determinations were obtained by
either diluting the commercial standard solutions to 10 g/mL (EtG,
EtS, M3G, DHEAS and OxaG) or dissolving the solid material in water
to make 50 g/mL stock solutions (TG, BS, NTS, DHEAG, AS and 17DHEAS). The M3G and OxaG standards in the sample mixture used

for chromatographic separation were at a concentration of 5 g/mL,

while the other metabolites were at 1 g/mL. The working solution
of the internal standard used in the recovery studies had a 50 g/mL
stock solution. All solutions and urine samples were stored in the
dark at 20 C.
2.4. PIESI-MS detection
The detection limit in PIESI-MS was obtained by serial dilution
of the standard solution until a signal-to-noise ratio (S/N) of three

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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.

was noted in 5 replicate injections of each sample. The S/N was

calculated by using a Genesis Peak Detection Algorithm with Xcalibur 2.0 software (Thermo Fishier Scientic, San Jose, CA, USA). The
PIESI-MS detection was performed both in the selected ion monitoring (SIM) mode and selected reaction monitoring (SRM) mode.
In the SIM mode, the m/z of the analyte/ion pairing reagent complex ion was monitored, while in the SRM mode the most abundant
MS/MS fragment ion from the collision induced dissociation (CID)
was monitored. The m/z width in the LOD determination study was
kept at 5 in both SIM and SRM mode.
2.5. Separation and detection of glucuro- and sulfoconjugated
drug metabolites by HPLC-PIESI-MS
Separations were performed on an Ascentis Express C18 column
(150 mm 2.1 mm, 2.7 m; Sigma Aldrich, St. Louis, MO, USA). The
mobile phase composition was (A) 0.1% formic acid in water, (B)
0.1% formic acid in methanol. The gradient program was optimized
as: 3% B, 05 min; 330% B, 56 min; 3080% B, 622 min. The MS
was operated in SIM mode and the chromatogram was recorded
with three separate SIM segments (segment 1, 05 min, m/z monitored: 449.2, 545.5, 784.4; segment 2, 512 min, m/z monitored:
785.4; segment 3, 1222 min, m/z monitored: 677.5, 689.4, 691.5,
693.6, 695.5, 787.5). The m/z width of the trapped parent complex
was set at 1 in this study due to the close m/z of many steroid
metabolites.
2.6. Sample preparation
A solid phase extraction (SPE) method was developed with a Discovery DSC-18 cartridge (1 g sorbent, 50 m particle size, Sigma
Aldrich) for the analysis of these metabolites in urine. The SPE
method allows for minimization of matrix effects as well as analyte

pre-concentration. The loading solution was composed of 500 L of

urine (obtained from a healthy male volunteer), 20 L of internal
standard stock solution, and 1480 L of 0.1% formic acid solution
spiked with metabolite standards. The extraction protocol was as
follows. The cartridges were rst washed with 5 mL of ACN and
5 mL of methanol respectively, and subsequently equilibrated with
10 mL of a 0.1% of formic acid aqueous solution. The sample solution
was then loaded onto the sorbent, and two steps followed. The cartridges were rst washed with 5 mL of HPLCMS grade water, and
then eluted with 8 mL of methanol. The eluent was subsequently
diluted with water in volumetric ask to obtain a nal volume of
10 mL sample solution prior to injection. Each sample was prepared
in triplicate. The nal concentrations of internal standard solutions
were kept at 100 ng/mL for all samples. In this study, only a small
amount of urine was needed, and it was diluted 20 times prior to
injection. The SPE protocol was employed to minimize urine matrix
effects.
2.7. Recovery study
The extraction efciency was investigated in terms of recovery.
HPLC-PIESI-MS was employed to test the recovery of the method.
The mobile phase consisted of methanol and water (60/40, v/v) containing 0.1% of formic acid. The PIESI-MS was operated in SRM mode
with the use of the best ion pairing reagent. The chromatogram
was recorded with three separate segments (segment 1, 05 min,
transition monitored: 785.4 658.3; segment 2, 510 min, transition monitored: 697.5 294.3; segment 3, 1025 min, transition
monitored: 693.6 294.3). In each segment, only the m/z of one
daughter ion was monitored. The m/z width of each daughter ion
monitored was set at 3. The two metabolites investigated in this
study were OxaG and AS. OxaG does not exist in the urine of individuals who have not consumed oxazepam, while AS is present as

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H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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%

OxaG: oxazepam glucuronide; AS: androsterone sulfate.

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

Absolute LODf (pg)

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].

3.2. PIESI-MS detection of drug metabolites with the use of

symmetrical ion pairing reagents
Table 3 summarizes the absolute LODs of eleven drug metabolites detected in the positive ion mode using symmetrical ion
pairing reagents and in the negative ion mode without using ion
pairing reagents. The best LOD for each compound is highlighted
in bold font (Table 3). Overall, the negative ion mode was not as
sensitive as the PIESI-MS. The detection limits obtained in the negative ion mode were 320 times worse. It should be noted that
the SRM analyses in the negative ion mode were not successfully
conducted due to unstable fragment ion signals. Moreover, it was
also observed that the analytes with small m/z ratios, such as EtS,
gave worse LODs (m/z: 125.0, LOD: 100 pg) than other, higher mass,
sulfated steroids (m/z: 350380, LOD: 10 pg to 20 pg) in the SIM
negative ion mode.
The positive ion mode was more suitable for the detection of
the tested metabolites than the negative ion mode. For example,
when EtS was detected in the SIM positive ion mode with the use
of C5 (bpyr)2 as the ion pairing reagent, the LOD could be reduced
from 100 pg (obtained in the SIM negative ion mode) to 5.0 pg. It
was also observed that with the optimal ion pairing reagents, the
PIESI-MS approach could improve the detection limit by several
fold in many cases. The performance of four ion pairing reagents
varied signicantly in detecting these drug metabolites. This suggests that the structure and/or geometry of ion pairing reagents
play an important role in the observed detection sensitivity for
anions. Overall, C5 (bpyr)2 showed the best overall performance
compared to the other three ion pairing reagents, with the majority of the LODs obtained using C5 (bpyr)2 falling below 37 pg. The
chemical properties of the analytes also affected the detection sensitivity. For example, C9 (mim)2 was considered one of the best ion
pairing reagents for detecting small organic and inorganic anions
in our previous studies [51,52]; however, it did not perform well
in detection of the drug metabolites in this study. Interestingly,
detection limits of the sulfated drugs (pKa < 1) were usually better
than the glucuronated drugs (pKa 4.5). This indicates that the pKa
of the analyte could also affect the observed detection limits. The
lower pKa of these sulfated conjugates results in more anions in

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

the solution phase, which could lead to more analyte/IPR complex

formation in solution. A similar phenomenon also was observed
in the negative ion mode. Moreover, M3G and OxaG always gave
poorer LODs compared to the other compounds. This could result
from the presence of a tertiary amine (pKa 10) and an imine group
(pKa 11) respectively in their structures. These could affect the
formation of analyte/ion pairing reagent complex.
The SRM mode often enhances analytical specicity and reduces
background noise, and as a result, better sensitivity can be often
achieved [58]. However, it was observed in this study that some
LODs obtained from the SRM mode were worse than that from the
SIM mode. For example, the LOD of NTS detected in the SRM mode
with C3 (triprp)2 (LOD: 1000 pg) was 400 times worse than the LOD
obtained in the SIM mode (LOD: 2.5 pg). This decreased sensitivity in SRM mode might be due to the fact that the product ions
generated during the CID process were not stable [58].
3.3. PIESI-MS detection of drug metabolites with the use of
unsymmetrical ion pairing reagents
The dicationic ion pairing reagents with unsymmetrical structures have been recently developed by our group, with the purpose
of further improving the sensitivity for anion detection by PIESI-MS
[59]. In this study, we evaluated the performance of two unsymmetrical ion pairing reagents, UDC 1 and UDC 2, on the detection
sensitivity of the drug metabolites (Table 4). As shown in Table 4,
the unsymmetrical ion pairing reagents provided improved detection sensitivity for more than half of the tested metabolites (6
out of 11) compared to the symmetrical ion pairing reagents. The
improvement in LODs from symmetrical to unsymmetrical ion pairing reagents ranged from two to seven times. It was found that
the unsymmetrical ion pairing reagents generally provided greater
detection improvements for sulfated steroids, while they gave similar detection limits for the glucuronated metabolites compared
to the symmetrical ion pairing reagents. For example, the LOD of
BS was 800 fg with the use of UDC2 in the SRM mode, which was
seven times better than the LOD obtained with the best symmetrical ion pairing reagent (5.5 pg by using C5 (bpyr)2 in SRM mode).
On the other hand, the LOD of TG was 8.5 pg and 6.0 pg with the
best unsymmetrical and symmetrical ion pairing reagent respectively, which shows a slightly worse detection limit when using
the unsymmetrical ion pairing reagent.
3.4. Chromatographic separation of the drug metabolites
The simultaneous separation of drug metabolites, in particular
structurally similar analytes, is essential for drug testing and analysis. Therefore, HPLC coupled with PIESI-MS was used to achieve
the separation and highly selective detection/quantication at the
same time. Gradient elution was performed in reversed phase
mode within 22 min (see Fig. 2). C5 (bpyr)2 was used for the HPLCPIESI-MS, since it was shown to have superior performance on
the detection of these drug metabolites and is also commercially
available. The MS was operated in SIM mode with three detection
segments. The drop in baseline at 5 min is caused by switching
from segment 1 to segment 2 (see Fig. 2). As shown in Fig. 2,
the selected drug metabolites were well separated except for two
pairs, EtS/EtG and BS/NTS. However, because of different m/z values for each pair, a complete chromatographic resolution was not
necessary.
3.5. Urine sample analysis
3.5.1. Recovery study
A SPE protocol was carried out with the use of human
urine matrix spiked with the drug metabolites at two different

23

concentrations (50 ng/mL and 400 ng/mL respectively). OxaG and

AS were selected as representatives of these drug metabolites. As
shown in the extracted ion chromatogram (EIC) of the urine blank
(Figs. 3 and 4), the interference peaks in the urine blank were negligible. The amount of AS naturally existing in urine was subtracted
from its total amount when calculating the concentrations of spiked
AS.
The results of the recovery study are shown in Table 5. The
extraction efciency of the internal standard was determined to
be 100%, and therefore it was directly used for the calibration
of OxaG and AS. The response factors of AS and OxaG in relation to the internal standard were measured to be 1.02 0.02
and 0.20 0.02 respectively. These values were reasonable considering the structural similarity between the AS and the internal
standard, as well as the structural difference between the OxaG
and the internal standard. The recovery yields of these two standards were obtained based on their response factors. As shown
in Table 6, the recoveries of OxaG and AS were 90% (RSD,
15%) and 107% (RSD, 7.9%) respectively at low concentrations,
while they were 89% (RSD, 4.5%) and 98% (RSD, 3.5%) respectively at high concentrations. The SPE protocol is suitable to be
employed for further quantitative analysis. The concentration of
AS that existed naturally in this urine sample was determined
to be 1.2 g/mL. This concentration is consistent with reported
AS concentrations in the urine of a healthy male (1.3 g/mL)
[50].

3.5.2. A comparison in detection sensitivity between PIESI-MS

and other HPLCMS methodologies
The determination of the detection limit of each drug metabolite in urine samples was performed in the same SPE, LC and MS
conditions as those of recovery study experiments. Table 6 compares the absolute LODs (pg) of steroid metabolites obtained using
the PIESI-MS approach with other reported HPLCMS methodologies [28,30,33,39,50]. Compared to other HPLCMS based methods,
PIESI-MS provided better detection limits from one to three
orders of magnitude for the detection of steroid glucuronides
and sulfates. It should be noted that most of the commonly
used HPLCMS methodologies employ sample pre-concentration
to obtain a higher sensitivity. However, this technique has no effect
on improving instrumental detection limits, which is the main
focus of this study. Moreover, the detection limits reported in this
study could be further enhanced with the use of more sensitive
mass analyzers and detectors (e.g., triple quadrupole mass analyzer).

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

H. Guo et al. / International Journal of Mass Spectrometry 389 (2015) 1425

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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