Determination of 4-hydroxy-3-methoxyphenylethylene glycol 4-sulfate in human urine using liquid chromatography-tandem mass spectrometry

Article abstract of DOI:10.1021/ac020101a

A major metabolite of norepinephrine (NE) in brain is 4-hydroxy-3-methoxyphenylethylene glycol (MHPG). In many species, a large fraction of MHPG formed in brain is converted to the sulfate conjugate. Consequently, MHPG sulfate has been proposed as a bioma

Full text of DOI:10.1021/ac020101a

Anal. Chem. 2002, 74, 5290-5296
Determination of
4-Hydroxy-3-methoxyphenylethylene Glycol
4-Sulfate in Human Urine Using Liquid
Chromatography-Tandem Mass Spectrometry
Peyton Jacob, III,* Margaret Wilson, Lisa Yu, John Mendelson, and Reese T. Jones
Division of Clinical Pharmacology, Building 100, Room 235, San Francisco General Hospital Medical Center,
Department of Medicine, and Drug Dependence Research Center, Department of Psychiatry, University of California,
San Francisco, California 94110
reuptake into nerve terminals might be expected to increase
norepinephrine turnover and increase production of MHPG.
As part of the clinical trials of the MAO inhibitor selegiline
(levodeprenyl) for treating cocaine addiction, we required a
method for measuring concentrations of MHPG sulfate in human
urine. Various methods for determination of MHPG sulfate have
been reported, including HPLC,4,5 GC,6 and GC/ MS7 methods.
All of the methods that we are aware of are indirect in that they
involve determination of MHPG released by cleaving the sulfate
conjugate, either chemically or enzymatically. This is time-
consuming, because free MHPG and the glucuronide conjugate
of MHPG are also present in urine. Therefore, in these methods,
the sulfate must either be separated from free MHPG and its
glucuronide or two determinations must be carried out on each
sample: one for free MHPG and one following enzymatic cleavage
of the sulfate to MHPG using an arylsulfatase. In the latter case,
concentrations of MHPG sulfate are calculated as the difference
of the two determinations,6 and in the former case, separation
using a selective solid-phase extraction procedure followed by
cleavage of the sulfate, either chemically or enzymatically, is
generally used.5,7
The advent of electrospray ionization (ESI) mass spectrometry
has made possible determination of low concentrations of a wide
range of readily ionizable, thermally labile substances in complex
matrixes. We found that MHPG sulfate can be detected with high
sensitivity using ESI-MS in the negative ion mode. Using a
deuterium-labeled analogue of MHPG sulfate as the internal
standard, we developed a liquid chromatography-tandem mass
spectrometry (LC-MS/ MS) method for determination of MHPG
sulfate in urine. The method is extremely simple to carry out and
appears to be the first reported truly direct method for quantitation
of MHPG sulfate in a biologic fluid.
A major metabolite of norepinephrine (NE) in brain is
4 -hydroxy-3 -methoxyphenylethylene glycol (MHP G). In
many species, a large fraction of MHP G formed in brain
is converted to the sulfate conjugate. Consequently,
MHP G sulfate has been proposed as a biomarker for NE
metabolism in the central nervous system. As part of the
clinical trials of the monoamine oxidase inhibitor sel-
egiline for treating cocaine addiction, we required a
method for measuring urine concentrations of MHP G
sulfate. Using a deuterium-labeled analogue as an internal
standard, we developed a liquid chromatography-elec-
trospray ionization tandem mass spectrometry (LC-MS/
MS) method for determination of MHP G sulfate in human
urine. Sample preparation involves simply diluting 5 0 µL
of urine with 1 mL of ammonium formate buffer and
adding the internal standard. The sample is centrifuged,
the supernate is transferred to an autosampler vial, and
1 0 µL is injected into the LC-MS/ MS system. Standard
curves from 5 0 to 1 0 0 0 0 ng/ mL are generated. Only one
sample of 2 7 7 clinical samples analyzed had a concentra-
tion outside of this range. P recision (coefficient of varia-
tion) ranged from 1 .9 to 9 .7 %, and accuracy ranged from
9 7 to 1 0 3 % of expected values for controls prepared by
spiking sulfatase-treated urine with MHP G sulfate.
A major metabolite of norepinephrine (NE) in brain is 4-hy-
droxy-3-methoxyphenylethylene glycol (MHPG; Figure 1). In
many species, a large fraction of MHPG formed in the brain is
converted to the sulfate conjugate. Consequently, determination
of MHPG sulfate in urine has been proposed as a measure of
norepinephrine metabolism in the cental nervous system.1,2 Since
one of the steps in the biotransformation of NE to MHPG is
catalyzed by monoamine oxidases (MAO), inhibitors of MAO
might decrease the excretion of MHPG and its sulfate conjugate
by shunting metabolism to other pathways.3 In contrast, drugs
that release norepinephrine from neuronal stores or block its
EXPERIMENTAL SECTION
Apparatus. LC-MS and LC-MS/ MS analyses were carried
out with a Hewlett-Packard (Palo Alto, CA) 1090 HPLC interfaced
with a Finnigan TSQ 7000 triple-stage quadrupole mass spectrom-
* To whom correspondence should be addressed. E-mail: peyton@itsa.ucsf.edu,
Phone: (415) 282-9495. Fax: (415) 206-5080.
(1) Peyrin, L. J. Neural Transm. Gen. Sect. 1 9 9 0 , 80, 51-65.
(2) Filser, J. G.; Spira, J.; Fischer, M.; Gattaz, W. F.; Muller, W. E. J. Psychiatr.
Res. 1 9 8 8 , 22, 171-81.
(4) Muller, H. U.; Riemann, D.; Berger, M.; Muller, W. E. Acta Psychiatr. Scand.
1 9 9 3 , 88, 16-20.
(5) Inagaki, H.; Hirata, K.; Minami, M. J. Chromatogr., B: Biomed. Sci. Appl.
1 9 9 8 , 707, 9-15.
(3) Nagatsu, T. Biochemistry of Catecholamines; University Park Press: Balti-
(6) Piletz, J. E.; Halaris, A. Clin. Chim. Acta 1 9 8 9 , 185, 165-76.
(7) Murray, S.; Baillie, T. A.; Davies, D. S. J. Chromatogr. 1 9 7 7 , 143, 541-51.
more, MD, 1973.
5290 Analytical Chemistry, Vol. 74, No. 20, October 15, 2002
10.1021/ac020101a CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/17/2002

Figure 1. Norepinephrine metabolic pathways. Abbreviations: MAO, monoamine oxidase; COMT, catecholamine-O-methyltransferase.
Figure 2. Synthesis of MHPG-d3 sulfate.
eter with an API2 ion source (Thermo-Finnigan, San Jose, CA).
A Phenomenex (Torrence, CA) Synergi Polar column, 250 mm
× 2 mm, with an Upchurch (Oak Harbor, WA) 2-mm C-18 guard
column, was used for the chromatography.
Synthesis of 4 -Hydroxy-3 -trideuteriomethoxyphenyleth-
ylene Glycol 4 -Sulfate P otassium Salt (MHP G-d 3 Sulfate).
MHPG-d3 was synthesized by the method of Markey et al.8 (Figure
2). The crude product was used without the further purification
(silica gel chromatography) described by the authors. A magneti-
cally stirred solution of 90 mg (0.5 mmoL) of MHPG-d3 in 4 mL
of dry THF, under an atmosphere of argon, was cooled in a dry
ice-acetone bath. A 1 M solution of potassium tert-butoxide in
THF (0.5 mL, 0.5 mmol) was added, which caused the mixture
to turn cloudy. Sulfur trioxide pyridine complex (80 mg, 0.5
mmol) was added in one portion, and the mixture was stirred
Reagents. MHPG sulfate potassium salt and arylsulfatase type
VI from Aerobacter aerogenes were purchased from Sigma Chemi-
cal Co. (St Louis, MO). The HPLC mobile phase was prepared
from HPLC grade water, HPLC grade methanol, and formic acid
(ACS reagent grade) obtained from Fisher Chemical Co. (Pitts-
burgh, PA). Anhydrous tetrahydrofuran (THF) was obtained from
Aldrich Chemical Co. (Milwaukee, WI). Sulfur trioxide pyridine
complex was obtained from Acros Organics/ Fisher Scientific
(Pittsburgh, PA). The internal standard, MHPG-d3 sulfate, was
synthesized as described below.
(8) Markey, S. P.; Powers, K.; Dubinsky, D.; Kopin, I. J. J. Labelled Compd.
Radiopharm. 1 9 8 0 , XVII, 103-14.
Analytical Chemistry, Vol. 74, No. 20, October 15, 2002 5291

for a few minutes with cooling in the dry ice-acetone bath. The
cooling bath was removed, and the mixture was stirred while
warming to room temperature, during which time a granular white
precipitate formed. After stirring 30 min at room temperature, 5
mL of anyhdrous ether was added, and the mixture was centri-
fuged. The supernate was removed, and the precipitate was
washed twice with 2-mL portions of ether. The product was then
dried with a stream of nitrogen at ∼50 °C to give 98 mg of white
powder. LC-MS analysis (ESI, negative ion mode) confirmed the
identity of the product: m/ z 266, M - K. LC-MS analysis in the
APCI mode indicated that the product was ∼50% pure, the major
impurity being vanillomandelic-d3 acid, a synthetic precursor of
MHPG-d3. From extracted ion chromatograms, it was estimated
that the product contained less than 0.2% MHPG-d0 sulfate. The
product was stored in a desiccator in a freezer, since a sample
turned brown after a few months at room temperature.
Cleavage of MHP G Sulfate in Urine with Arylsulfatase. The
pH of a pooled urine specimen (36 mL) from four people was
adjusted to 7 with 4 mL of 1 M sodium acetate followed by
concentrated sodium hydroxide added dropwise. To this was
added 1.05 mL (18 units) of arylsulfatase type VI from A. aerogenes,
and the mixture was incubated at 37 °C for 20 h in a stoppered
flask. The flask was then heated in a boiling water bath for 30
min to inactivate the enzyme. Absence of MHPG sulfate was
verified by LC-MS/ MS analysis (see below).
MHPG sulfate and m/ z 266 (M - H) to 168 for the internal
standard, using a collision energy of 25 eV, were monitored for
confirmation.
Data Analysis. Finnigan XCalibur/ LC Quan software was used
to generate calibration curves (linear regression, equal weighting)
and calculate concentrations using peak area ratios of analyte/
internal standard. Standard curves were generated from 0 to 2000
ng/ mL and from 0 to 10 000 ng/ mL. The latter range was used
for concentrations above 2000 ng/ mL.
Clinical Study. The protocol involved 12 human volunteers,
all of whom had previously used cocaine but were not cocaine
dependent, studied for 14 days on a research ward at the
University of California, San Francisco. Urine was collected in 24-h
blocks. On the first day, the subjects were given an intravenous
infusion of cocaine, 2.5 mg/ kg over 4 h. On the fourth day through
the thirteenth day, the subjects were administered transdermal
selegiline, one STS patch (20 mg) per day. On the tenth day,
subjects were again administered cocaine by intravenous infusion,
2.5 mg/ kg over 4 h. Urine collection continued for 3 days following
this second infusion.
RESULTS AND DISCUSSION
AP I Mass Spectrometry of MHP G Sulfate. Initially, analysis
using atmospheric pressure chemical ionization (APCI) was
attempted. This was done because APCI is relatively insensitive
to suppression of ionization by substances derived from the sample
matrix.9 The APCI spectrum (negative ion mode) of MHPG sulfate
is shown in Figure 3. The relative abundance of the pseudomo-
lecular ion (M - H)-, m/ z 263, was only 3% and the base peak in
the spectrum was m/ z 245, resulting from loss of water. Collision-
induced dissociation of m/ z 245 resulted in the formation of two
major product ions, m/ z 165 and 150 (Figure 3). Using the
transitions of m/ z 245 to 165 and m/ z 245 to 150, an attempt was
made to develop a quantitative method for determination of MHPG
sulfate, but sensitivity was inadequate to measure the concentra-
tions found in human urine, which have been reported to average
∼500 ng/ mL.1,2 This lack of sensitivity was presumably due to
extensive decomposition of MHPG sulfate in the heated vaporizer
of the APCI source.
Although more prone to suppression of ionization by sub-
stances derived from the sample matrix than is APCI, ESI is often
applicable to thermally labile, ionic substances such as MHPG
sulfate. Indeed, ESI has been used for mass spectral determination
of low concentrations of steroidal sulfates.10 In the ESI mode, the
negative ion spectrum of MHPG sulfate displayed one major ion,
the pseudomolecular ion m/ z 263. CID of this ion produced two
major ions, m/ z 165 and 150, the same two product ions observed
from CID of the APCI-generated precursor ion m/ z 245 (Figure
4). The m/ z 165 ion presumably results from loss of SO3 and H2O
and m/ z 150 from additional loss of the methyl group (Figure 5).
Using ESI and operating the mass spectrometer in the SRM
mode, experiments were conducted to determine whether ad-
equate sensitivity could be obtained for the determination of
MHPG sulfate in human urine. Injection of low-picogram amounts
produced a peak significantly above noise level. Consequently,
P reparation of Standards and Controls. Standards were
prepared by diluting a stock aqueous solution of MHPG sulfate
with HPLC grade water to concentrations of 50, 100, 200, 500,
1000, 2000, 5000, and 10 000 ng/ mL. Controls were prepared from
(1) the pooled urine specimen that was treated with arylsulfatase
(see above), (2) the pooled urine specimen spiked with MHPG
sulfate, or (3) the pooled urine specimen diluted with HPLC grade
water.
Sample P reparation. Fifty microliters of urine sample,
standard, or control was added to 1 mL of 0.1% formic acid in 10
mM ammonium formate. The internal standard, MHPG-d3 sulfate,
∼100 ng (assuming the preparation was ∼50% pure) in 100 µL of
water, was added. The solution was briefly mixed, and centrifuged,
and the supernate was transferred to an autosampler vial for LC-
MS/ MS analysis.
Liquid Chromatography. The instrument was operated in the
isocratic mode, with a flow rate of 0.2 mL/ min at ambient
temperature. The mobile phase consisted of 95% 0.1% aqueous
formic acid and 5% methanol, degassed by sparging with helium.
The injection volume was 10 µL. A divert valve was programmed
to shunt the eluate to waste prior to and following elution of the
analyte.
Mass Spectrometry. The electrospray voltage was 4.5 kV,
and the heated capillary was set at 250 °C. The sheath gas and
auxiliary gas (both nitrogen) were set at 80 psi and 40 arbitrary
units, respectively. For collision-induced dissociation (CID), the
collision gas (argon) pressure in the second (rf-only) quadrupole
was set at ∼2.5 mTorr. The resolution was set at 0.6 amu.
Quantitative analyses were carried out using selected reaction
monitoring (SRM). For quantitation, the SRM transitions moni-
tored were m/ z 263 (M - H) to 150 for MHPG sulfate and m/ z
266 (M - H) to 150 for the internal standard, using a collision
energy of 30 eV. The transitions m/ z 263 (M - H) to 165 for
(9) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem.
1 9 9 8 , 70, 882-9.
(10) Murphy, R. C.; Fiedler, J.; Hevko, J. Chem. Rev. 2 0 0 1 , 101, 479-526.
5292 Analytical Chemistry, Vol. 74, No. 20, October 15, 2002

Figure 3. APCI mass spectra of MHPG sulfate.
Figure 4. ESI mass spectra of MHPG sulfate.
efforts were directed toward developing a quantitative method
using ESI.
had only baseline noise at the retention time of MHPG sulfate.
These results indicated that MHPG sulfate was being detected
without significant interference.
Sample P reparation. Initially, solid-phase anion exchange
extraction procedures were explored, since the high polarity of
MHPG sulfate suggested that liquid-liquid extraction would result
in poor recoveries and that sample cleanup might be necessary
to remove matrix impurities that could interfere directly or cause
suppression of ionization. Although these experiments were
encouraging, elution of MHPG sulfate from anion exchange
columns required solvents with a high ionic strength containing
nonvolatile salts, and such eluates would not be desirable for
injection into an LC-MS system.
It occurred to us that if there was sufficient retention of MHPG
sulfate on the HPLC column, it might be possible to dilute urine
with HPLC mobile phase, inject without extraction, and achieve
sufficient separation from matrix impurities for quantitation. Using
a Phenomenex Synergi Polar column, which contains a phenox-
ypropylsilane group to facilitate retention of polar compounds,
MHPG sulfate was well retained and eluted with good peak
symmetry. A mobile phase of 10 mM ammonium formate in 95:5
water/ methanol was used, and the mass spectrometer was
operated in the SRM mode, monitoring the transitions m/ z 263
to 165 and m/ z 263 to 150. Injecting a pooled (four persons) urine
specimen that had been diluted with mobile phase gave a peak
with the same retention time as an MHPG sulfate standard. The
ratio of intensities of the two transitions was ∼0.5 for both the
urine sample and the standard. Furthermore, injection of a sample
of the pooled urine that had been treated with arylsulfatase in
order to hydrolyze MHPG sulfate resulted in a chromatogram that
To investigate the possibility of matrix suppression of ioniza-
tion, the arylsulfatase-treated urine was heated to ∼100 °C to
inactivate the enzyme and then spiked with 1000 ng/ mL MHPG
sulfate. The sample was analyzed, and the peak area was compared
to the peak area of an aqueous standard containing the same
concentration of MHPG sulfate. This experiment indicated that
the matrix suppressed ionization by ∼50%.
Synthesis of MHP G-d 3 Sulfate. The use of a stable isotope-
labeled internal standard is generally advantageous in any
quantitative mass spectrometric method, but in the present case,
it was considered to be essential in order to correct for matrix
effects. Coeluting with the analyte and having essentially the same
ionization characteristics as the analyte, the internal standard
should undergo the same degree of supression of ionization.
Therefore, the ratio of signal intensities for analyte/ internal
standard should be independent of the degree of ionization
suppression (which might vary among different samples and
standards) and provide a reliable basis for quantitation. Conse-
quently, we undertook the synthesis of a deuterium-labeled
analogue of MHPG sulfate.
MHPG sulfate, deuterium-labeled on the side chain, has been
synthesized previously, in eight steps, and used in a GC/ MS
method for determination of MHPG sulfate in urine.7 A consider-
ably shorter synthesis of a deuterium-labeled analogue appeared
to be possible based on the synthesis of methoxy-labeled MHPG
reported by Markey et al.8 We found that, indeed, methoxy-labeled
Analytical Chemistry, Vol. 74, No. 20, October 15, 2002 5293

Figure 5. Proposed fragmentation pathways for MHPG sulfate.
Matrix Suppression of Ionization: Effect of HP LC Mobile
P hase. Using a 2 mm × 250 mm Phenomenex Synergi column
with a mobile phase consisting of 10 mM ammonium formate in
95:5 water/ methanol, suppression of ionization was ∼50%, based
on the peak area for the internal standard in urine specimens
compared to that in aqueous standards. A proposed mechanism
for matrix effects is competition between the analyte and other
ionic species during ion evaporation.11 It was reasoned that, if this
were the case, it might be possible to diminish the matrix effect
by lowering the pH of the HPLC mobile phase. At the pH of
ammonium formate (∼6), a wide range of weak acids, likely to
be present in urine, would be expected to be ionized. Lowering
the pH should convert many weak acids to neutral species. But,
since MHPG sulfate is a fairly strong acid, it should remain ionized
at pH values of ∼2-3, a pH that could be tolerated by reversed-
phase HPLC columns and would protonate the anions derived
from many weak acids.
To test this hypothesis, a mobile phase consisting of 0.1%
formic acid in 95:5 water/ methanol was employed, and the internal
standard peak area for aqueous standards was compared to the
internal standard peak area for urine samples. Under these
conditions, the matrix effect was virtually eliminated! Comparing
peak areas for the internal standard in chromatograms from
clinical study samples with those in aqueous standards, there was
only ∼7% suppression of ionization. However, from this result we
cannot say with certainty that lowering the pH per se was the
mechanism for elimination of the matrix effect: the retention time
for the analyte was lengthened compared to that using the
ammonium formate mobile phase, and more effective separation
from matrix components is another possible mechanism. Regard-
less of the mechanism, this result suggested that use of the formic
acid-containing mobile phase would be advantageous.
Figure 6. ESI mass spectra of MHPG-d3 sulfate.
MHPG prepared by this method could be readily converted to
the sulfate ester by treatment with potassium tert-butoxide
followed by sulfur trioxide pyridine complex (Figure 2). The
labeled analogue was characterized by its ESI-MS and MS/ MS
spectra (Figure 6). The MS/ MS spectrum confirms that the m/ z
150 ion results from loss of the methyl group, since m/ z 150 is
formed from both MHPG-d0 sulfate and MHPG-d3 sulfate (Figure
4). Although the product was obtained in only ∼50% purity (a
major impurity being vanillomandelic-d3 acid, one of the synthetic
intermediates), it was suitable for use as an internal standard, since
there was no significant formation of m/ z 263, the pseudomo-
lecular ion of the analyte, formed under ESI conditions.
Method Validation. SRM chromatograms from a human urine
specimen and an arylsulfatase-treated pooled (four persons) urine
are shown in Figure 7. For the arylsulfatase-treated urine, the SRM
chromatogram of the transitions m/ z 263 to 165 and m/ z 263 to
150 (MHPG-d0 sulfate) revealed only background noise at the
retention time of MHPG sulfate, as did the m/ z 266 to 150
(11) Niessen, W. M. J. Chromatogr., A 1 9 9 9 , 856, 179-97.
5294 Analytical Chemistry, Vol. 74, No. 20, October 15, 2002

Table 1. Intraday Precision and Accuracy
actual concn
(ng/ mL)
measd mean
(ng/ mL)
accuracya
(%)
precision
(% CV)
replicate
analyses
0b
50c
8.5
56.9
107
2
6
6
6
6
6
6
97
99
4.7
6.4
2.4
2.9
4.4
3.2
100c
d
245
1000c
e
10000c
1005
1722
9953
100
99
a
Determined from blank-corrected mean. b Arylsulfatase-treated
pooled (n ) 4) urine. c Arylsulfatase-treated pooled urine spiked with
MHPG sulfate. d Pooled (n ) 4) urine diluted 1/ 5 with water. e Pooled
urine spiked with 500 ng/ mL MPHG sulfate.
Table 2. Interday Precision and Accuracy
actual concn
(ng/ mL)
measd mean
(ng/ mL)
accuracya
(%)
precision
(% CV)
replicate
analyses
0b
100c
d
6.4
109
257
991
1776
4953
6
6
6
6
6
4
103
99
9.7
3.4
1.9
3.6
5.6
10002
c
5000c
99
a
Determined from blank-corrected mean. b Arylsulfatase-treated
pooled (n ) 4) urine. c Arylsulfatase-treated pooled urine spiked with
MHPG sulfate. d Pooled (n ) 4) urine diluted 1/ 5 with water. e Pooled
urine spiked with 500 ng/ mL MPHG sulfate.
(percent coefficient of variation) ranged from 2.4 to 4.7%, and
intraday accuracy (percent of expected values) ranged from 97
to 100% (Table 1). Between-run (4 or 6 days) precision and
accuracy was determined from QC samples analyzed along with
clinical study samples (Table 2). Precision ranged from 1.9 to 9.7%,
and accuracy ranged from 99 to 103%. The concentrations used
for the QC samples were chosen to span the expected range based
on published studies,1,2 and the results of our clinical study,
discussed below, confirmed that this was the case.
Figure 7. SRM chromatograms of untreated urine (upper panel)
and arylsulfatase-treated urine (lower panel). No internal standard
added.
transition at the retention time of the internal standard, MHPG-
d3 sulfate. However, there was a significant peak at the retention
time of MHPG sulfate in the SRM chromatogram for m/ z 266 to
168 transition of the internal standard in both the sulfatase-treated
and untreated urine specimens (Figure 7). Therefore, the transi-
tions m/ z 263 to 150 for MHPG sulfate and m/ z 266 to 150 for
the internal standard were used for quantitation.
Clinical Study. The method was used to determine MHPG
sulfate concentrations in 277 urine specimens from a clinical study
of the MAO inhibitor selegiline (levodeprenyl), which is being
investigated as a pharmacotherapy for treating cocaine depen-
dence. Concentrations ranged from <50 to 5655 ng/ mL with a
mean of 962 ng/ mL. These specimens included urine collections
prior to drug treatment and following administration of cocaine
and selegiline. Only one of the 277 samples analyzed had a
concentration below the LOQ of 50 ng/ mL. To verify that cocaine,
selegiline, and their metabolites do not interfere with the assay,
15 urine specimens collected from subjects following administra-
tion of cocaine and selegiline were treated with arylsulfatase to
hydrolyze MHPG sulfate prior to LC-MS/ MS analysis. Only
baseline noise was observed at the retention time of MHPG sulfate
in the SRM chromatograms of the transition m/ z 263 to 150
(MHPG-d0) and m/ z 266 to 150 (MHPG-d3).
Standard curves were generated from 0 to 2000 ng/ mL and
from 0 to 10 000 ng/ mL, using aqueous standards. The latter
range was used for concentrations above 2000 ng/ mL. Equations
for typical standard curves are Y ) -0.0115 + 0.00107X, r2
)
0.9972 (0-2000 ng/ mL); Y ) 0.0255 + 0.000981X, r2 ) 0.9992
(0-10 000 ng/ mL). The concentrations of the standards and the
back-calculated values (in parentheses) for this run were as follows
(in ng/ mL): 50 (52.2, 64.5); 100 (108, 111); 200 (212, 195); 500
(518, 452); 1000 (943, 971); 2000 (2090, 1960); 5000 (4950, 4800);
10 000 (10 100, 9950).
To determine precision and accuracy, sulfatase-treated urine
was spiked with concentrations of MHPG sulfate (following heat
inactivation of the enzyme) that spanned the expected range, and
replicate samples were analyzed. In addition, a urine pool obtained
from four individuals was analyzed as such, diluted 1:2 with water,
1:5 with water, and spiked with 500 ng/ mL. Six replicate analyses
were carried out on all urine specimens. Intraday precision
A SRM chromatogram from a clinical study subject is shown
in Figure 8. The objective was to investigate the effect of cocaine,
in the presence of and in the absence of selegiline, on urinary
Analytical Chemistry, Vol. 74, No. 20, October 15, 2002 5295

HPG sulfate excretion was increased above baseline levels by
∼50% (p < 0.01) for at least 3 days following cocaine infusion in
the absence of selegiline. In contrast, there was no significant
change in MHPG sulfate excretion following cocaine infusion in
the presence of selegiline. Presumably, inhibition of MAO by
selegiline shunts norepinephrine metabolism to other pathways,
thereby diminishing MHPG formation to levels similar to those
formed in the absence of cocaine.
CONCLUSIONS
An electrospray ionization LC-MS/ MS method for determi-
nation of MHPG sulfate in human urine has been developed.
Sample preparation simply involves diluting a small volume of
urine with a buffer, adding the internal standard, centrifuging to
remove particulates, and transferring the sample to an autosampler
for analysis. The run time is 9 min, allowing ∼150 samples to be
injected during a 24-h period. This method appears to be the first
truly direct method for determination of MHPG sulfate in human
biofluids to be reported.
ACKNOWLEDGMENT
The authors are grateful to Peter Shwonek for data analysis,
Kaye Welch for editorial assistance, and the staff of the Drug
Dependence Research Center, Langley Porter Psychiatric Institute
for assistance in carrying out the clinical study. Financial support
from the National Institute on Drug Abuse, National Institutes of
Health (Contracts N01DA-4-8306 and N01DA-0-8807 and Grants
DA12393 and DA00053) is gratefully acknowledged.
Figure 8. Effects of intravenous cocaine and transdermal selegiline
on MHPG sulfate excretion, and SRM chromatogram from clinical
study subject’s urine.
Received for review February 15, 2002. Accepted August
2, 2002.
excretion of MHPG sulfate as a measure of central norepinephrine
metabolism. The data are presented in Figure 8. Interestingly,M-
AC020101A
5296 Analytical Chemistry, Vol. 74, No. 20, October 15, 2002