Excretion and metabolism of milnacipran in humans after oral administration of milnacipran hydrochloride

Article abstract of DOI:10.1124/dmd.112.045120

The pharmacokinetics, excretion, and metabolism of milnacipran were evaluated after oral administration of a 100-mg dose of [¹⁴C] milnacipran hydrochloride to healthy male subjects. The peak plasma concentration of unchanged milnacipran (～240 ng/ml) was attained at 3.5 h and was lower than the peak plasma concentration of radioactivity (～679 ng Eq of milnacipran/ml) observed at 4.3 h, indicating substantial metabolism of milnacipran upon oral administration. Milnacipran has two chiral centers and is a racemic mixture of cis isomers: d-milnacipran (1S, 2R) and l-milnacipran (1R, 2S). After oral administration, the radioactivity of almost the entire dose was excreted rapidly in urine (approximately 93% of the dose). Approximately 55% of the dose was excreted in urine as unchanged milnacipran, which contained a slightly higher proportion of d-milnacipran (～31% of the dose). In addition to the excretion of milnacipran carbamoyl O-glucuronide metabolite in urine (～19% of the dose), predominantly as the l-milnacipran carbamoyl O-glucuronide metabolite (～17% of the dose), approximately 8% of the dose was excreted in urine as the N-desethyl milnacipran metabolite. No additional metabolites of significant quantity were excreted in urine. Similar plasma concentrations of milnacipran and the l-milnacipran carbamoyl O-glucuronide metabolite were observed after dosing, and the maximum plasma concentration of l-milnacipran carbamoyl O-glucuronide metabolite at 4 h after dosing was 234 ng Eq of milnacipran/ml. Lower plasma concentrations (<25 ng Eq of milnacipran/ml) of N-desethyl milnacipran and d-milnacipran carbamoyl O-glucuronide metabolites were observed. Copyright

Full text of DOI:10.1124/dmd.112.045120

1521-009X/12/4009-1723–1735$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD 40:1723–1735, 2012
Vol. 40, No. 9
45120/3787064
Excretion and Metabolism of Milnacipran in Humans after Oral
Administration of Milnacipran Hydrochloride
Fanying Li, Christina Chin, Julie Wangsa, and John Ho
Forest Research Institute, Farmingdale, New York
Received February 15, 2012; accepted May 31, 2012
ABSTRACT:
The pharmacokinetics, excretion, and metabolism of milnacipran tion of d-milnacipran (ϳ31% of the dose). In addition to the excre-
were evaluated after oral administration of a 100-mg dose of tion of milnacipran carbamoyl O-glucuronide metabolite in urine
14C]milnacipran hydrochloride to healthy male subjects. The peak (ϳ19% of the dose), predominantly as the l-milnacipran carbamoyl
plasma concentration of unchanged milnacipran (ϳ240 ng/ml) was O-glucuronide metabolite (ϳ17% of the dose), approximately 8%
attained at 3.5 h and was lower than the peak plasma concentra- of the dose was excreted in urine as the N-desethyl milnacipran
[
tion of radioactivity (ϳ679 ng Eq of milnacipran/ml) observed at metabolite. No additional metabolites of significant quantity were
4.3 h, indicating substantial metabolism of milnacipran upon oral excreted in urine. Similar plasma concentrations of milnacipran
administration. Milnacipran has two chiral centers and is a racemic and the l-milnacipran carbamoyl O-glucuronide metabolite were
mixture of cis isomers: d-milnacipran (1S, 2R) and l-milnacipran observed after dosing, and the maximum plasma concentration of
(1R, 2S). After oral administration, the radioactivity of almost the l-milnacipran carbamoyl O-glucuronide metabolite at 4 h after dos-
entire dose was excreted rapidly in urine (approximately 93% of the ing was 234 ng Eq of milnacipran/ml. Lower plasma concentrations
dose). Approximately 55% of the dose was excreted in urine as
(<25 ng Eq of milnacipran/ml) of N-desethyl milnacipran and d-
unchanged milnacipran, which contained a slightly higher propor- milnacipran carbamoyl O-glucuronide metabolites were observed.
Introduction
of [14C]milnacipran hydrochloride to healthy male subjects. In addi-
tion to determining the pharmacokinetics of milnacipran and radioac-
tivity, the plasma concentrations of milnacipran metabolites were
measured. The excretion of radioactivity, milnacipran, and its metab-
olites were also measured. Furthermore, the chemical structures of
milnacipran metabolites were identified.
Milnacipran is a serotonin and norepinephrine reuptake inhibitor,
and milnacipran hydrochloride is approved in the United States for
management of fibromyalgia (Savella, Forest Laboratories, Inc.,
2009) and in other countries for the treatment of depression (Puech et
al., 1997). Milnacipran (Z-2-aminomethyl-1-phenyl-N,N-diethylcy-
clopropane carboxamide) has two chiral centers and is a racemic
mixture of cis isomers: d-milnacipran (1S, 2R) and l-milnacipran (1R,
2S). Results from previous pharmacokinetic studies in humans
(Puozzo and Leonard, 1996; Puozzo et al., 2002) have shown that
milnacipran is rapidly absorbed from the gastrointestinal tract with a
median Tmax of 2 h. The absolute oral bioavailability of milnacipran
is high (ϳ85%), and absorption is not affected by food intake. The
plasma half-life of milnacipran is approximately 8 h, and approxi-
mately 50 to 60% of the dose is excreted in urine as unchanged
milnacipran.
Materials and Methods
Chemicals. [14C]Milnacipran hydrochloride with radiochemical purity of
99.0% was synthesized at GE Healthcare (Chalfont St. Giles, Buckingham-
shire, UK). Milnacipran, d- and l-milnacipran, and N-desethyl milnacipran
standards were obtained from Forest Research Institute (Commack, NY).
Glusulase containing 90,000 units/ml ␤-glucuronidase and 19,000 units/ml
sulfatase was purchased from PerkinElmer Life and Analytical Sciences
(Waltham, MA). Magnesium chloride hexahydrate, L-␣-phosphatidylcholine,
D-saccharic acid 1,4-lactone, and UDP-glucuronic acid (UDPGA) trisodium
salt were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile, deion-
ized water, glacial acetic acid, 12 N hydrochloric acid, methanol, potassium
phosphate monobasic, and 10 N sodium hydroxide were purchased from
Thermo Fisher Scientific (Waltham MA). Anhydrous sodium acetate and
sodium phosphate dibasic were purchased from Mallinckrodt Baker, Inc.
(Phillipsburg, NJ). Ammonium acetate and ammonium formate were pur-
chased from Fluka Chemical (St. Louis, MO). Human hepatic microsomes
were purchased from BD Gentest (Woburn, MA). Carbon dioxide was pur-
chased from the BOC Group, Inc. (Murray Hill, NJ). All chemicals were
analytical grade or better, unless stated otherwise.
The objective of this study was to investigate the metabolism of
milnacipran in humans after the oral administration of a 100-mg dose
This work was supported by Forest Research Institute. Medical writing and
editorial assistance was also supported by Forest Research Institute.
The clinical phase of this study was conducted by Quintiles Inc. (Overland
Park, KS). The authors confirmed that the article is an accurate representation of
the study results.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
Human Mass Balance Study. In this single-center open-label study, six
http://dx.doi.org/10.1124/dmd.112.045120.
healthy male subjects received a single oral dose of 100 mg of [14C]milnaci-
ABBREVIATIONS: UDPGA, UDP-glucuronic acid; LC, liquid chromatography; MS/MS, tandem mass spectrometry; HPLC, high-performance
liquid chromatography; SRM, selected reaction monitoring; LSC, liquid scintillation counting; HRMS, high-resolution mass spectrometry; AUC,
area under the curve; dpm, disintegrations per minute.
1723

1724
LI ET AL.
over a 5-min period using the Packard Tri-Carb 3100 TR Liquid Scintillation
TABLE 1
Analyzer and then converted to milnacipran concentration (nanogram equiv-
alents of milnacipran per gram) on the basis of the specific activity of the dose.
The oxidizer recovery was determined by combustion of 14C standards in an
identical manner. The amounts of radioactivity (expressed in milnacipran)
excreted in urine and feces were determined and used to evaluate mass balance
of the dose.
Plasma pharmacokinetics of 14C radioactivity and milnacipran after oral
administration of a 100-mg dose of [14C]milnacipran hydrochloride to four
healthy male subjects
Data are presented as means Ϯ S.D.
Pharmacokinetic Parameters
and Units
Milnacipran (% 14C
Radioactivity)
14C Radioactivitya
Milnacipran
Analysis of Unchanged Milnacipran. The plasma concentration of un-
changed milnacipran was determined by a validated liquid chromatography-
tandem mass spectrometry (LC-MS/MS) method. After the supernatant of the
plasma sample (or standard) was mixed with the [2H10]milnacipran internal
standard and mobile phase [120 mM formic acid-water-methanol (200:1200:
600, v/v/v)], the components in the mixture were separated using a Symmetry
C8 column (100 ϫ 2.1 mm, 3.5 ␮m particle size; Waters, Milford, MA) with
an isocratic elution of mobile phase at 0.25 ml/min. Temperatures of the
autosampler and the high-performance liquid chromatography (HPLC) column
were kept at 15 and 30°C, respectively. The LC system (Agilent 1100; Agilent
Technologies, Waldbronn, Germany) was interfaced to an API 3000 triple
quadruple mass spectrometer (AB Sciex, Toronto, ON, Canada). Electrospray
ionization of the mass spectrometer was set to positive ion multiple reaction
monitoring mode (precursor ion 3 product ion) as follows: milnacipran, m/z
247.1 3 230.2; internal standard, m/z 257.1 3 240.2. Protonated molecular
ions of milnacipran and the internal standard were the precursor ions for this
analysis. The ratio of milnacipran product ion peak area to that of its internal
standard (peak area ratio of milnacipran) was the response used for quantifi-
cation. The method was linear over a milnacipran concentration range of 5 to
2000 ng/ml with a lower limit of quantification of 5 ng/ml in 25 ␮l of human
plasma. Quality control samples containing 15, 500, and 1600 ng/ml milnaci-
pran were also analyzed. Unless stated otherwise, the concentration of milnaci-
pran determined by LC-MS/MS analysis is expressed as the amount of mil-
nacipran free base per unit volume.
Metabolite Profile Analysis. To acquire sufficient volumes for qualitative
and quantitative analyses of the plasma metabolites, samples from the four
subjects who did not vomit were used to prepare 14 plasma pools according to
time of collection (0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 18, and 24 h
postdose). One milliliter of plasma from each subject was used to prepare the
pool. Unless stated otherwise, the analysis of the plasma pools was simply
described as the analysis of the plasma. Urine samples (0–4, 4–8, 8–12,
12–24, 24–48, 48–72, and 72–96 h postdose) from the four subjects were also
analyzed to establish the metabolite profile. For the chiral LC-selected reaction
monitoring (LC-SRM) analysis of milnacipran enantiomers, a 0 to 96 h urine
pool was prepared for each subject by mixing 0.1% (by volume) of each urine
sample of the subject collected in 96 h after dosing.
Cmax, ng/ml
AUC0–t, ng ⅐ h/ml
AUC0–ϱ, ng ⅐ h/ml
Tmax, h
679.0 Ϯ 112.6
6446.3 Ϯ 666.3 2342.6 Ϯ 252.0
7325.3 Ϯ 622.1 2766.5 Ϯ 413.2
239.6 Ϯ 33.2
35.3
36.3
37.8
4.3 Ϯ 0.5
7.7 Ϯ 1.0
3.5 Ϯ 1.0
8.9 Ϯ 3.2
t1/2, h
a Units for Cmax are nanogram equivalents of milnacipran times milliliter; units for AUC0–t
and AUC0–ϱ are nanogram equivalents of milnacipran times hour per milliliter.
pran hydrochloride solution (5 ml of 20 mg/ml) at 8:00 AM after breakfast at
7:30 AM. The specific activity of the dose was 1 ␮Ci/mg milnacipran hydro-
chloride. After oral administration of [14C]milnacipran hydrochloride to pig-
mented mice, rats, and monkeys, radioactivity (milnacipran and/or metabo-
lites) was distributed into the tissues of the animals (data on file, company
study reports). Although radioactivity was observed in the uveal tract (a
melanin-rich tissue) of the pigmented mice and monkeys, the elimination of
radioactivity from the uveal tract of the animals’ eyes indicated that a metab-
olism study could be safely conducted in humans using a 100 ␮Ci dose of
[
14C]milnacipran hydrochloride.
Blood samples were collected in prechilled tubes containing tripotassium
EDTA before dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 18, 24, 48,
72, 96, and 120 h postdose. Within 30 min from time of collection, plasma was
harvested after the centrifugation of blood samples at Ն2500g for 10 min at
4°C. Urine samples were collected and pooled from Ϫ2 to 0 h predose and at
0 to 4, 4 to 8, 8 to 12, 12 to 24, 24 to 48, 48 to 72, 72 to 96, and 96 to 120 h
postdose. Feces samples were collected from day Ϫ1 to day 6 and were pooled
into 24-h samples (0–24, 24–48, 48–72, 72–96, and 96–120 h); a predose
feces sample was collected and pooled from Ϫ12 to 0 h. After collection, all
samples (plasma, urine, and feces) were kept frozen at Ϫ80°C until analysis.
Subjects were released from the study on day 6 after one of the following
criteria had been met: 1) two consecutive urine and feces samples contained
Ͻ3 times the radioactivity of the background or 2) the radioactivity excreted
in a day was Ͻ1% of the radioactivity in the dose (expressed simply as
percentage of the dose).
After oral administration of [14C]milnacipran hydrochloride, two of the six
subjects vomited (data on file, company study report). Pharmacokinetic anal-
ysis and the evaluation of mass balance and metabolite profile were conducted
on samples collected from the remaining four subjects. However, the metab-
olite profile in feces was not evaluated because Ͻ4% of the dose was excreted
in feces.
All subjects completed an informed consent form at prescreening for the
study. The study was approved by the Heartland Institutional Review Board
and conducted at Quintiles Inc. (Kansas City, MO) in accordance with the
International Conference on Harmonisation Guidance on General Consider-
ations for Clinical Trials, Nonclinical Safety Studies for the Conduct of Human
Clinical Trials for Pharmaceuticals, and Good Clinical Practice: Consolidated
Guidance.
Sample preparation. After 3 ml of acetonitrile was mixed with 1 ml of
plasma sample by vortex for approximately 30 s, the mixture was kept at room
temperature for approximately 10 min and then centrifuged at 14,000 rpm for
15 min. The supernatant was transferred to a clean test tube and evaporated to
dryness in a TurboVap evaporator under a gentle flow of nitrogen. The sample
residue was reconstituted in 400 ␮l of 1 M ammonium acetate (pH 5)-water-
TABLE 2
Cumulative excretion after oral administration of a 100-mg dose of
[
14C]milnacipran hydrochloride to four healthy male subjects
Radioactivity Analysis of Plasma, Urine, and Feces Samples. After an
aliquot of plasma or urine sample was mixed with 10 ml of Ultima Gold
scintillant (PerkinElmer Life and Analytical Sciences), the radioactivity con-
centration of the sample was measured for 5 min using a Packard Tri-Carb
3100 TR Liquid Scintillation Analyzer (PerkinElmer Life and Analytical
Sciences). The radioactivity concentration was converted to milnacipran con-
centration (nanogram equivalents of milnacipran per milliliter) based on the
specific activity of the dose. Feces samples were homogenized in water using
a Tekmar Lab Blender (Teledyne Technologies, Inc., Mason, OH). Weighed
aliquots, in triplicate, of each homogenized feces sample (simply expressed as
feces samples unless stated otherwise) were combusted in a Packard Sample
Oxidizer model 307 (PerkinElmer Life and Analytical Sciences). After the
addition of Carbo-Sorb E and Permafluor E scintillant (10 ml each; PerkinEl-
mer Life and Analytical Sciences) to the combustion products of the feces
samples, the radioactivity concentration of the feces samples was measured
Data are presented as means Ϯ S.D.
Excretion
Period
Urine
Feces
Total
% dose
0–4 h
0–8 h
24.8 Ϯ 1.9
48.5 Ϯ 5.1
60.8 Ϯ 6.7
79.4 Ϯ 4.6
89.3 Ϯ 2.8
92.1 Ϯ 2.2
92.8 Ϯ 1.7
93.1 Ϯ 1.5
93.3 Ϯ 1.4
N.A.
N.A.
N.A.
24.8 Ϯ 1.9
48.5 Ϯ 5.1
60.8 Ϯ 6.7
79.6 Ϯ 4.8
92.2 Ϯ 3.2
95.4 Ϯ 2.0
96.5 Ϯ 1.1
96.8 Ϯ 0.7
96.9 Ϯ 0.6
0–12 h
0–24 h
0–48 h
0–72 h
0–96 h
0–120 h
0–144 h
0.407 Ϯ 0.2
2.83 Ϯ 0.9
3.27 Ϯ 1.5
3.62 Ϯ 1.1
3.65 Ϯ 1.1
3.65 Ϯ 1.1
N.A., not applicable because no feces samples were collected.

EXCRETION AND METABOLISM OF MILNACIPRAN IN HUMANS
1725
FIG. 1. HPLC chromatogram of the 1.5-h
plasma pool (A) and the 4 to 8 h urine
sample (B) of subject 3 after oral adminis-
tration of a 100-mg dose of [14C]milnaci-
pran hydrochloride. ء, the retention times
of the chromatographic peaks observed in
LSC of the HPLC fraction (i.e., procedure
for metabolite profile of plasma) was ap-
proximately 1 to 2 min longer than those
observed in the radioactivity flow detector
because of the extra tubing used for collect-
ing the HPLC fractions.
methanol (2:62.4:35.6, v/v/v) to become the processed plasma sample for elution with flow rate of the mobile phase set at 1 ml/min. Mobile phase A was
analysis. Urine samples were analyzed directly without sample preparation.
Sample analysis. After injection of the urine or processed plasma sample (in
200-␮l aliquots) into an Alliance 2690 Separations Module HPLC system
(Waters), the components of the sample were separated on a Luna Phenyl-
1 M ammonium acetate (pH 5)-water-methanol (20:930:50, v/v/v); mobile
phase B was 1 M ammonium acetate (pH 5)-water-methanol (20:30:950,
v/v/v). The gradient elution was initiated at 34% of mobile phase B for 29 min,
ramped to 90% over 11 min, maintained at 90% for 5 min, and returned to the
Hexyl column (250 ϫ 4.6 mm, 5 ␮m particle size; Phenomenex, Torrance, initial condition over 1 min. The column was equilibrated for 9 min at the
CA) with an Eclipse XDB-C18 guard column (12.5 ϫ 4.6 mm, 5 ␮m particle initial condition between injections. After injection of the processed plasma
size; Agilent Technologies). Temperatures of the autosampler and HPLC sample onto the HPLC column, the HPLC eluent was collected in 0.5-min
column were kept at 10 and 35°C, respectively. The analysis used a gradient
fractions over a period of 54 min, totaling 108 fractions collected from each
sample. The radioactivity of each fraction was determined by LSC. The HPLC
TABLE 3
TABLE 4
Plasma concentration of milnacipran and its metabolites after oral
administration of a 100-mg dose of [14C]milnacipran hydrochloride to four
healthy male subjects
Metabolite profiles of urine after oral administration of a 100-mg dose of
[
14C]milnacipran hydrochloride to four healthy male subjects
Plasma Concentration
Data are mean values, calculated on the basis of the number of samples that contained a
peak.
Peak 3
Peak 4
Time
Peak 1
(l-Milnacipran
Carbamoyl
(d-Milnacipran
Carbamoyl
O-Glucuronide)
Peak 5
(Milnacipran)
Excretion
(N-Desethyl
Milnacipran)
Peak 2
Period
O-Glucuronide)
Peak 1
Peak 2
Peak 3
% dose
Peak 4
Peak 5
Total
ng Eq of milnacipran/ml
0.5 h
1 h
1.5 h
2 h
3 h
4 h
BLOD
BLOD
BLOD
BLOD
24.60
19.63
23.46
13.67
BLOD
26.57
101.66
161.30
190.14
195.72
234.38
140.79
50.79
BLOD
BLOD
BLOD
BLOD
16.42
17.49
BLOD
BLOD
33.92
83.17
0–4 h
4–8 h
0.92
1.35
1.26
2.06
1.36
0.73
0.06
7.73
0.43
0.43
0.21
0.28
0.13
BLOD
0.07
1.33
6.26
5.39
2.36
2.36
0.76
0.19
BLOD
17.23
0.55
0.64
0.37
0.58
0.28
BLOD
BLOD
2.14
15.54
14.42
7.17
10.87
5.45
1.47
0.30
54.71
BLOD
BLOD
9.12
156.73
178.93
208.01
214.21
186.82
87.22
8–12 h
12–24 h
24–48 h
48–72 h
72–96 h
0–96 h
12.53
BLOD
BLOD
BLOD
6 h
12 h
84.26
BLOD, below limit of detection (i.e., concentration Ͻ7.8 ng Eq of milnacipran/ml).
BLOD, below limit of detection for on-column analysis (approximately 396 cpm/200 ␮l).

1726
LI ET AL.
Biosynthesis of d- and l-milnacipran carbamoyl O-glucuronide. Based on
TABLE 5
modifications of a published method (Delbressine et al., 1990), d-milnacipran
and l-milnacipran were incubated with carbon dioxide, human hepatic micro-
somes, and UDPGA. The resulting d- and l-milnacipran carbamoyl O-glucu-
ronides were purified by HPLC separation and became the biosynthetic stan-
dards. The glucuronides were not produced in the absence of carbon dioxide,
microsomes, or UDPGA from the incubation. The chemical structures of the
biosynthetic standards were characterized by LC-MS/MS, LC-HRMS, and
hydrolysis.
LC-MS/MS. Metabolites were identified on an Alliance 2795 Separations
Module system (Waters) set at the HPLC conditions described above for the
metabolite profile analysis. The LC system was interfaced to a TSQ 7000 triple
quadruple mass spectrometer (Thermo Fisher Scientific) with the atmosphere
pressure chemical ionization source set at positive ion mode. Metabolites
were initially characterized by comparing predose with postdose samples using
a full scan (from m/z 100 to 650) and profile mode set at 1 s of scan time.
Vaporization and capillary temperature were maintained at 500 and 200°C,
respectively. Sheath gas (nitrogen) was 60 psi, and corona discharge was 5 ␮A.
The electron multiplier was adjusted to obtain the optimum signals. Structural
information of the metabolites was further generated from the product ion
spectra of their respective protonated molecular ions.
Urinary excretion of milnacipran enantiomers after oral administration of a 100-
mg dose of [14C]milnacipran hydrochloride to four healthy male subjects
Excretion in 0–96 h Urine Sample
Subject
l-Milnacipran
d-Milnacipran
Milnaciprana
% dose
33.6
28.6
32.4
30.4
31.2
1
2
3
22.1
24.4
23.8
23.6
23.5
55.7
53.0
56.2
54.0
54.7
4
Mean
a Data are from metabolite profiles of urine (Table 4).
eluent from analysis of urine sample was monitored by a radioactivity flow
detector with a detection cell that contained solid scintillant (high-pressure lithium
glass-packed, 150 ␮l; IN/US, Tampa, FL). The radioactivity detector was set at
6 s for dwell time and 9 s for time of flight. The HPLC eluent was also
monitored by a UV detector. The wavelength of the UV detector was set at
220 nm with a filter of 1 s. The nonradioactive standards were monitored
by the UV detector.
LC-SRM. To improve sensitivity, identification of plasma metabolites was
conducted by SRM of the metabolites based on their product ion spectra. The SRM
was set to positive ion mode (precursor ion 3 product ion) as follows: milnacipran
Identification of Metabolites. Five chromatographic peaks with measur-
able radioactivity were found in the plasma and urine metabolite profiles. The
metabolites eluted in these peaks were identified using LC-MS/MS, LC-SRM,
and LC-high-resolution mass spectrometry (LC-HRMS) methods. Biosyn- carbamoyl O-glucuronide, m/z 467 3 230; milnacipran, m/z 247 3 230. Proton-
thetic standards were prepared as described below.
ated molecular ions of milnacipran carbamoyl O-glucuronide and milnacipran
FIG. 2. LC-MS/MS analysis of N-des-
ethyl milnacipran metabolite (peak 1, pre-
cursor ion ϭ protonated molecular ion,
m/z 219) in the 0 to 4 h urine sample of
subject 1. A, protonated molecular ion of
N-desethyl milnacipran metabolite. B,
product ion spectrum produced by the
protonated molecular ion of N-desethyl
milnacipran metabolite.

EXCRETION AND METABOLISM OF MILNACIPRAN IN HUMANS
1727
were the precursor ions for this analysis. The purpose of measuring milnacipran ammonium acetate (pH 5)-water-methanol (5:945:50, v/v/v); mobile phase B,
was to evaluate the potential degradation of the glucuronide to milnacipran in the
ionization source of the mass spectrometer (i.e., in-source degradation).
1 M ammonium acetate (pH 5)-water-methanol (5:45:950, v/v/v). Postcolumn
flow was split such that the mobile phase was introduced into the mass
spectrometer via an electrospray interface at a rate of 100 ␮l/min; the remain-
ing flow was diverted as waste. The protonated molecular ion of the metabolite
was obtained by time-of-flight scan (m/z 100-1000 at an accumulation time of
1 s). The source temperature was maintained at 220°C, and the IonSpray
voltage was set at 5500 V. Curtain gas, ion source gas 1, and ion source gas
2 were set at 20, 30, and 10, respectively. Declustering potential 1, declustering
potential 2, and focusing potential were set at 30, 15, and 80 V, respectively.
Both ion release delay and ion release width were set at 10. The electron
multiplier was adjusted to obtain optimum signals.
Chiral LC-SRM analysis, conducted on an Alliance 2795 Separations Mod-
ule (Waters), was used to analyze milnacipran enantiomers in plasma and urine
samples. The milnacipran enantiomers were separated on a Chirobiotic V
column (150 ϫ 4.6 mm, 5-␮m particle size; Advanced Separation Technolo-
gies, Inc., Whippany, NJ) with a Chirobiotic V guard column (20 ϫ 4.0 mm;
Advanced Separation Technologies, Inc.); the column was eluted with a
mobile phase of 1 M ammonium formate-methanol (1:799, v/v) at a flow rate
of 1 ml/min. Temperatures of the autosampler and HPLC column were kept at
10 and 20°C, respectively. The SRM setting of milnacipran described above
was used for the SRM of milnacipran enantiomers (m/z 247 3 230).
Hydrolysis of Urine Metabolites. After isolation of peak 3 and peak 4 from
LC-HRMS. Structure characterization of metabolites was conducted using the 0 to 4 h urine samples of two subjects, the hydrolytic properties of the
an Agilent 1100 series HPLC system (Agilent Technologies) set at the HPLC
conditions described above for the metabolite profile analysis. The LC system
was interfaced to a QSTAR XL mass spectrometer (AB Sciex). Mobile phases
metabolites eluted in these peaks were established to support the structures of
the milnacipran metabolites.
Isolation. Peak 3 and peak 4 in 1 ml of the 0 to 4 h urine sample collected
were modified as follows to minimize ion suppression: mobile phase A, 1 M from subject 1 were isolated after HPLC analysis of the urine sample as five
FIG. 3. The LC-MS/MS chromatogram
from the analysis of chemically synthe-
sized and biosynthetic standards. The re-
tention times of the chromatographic
peaks observed in LC-MS/MS analysis
were approximately 1 to 2 min different
from those observed in HPLC-radioactiv-
ity flow detector analysis because of the
different HPLC instrument used. ء,
chemically synthesized standard; ءء, bio-
synthetic standard.

1728
LI ET AL.
200-␮l aliquots. The HPLC eluent collected between 22 to 25 min was
evaporated to dryness in a TurboVap under a gentle flow of nitrogen at 30°C,
obtain retention times and maximum signals of the chromatographic peaks
(peak area in counts per minute). The background radioactivity was estimated
and the dry residue was then reconstituted by sonification in 250 ␮l of water from a blank region of the chromatogram selected arbitrarily after the elution
and 20 ␮l of acetonitrile to produce isolated peak 3 of subject 1. The HPLC
eluent collected between 26.5 to 28.5 min was also processed to produce the
isolated peak 4 of subject 1. Likewise, isolated peak 3 and isolated peak 4 of
subject 3 were prepared.
of all chromatographic peaks. The net amount of radioactivity in the chro-
matographic peak was calculated by subtracting the background radioactivity
from the radioactivity in the chromatographic peak. Likewise, the net amount
of radioactivity in a HPLC fraction of a plasma sample was calculated by
subtracting the background radioactivity of a blank HPLC fraction from the
radioactivity in the fraction.
Plasma metabolite profile. The plasma concentration of a metabolite eluted
as a chromatographic peak in the metabolite profile of a plasma sample pool
was calculated as follows:
Acid hydrolysis. After the sample (40 ␮l of isolated peak 3 or peak 4) or
biosynthetic standard was mixed with 40 ␮l of 2 N hydrochloric acid in a glass
test tube, the test tube was capped tightly and kept on a heating block at 70°C
for 1 h. The samples were then cooled to ambient room temperature and
partially neutralized by addition of 24 ␮l of 2 N sodium hydroxide. These
partially neutralized samples were mixed gently by vortex for approximately
30 s, transferred to HPLC vials, and mixed with 24 ␮l of water and 52 ␮l of
1 M ammonium acetate (pH 5)-water-methanol (2:62.4:35.6, v/v/v). After
centrifugation of the HPLC vials for approximately 5 min, 20 ␮l of the clear
supernatant (hydrolyzed samples) were analyzed by LC-SRM. A nonhydro-
lyzed control sample [40 ␮l of sample, 52 ␮l of 1 M ammonium acetate (pH
5)-water-methanol (2:62.4:35.6, v/v/v), 24 ␮l of water, and 64 ␮l of a reagent
containing 40 ␮l of 2 N hydrochloric acid and 24 ␮l of 2 N sodium hydroxide]
was similarly analyzed.
Plasma concentration
of the metabolite in
the plasma sample pool
(ng Eq of
Net amount of radioactivity
in chromatographic peak
of a metabolite
(% total radioactivity
in the chromatogram)
Concentration of
radioactivity in the
plasma sample pool
(ng Eq of
ϭ
ϫ
milnacipran/ml)
milnacipran/ml)
The concentration of radioactivity in the plasma sample pool (nanogram
equivalents of milnacipran per milliliter) was calculated on the basis of the
concentration of radioactivity in the plasma samples (data on file, company
Enzyme hydrolysis. After the sample (40 ␮l of isolated peak 3 or peak 4)
or biosynthetic standard was mixed with 60 ␮l of 0.2 M sodium acetate buffer
at pH 5, 10 ␮l of water, and 10 ␮l of Glusulase in a glass test tube, the sample
was incubated in a water bath at 37°C for 1 h. The samples were then cooled
to ambient room temperature, transferred to HPLC vials, and mixed with 8 ␮l
of water and 52 ␮l of 1 M ammonium acetate (pH 5)-water-methanol (2:62.4:
35.6, v/v/v). After centrifugation of the HPLC vials for approximately 5 min,
20 ␮l of the clear supernatant (hydrolyzed samples) was analyzed by LC-SRM.
A control sample without hydrolysis by Glusulase was analyzed similarly. The
inhibition of hydrolysis was evaluated by replacing the water in the enzyme
hydrolysis procedure with 100 mg/ml D-saccharic acid 1,4-lactone.
Data Analysis. Pharmacokinetics and mass balance. Radioactivity concen-
trations in plasma, urine, and feces samples were determined at the clinical site
using LSC, with combustion as necessary. Raw data (weight/volume and
radioactivity concentration) of urine and feces samples collected from each
subject were captured in the Debra data capture system (LabLogic Systems,
Ltd., Sheffield, UK). Descriptive statistics were used to analyze the excretion
of radioactivity in urine and feces (percentage of the dose), as well as the
pharmacokinetic parameters of milnacipran and radioactivity.
Pharmacokinetic assessments [area under the plasma concentration-time
curve from time 0 up to the last measurable concentration (AUC0–t), AUC
from time 0 up to infinity (AUC0–ϱ), maximum plasma concentration (Cmax),
time to maximum plasma concentration (Tmax), terminal elimination half-life
(t1/2), and terminal rate constant (␭z)] were derived using noncompartmental
analysis (Gibaldi and Perrier, 1975) with WinNonlin software (version 4.1 or
higher; Pharsight, Mountain View, CA). Plasma concentrations below the limit
of quantification were treated as zero for pharmacokinetic calculations with
actual sampling times.
The amount of radioactivity excreted in a sample [disintegrations per minute
(dpm)] was calculated by multiplying the radioactivity concentration of the
sample (disintegrations per minute per milliliter, urine; disintegrations per
minute per gram, feces) by the amount of the sample (milliliters, urine; grams,
feces). The amount of the radioactivity excreted in a sample (percentage of the
dose) was calculated by the following equation:
Amount of radioactivity
Amount of radioactivity
excreted in a sample (dpm)
excreted in a sample
(% dose)
ϭ
ϫ 100%
Amount of radioactivity
in the dose (dpm)
The cumulative excretion in urine and the cumulative excretion in feces for
each subject were determined by summing up the excretion at each time
interval. Mass balance (the total recovery of radioactivity in the dose) of
milnacipran in each subject was the sum of the cumulative excretions in urine
and in feces (percentage of the dose) for that subject.
Metabolite profile. The chromatograms of urine samples were recorded and
processed by Laura 3 software (version 3.096.119; LabLogic Systems Ltd.) to
FIG. 4. Proposed fragmentation pattern for the protonated molecular ion of N-
desethyl milnacipran metabolite, [M ϩ H]ϩ, m/z 219.

EXCRETION AND METABOLISM OF MILNACIPRAN IN HUMANS
1729
study report) used for the preparation of the pool. The net amount of
Proportion of
milnacipran
enantiomer
Amount of
milnacipran
enantiomer
radioactivity in the chromatographic peak of the metabolite is expressed as
a percentage of total radioactivity found in the chromatogram.
Urine metabolite profile. The excretion (percentageof the dose) of a me-
tabolite eluted as a chromatographic peak in the metabolite profile of a urine
sample was calculated as follows:
Amount of
milnacipran
in the
excreted in the
ϭ
ϫ
excreted in the
0–96 h urine pool
(% dose)
0–96 h urine pool
(% milnacipran in
the urine pool)
0–96 h urine pool*
(% dose)
ءData on file, company study report.
Net amount of
Amount of
The proportion of milnacipran enantiomer (d- and l-milnacipran) in the 0 to
96 h urine pool (% of milnacipran in the urine pool) was calculated for each
subject using the following set of equations:
Amount of the
metabolite excreted
in the urine
radioactivity in
chromatographic
radioactivity
excreted in the
urine sample*
(% dose)
ϭ
ϫ
peak of a metabolite
(% total radioactivity
in the chromatogram)
sample (% dose)
Proportion of
l-milnacipran
Peak area of l-milnacipran ϫ 100%
enantiomer in
the 0–96 h
urine pool
ϭ
ءData on file, company study report.
Peak area of
l-milnacipran
Peak area of
d-milnacipran of urine standard
Peak area ratio
ϩ
ϫ
ͩ
ͪ
The net amount of radioactivity in the chromatographic peak of the me-
tabolite is expressed as a percentage of total radioactivity found in the
chromatogram.
Enantiomer composition of milnacipran in urine sample. After chiral
LC-SRM analysis of the 0 to 96 h urine pool or urine standard, detector output
was recorded and analyzed using Xcalibur software (version 1.1; Thermo
Fisher Scientific). Each chromatogram was analyzed to obtain retention times
and maximum signals (peak areas) for d- and l-milnacipran. The amount of
milnacipran enantiomer (d- or l-milnacipran) excreted in the 0 to 96 h urine
pool (% of the dose) of an individual subject was calculated as follows:
Peak area of l-milnacipran in urine standard
Peak area ratio
of urine standard*
ϭ
Peak area of d-milnacipran in urine standard
ءUrine standard was a racemic mixture with equal amounts of d- and l-milnacipran.
Proportion of
Proportion of
l-milnacipran enantiomer
d-milnacipran enantiomer
ϭ 100% Ϫ
in the 0–96 h
urine pool
in the 0–96 h urine pool
FIG. 5. LC-MS/MS analysis of l-milnaci-
pran carbamoyl O-glucuronide metabolite
(peak 3, precursor ion ϭ protonated molec-
ular ion, m/z 467). A, protonated molecular
ion of l-milnacipran carbamoyl O-glucu-
ronide metabolite in the 0 to 4 h urine
sample of subject 1. B, product ion spec-
trum produced by the protonated molecular
ion of l-milnacipran carbamoyl O-glucu-
ronide metabolite in the 0 to 4 h urine
sample of subject 3.

1730
LI ET AL.
Results
concentration and half-life were 3.5 and 8.9 h, respectively, for
unchanged milnacipran, compared with 4.3 and 7.7 h for 14C radio-
activity, respectively.
Pharmacokinetics and Mass Balance. After oral administration of
a 100-mg dose of [14C]milnacipran hydrochloride, the average peak
plasma concentration among the four male healthy subjects without
emesis was 239.6 ng/ml for unchanged milnacipran and 679.0 ng Eq
of milnacipran/ml for 14C radioactivity (Table 1), indicating that
unchanged milnacipran contributed approximately 35% of the plasma
radioactivity at the peak plasma concentration. The AUC0–t of un-
changed milnacipran was 2342.6 ng ⅐ h/ml and similarly contributed
approximately 36% of the AUC0–t of 14C radioactivity (6446.3 ng Eq
of milnacipran ⅐ h/ml). The results indicate that milnacipran metabo-
lites were produced and observed in plasma after the oral administra-
Approximately 97% of the radioactivity in the dose was recovered
from the excreta (Table 2). On average, 93.3% of the dose (range,
91.3–94.3%) was excreted in urine, whereas 3.65% of the dose (range,
2.8–5.3%) was excreted in feces, indicating that urinary excretion was
the principal route of elimination.
Metabolite Profile. A brief validation of the metabolite profile
procedure was conducted to evaluate the sample processing recovery,
chromatography resolution, HPLC column recovery, linearity of the
analysis, and counting efficiency of the detector. The high recovery of
tion of milnacipran hydrochloride. The average time to peak plasma radioactivity from plasma (average 81.0% of the plasma radioactivity)
FIG. 6. The chromatograms from the anal-
ysis of the isolated peak 3 from the 0 to 4 h
urine sample of subject 3 before hydrolysis
(A), after treatment with 2 N hydrochloric
acid at 70°C for 1 h (B), after treatment
with Glusulase at 37°C for 1 h (C), and
after treatment with Glusulase and the in-
hibitor, D-saccharic acid 1,4-lactone, at
37°C for 1 h (D). Milnacipran, m/z 247 3
230, retention time ϳ34 min; l-milnacipran
carbamoyl O-glucuronide metabolite (peak
3), m/z 467 3 230, retention time ϳ23 min.

EXCRETION AND METABOLISM OF MILNACIPRAN IN HUMANS
1731
in sample processing, along with the high HPLC column recovery of The majority of milnacipran in the dose was excreted unchanged in
radioactivity from the sample (average 99.1% of the sample radioac- urine (peak 5, average 55% of the dose), with peak 3 (average 17% of
tivity) in the HPLC analysis provided a thorough analysis of metab- the dose) and peak 1 (average 7.7% of the dose) representing the
olites in the metabolite profile.
major metabolites excreted in urine. Small amounts of peak 2 and
Plasma metabolite profiles. A total of five chromatographic peaks peak 4 (neither exceeding 2.1% of the dose) were also excreted in
with measurable radioactivity were observed in the metabolite profile urine. No other chromatographic peaks with significant radioactivity
of plasma (Fig. 1A). Peak 3 was the major metabolite in plasma, with were observed in the urine metabolite profile. Chiral LC-SRM anal-
a maximum plasma concentration of approximately 234 ng Eq of ysis of the 0 to 96 h urine pools showed that the renal excretion of
milnacipran/ml (Table 3). The plasma concentration of peak 3 was d-milnacipran (31% of the dose) was slightly higher than that of
similar to that of milnacipran (peak 5) for all plasma samples from 0.5 l-milnacipran (24% of the dose) (Table 5).
to 12 h. The plasma concentration of each remaining peak of the
Identification of Milnacipran and Its Metabolites. The chemical
plasma metabolite profile was relatively low and did not exceed 25 ng structures of unchanged milnacipran (peak 5) and milnacipran metab-
Eq of milnacipran/ml. No other chromatographic peaks with signifi- olites (peaks 1, 3, and 4) were identified using LC-MS/MS, LC-SRM,
cant radioactivity were observed in the plasma metabolite profile.
and LC-HRMS methods. Identification of the metabolite in peak 2
Urine metabolite profiles. Five distinct chromatographic peaks was not attempted because Ͻ2% of the dose was excreted as this peak.
were also observed in the urine metabolite profiles (Fig. 1B; Table 4). For LC-MS/MS and LC-SRM analyses, the chemical structure of a
FIG. 7. The chiral LC-SRM analysis of ra-
cemic milnacipran standard (A), the l-mil-
nacipran produced from hydrolysis of the
isolated l-milnacipran carbamoyl O-glucu-
ronide metabolite (peak 3) (B), and the d-
milnacipran produced from hydrolysis of
the isolated d-milnacipran carbamoyl O-
glucuronide metabolite (peak 4) (C). Both
metabolites were isolated from the 0 to 4 h
urine sample of subject 1. l-Milnacipran,
m/z 247 3 230, retention time ϳ15 min;
d-milnacipran, m/z 247 3 230, retention
time ϳ16 min.

1732
LI ET AL.
metabolite was identified when retention time, protonated molecular proposed structure for the fragment of m/z 174. The fragment of m/z
ion, and mass spectrum (LC-MS/MS) or product ion (LC-SRM) were 131 was produced by addition of a hydrogen molecule (2 Da) to the
identical to those of the standard (biosynthetically or chemically fragment generated by neutral losses of ammonia (NH3, 17 Da) and
synthesized). For the LC-HRMS analysis, the chemical structure was carbon monoxide (CO, 28 Da) from the fragment of m/z 174. The
characterized when the protonated molecular ion of the metabolite carbonium ion of 1-methyl-2-phenyl-cyclopropane was the proposed
was within Ϯ15 ppm of the theoretical protonated molecular ion of structure for the fragment of m/z 131. The stability of the carbonium
the metabolite. Acid hydrolysis and enzyme hydrolysis were also ion was probably provided by the electrons of the phenyl ring. The
conducted to confirm glucuronide metabolites in peaks 3 and 4 of the protonated molecular ion (m/z 219.1478) of the metabolite observed in
urine samples from two subjects.
the LC-HRMS analysis was accurate (6.3 ppm from theoretical) and
Peak 1. The chemical structure of the metabolite eluted as peak 1 supported the proposed structure. Peak 1 in the metabolite profile of
in the metabolite profile of urine was identified as N-desethyl mil- plasma was also identified as the N-desethyl milnacipran metabolite
nacipran, which has a molecular weight of 218.29 and a monoisotopic using a similar procedure.
molecular weight of 218.14. The retention time (11 min), protonated
Peak 3. The chemical structure of the metabolite eluted as peak 3
molecular ion (m/z 219) (Fig. 2A), and mass spectrum (Fig. 2B) of the in the metabolite profile of urine was identified as l-milnacipran
metabolite observed in the LC-MS/MS analysis matched those of carbamoyl O-glucuronide, which has a molecular weight of 466.48
N-desethyl milnacipran standard (Fig. 3). A fragmentation scheme and a monoisotopic molecular weight of 466.20. The retention time
(Fig. 4) was proposed on the basis of the major fragments observed in (22 min), protonated molecular ion (m/z 467) (Fig. 5A), and mass
the mass spectrum (m/z 202, m/z 174, and m/z 131) (Fig. 2B). It spectrum (Fig. 5B) of the metabolite observed in the LC-MS/MS
appeared that the amine function of the molecule was protonated to analysis matched those of the l-milnacipran carbamoyl O-glucuronide
produce the protonated molecular ion (m/z 219). The fragment of m/z biosynthetic standard (Fig. 3). Major fragments of m/z 291, m/z 247,
202 was produced after a neutral loss of ammonia (NH3, 17 Da) from and m/z 230 were observed in the mass spectrum (i.e., product ion
the protonated molecular ion. The fragment of m/z 174 was produced spectrum) after the fragmentation of the protonated molecular ion. In
after an additional neutral loss of ethene (C2H4, 28 Da) from the addition, a minor fragment of m/z 449 was observed in the spectrum.
fragment of m/z 202. The protonated form of a lactam was the It appeared that a nitrogen atom of the molecule was protonated to
FIG. 8. LC-MS/MS analysis of unchanged
milnacipran (peak 5, precursor ion ϭ pro-
tonated molecular ion, m/z 247) in the 0 to
4 h urine sample of subject 1. A, protonated
molecular ion of unchanged milnacipran.
B, product ion spectrum produced by the
protonated molecular ion of unchanged
milnacipran.

EXCRETION AND METABOLISM OF MILNACIPRAN IN HUMANS
1733
produce the protonated molecular ion. The fragment of m/z 449 nacipran, which has a molecular weight of 246.35 and a monoisotopic
represented a neutral loss of a water molecule (H2O, 18 Da) from the molecular weight of 246.17. The retention time (36 min), protonated
protonated molecular ion (m/z 467). The fragment of m/z 291 was molecular ion (m/z 247) (Fig. 8), and mass spectrum of the compound
produced by a neutral loss of dehydrated glucuronic acid moiety (176 observed in the LC-MS/MS analysis matched those of the milnacipran
Da) from the protonated molecular ion. The fragment of m/z 247 was standard (Fig. 3). The fragments of m/z 230, m/z 202, m/z 174, m/z
produced after an additional neutral loss of carbon dioxide (44 Da) 157, and m/z 129 observed in the mass spectrum after the fragmen-
from the fragment of m/z 291. The fragment of m/z 230 was produced tation of the protonated molecular ion were similar to those observed
after a neutral loss of ammonia (17 Da) from the fragment of m/z 247. in the fragmentation of the N-desethyl milnacipran metabolite (Fig. 4).
The protonated molecular ion (m/z 467.1974) of the metabolite ob- It appeared that the amine function of the molecule was protonated to
served in the LC-HRMS analysis was accurate (10.7 ppm from produce the protonated molecular ion (m/z 247). The fragment of m/z
theoretical) and supported the proposed structure.
230 was produced after a neutral loss of ammonia (NH3, 17 Da) from
In addition, the hydrolysis of the urine metabolite in peak 3 to the protonated molecular ion and was the predominant fragment
milnacipran (Fig. 6) by ␤-glucuronidase (selective hydrolysis) and observed in the mass spectrum. The fragment of m/z 202 was pro-
acid (partial hydrolysis) was similar to the hydrolysis of the l- duced after a neutral loss of ethene (C2H4, 28 Da) from the fragment
milnacipran carbamoyl O-glucuronide biosynthetic standard. The en- of m/z 230. The fragment of m/z 174 was produced after an additional
zymatic hydrolysis was inhibited by D-saccharic acid 1,4-lactone, neutral loss of ethene (C2H4, 28 Da) from the fragment of m/z 202.
indicating that the metabolite was a ␤-glucuronide. It appeared that The protonated form of a lactam was the proposed structure for the
the aglycone produced from hydrolysis of the urine metabolite was a fragment of m/z 174. The fragment of m/z 157 was produced after a
milnacipran carbamic acid, which underwent facile decarboxylation neutral loss of ammonia (NH3, 17 Da) from the fragment of m/z 174.
and appeared as milnacipran.
The fragment of m/z 129 was produced after a neutral loss of carbon
The l-isomer of milnacipran, l-milnacipran, was the enantiomer monoxide (CO, 28 Da) from the fragment of m/z 157. The protonated
produced from the hydrolysis of isolated peak 3. After enzymatic molecular ion (m/z 247.1778) of the compound observed in the
hydrolysis of isolated peak 3 in urine samples from subject 1 and LC-HRMS analysis was accurate (10.9 ppm from theoretical) and
subject 3, l-milnacipran was the sole milnacipran enantiomer observed supported the proposed structure. Peak 5 in the metabolite profile of
in the chiral analysis of the hydrolyzed product (Fig. 7B). These plasma was also identified as unchanged milnacipran using a similar
results indicate that l-milnacipran was metabolized and excreted in procedure.
urine as l-milnacipran carbamoyl O-glucuronide metabolite (peak 3).
Peak 3 in the metabolite profile of plasma was also identified as the
l-milnacipran carbamoyl O-glucuronide metabolite using similar ap-
proaches with LC-SRM and chiral LC-SRM analyses.
Discussion
After oral administration of a 100-mg dose of [14C]milnacipran
hydrochloride to healthy male volunteers, the radioactivity of almost
Peak 4. The chemical structure of the metabolite eluted as peak 4 in the entire dose (approximately 93% of the dose) was excreted in urine
the metabolite profile of urine was identified as d-milnacipran carbamoyl within 96 h, signifying the excellent oral absorption of milnacipran.
O-glucuronide. The retention time (24 min), protonated molecular ion Renal excretion of unchanged milnacipran in humans appeared to be
(m/z 467), and mass spectrum of the metabolite observed in the LC- the major elimination route of milnacipran, accounting for approxi-
MS/MS analysis matched those of the d-milnacipran carbamoyl O-glucu- mately 55% of the dose. This study also found that the renal excretion
ronide biosynthetic standard (Fig. 3). Because the d- and l-milnacipran of d-milnacipran (31% of the dose) was slightly higher than that of
carbamoyl O-glucuronides are diastereomers, similar fragments (m/z 449, l-milnacipran (24% of the dose).
291, 247, and 230) were observed in the mass spectra of both glucu-
A major milnacipran metabolite, previously reported as milnacipran
ronides. The protonated molecular ion (m/z 467.1968) of the peak 4 N-glucuronide (Puozzo et al., 1998), was ultimately identified in this
metabolite observed in the LC-HRMS analysis was accurate (12.0 ppm study as l-milnacipran carbamoyl O-glucuronide. This major metab-
from theoretical) and also supported the proposed structure.
olite was excreted primarily in urine and accounted for approximately
In addition, the hydrolysis of the urine metabolite in peak 4 to 17% of the dose. The d-milnacipran enantiomer was similarly con-
milnacipran by ␤-glucuronidase (selective hydrolysis) and acid (par- verted to d-milnacipran carbamoyl O-glucuronide metabolite and ex-
tial hydrolysis) was similar to the hydrolysis of the d-milnacipran creted in urine, but it accounted for only 2% of the dose. This study
carbamoyl O-glucuronide biosynthetic standard. The enzymatic hy- also found that approximately 8% of the dose was excreted in urine as
drolysis was inhibited by D-saccharic acid 1,4-lactone, indicating that the N-desethyl milnacipran metabolite. No other metabolites with
the metabolite was a ␤-glucuronide. It appeared that the aglycone significant radioactivity were observed in urine. The biotransforma-
produced from the hydrolysis of the urine metabolite was a milnaci- tion of milnacipran in humans is summarized in Fig. 9.
pran carbamic acid, which underwent facile decarboxylation and
appeared as milnacipran.
Because unchanged milnacipran accounted for only 36.3% of the
AUC0–t for plasma radioactivity (on average), it was expected that
The d-isomer of milnacipran, d-milnacipran, was the enantiomer milnacipran metabolite(s) would be present in plasma after the oral
produced from the hydrolysis of isolated peak 4. After the enzymatic administration of milnacipran hydrochloride. The milnacipran metab-
hydrolysis of isolated peak 4 from subject 1 and subject 3, d- olites excreted in urine were also observed in plasma. The maximum
milnacipran was the sole milnacipran enantiomer observed by chiral concentration of the major plasma metabolite, the l-milnacipran car-
analysis of the hydrolyzed product (Fig. 7C). Therefore, the results bamoyl O-glucuronide metabolite, was approximately 234 ng Eq of
supported the fact that d-milnacipran was metabolized and excreted in milnacipran/ml. In addition, the concentration of this metabolite was
urine as d-milnacipran carbamoyl O-glucuronide metabolite (peak 4). similar to that of milnacipran for all plasma samples collected during
Peak 4 in the metabolite profile of plasma was also identified as the the 0.5 to 12 h after dosing. Therefore, the AUC of l-milnacipran
d-milnacipran carbamoyl O-glucuronide metabolite using a similar carbamoyl O-glucuronide metabolite also accounted for approxi-
approach with LC-SRM analysis.
mately 36% of the AUC0–t of plasma radioactivity. The plasma
Peak 5. The chemical structure of the compound eluted as peak 5 concentrations of the N-desethyl milnacipran and the d-milnacipran
in the metabolite profile of urine was identified as unchanged mil- carbamoyl O-glucuronide metabolites were relatively low and did not

1734
LI ET AL.
FIG. 9. Proposed metabolic transformation of milnacipran in
humans.
exceed 25 ng Eq of milnacipran/ml. Thus, the AUCs of these two neutral losses (220 and 176 Da) from the protonated molecular ions
metabolites were not determined. (m/z 467) of these metabolites. The mass spectra results also sup-
Similarly to other carbamoyl O-glucuronide metabolites (Tremaine ported the chemical structure of milnacipran carbamoyl O-glucu-
et al., 1989; Delbressine et al., 1990; Schaefer, 1992; Schaefer et al., ronide metabolites.
1998), the milnacipran carbamoyl O-glucuronide metabolites were
A significant in-source (i.e., in the ionization source of the mass
probably formed by a nonenzymatic reversible reaction of the primary spectrometer) degradation of both d- and l-milnacipran carbamoyl
amine function of milnacipran with carbon dioxide to form a mil- O-glucuronide metabolites to milnacipran was observed when the
nacipran carbamic acid intermediate, which was subsequently conju- biosynthetic standards, isolated peak 3, or isolated peak 4 were
gated to glucuronic acid. In this study, the milnacipran carbamoyl analyzed by LC-MS/MS and LC-SRM. Milnacipran generated in-
O-glucuronide metabolites were partially hydrolyzed by acid and source from milnacipran carbamoyl O-glucuronide metabolite should
selectively hydrolyzed by ␤-glucuronidase to its aglycone. The agly- have the retention of milnacipran carbamoyl O-glucuronide metabo-
cone produced from hydrolysis of the glucuronide was a milnacipran lite. However, the retention time of milnacipran was much longer than
carbamic acid, which underwent facile decarboxylation and appeared that of milnacipran carbamoyl O-glucuronide metabolite in the anal-
as milnacipran. Similar hydrolytic properties have been reported for yses. Hence, the milnacipran generated in-source was easily distin-
the carbamoyl O-glucuronide metabolite of numerous compounds guished from the milnacipran presented in the sample.
(Tremaine et al., 1989; Kwok et al., 1990; Schaefer, 1992).
Milnacipran is not transformed to its trans-isomer in humans. The
In addition to the common neutral loss of 176 Da (i.e., dehydrated stereochemistry of milnacipran shows it to be a cis-isomer composed
glucuronic acid moiety) from the protonated molecular ion of regular of a pair of enantiomers: d-milnacipran (1S, 2R) and l-milnacipran
glucuronide metabolites, a unique neutral loss of 220 Da (i.e., the sum (1R, 2S). The trans-isomer of milnacipran is also a pair of enantio-
of dehydrated glucuronic acid moiety and carbon dioxide, 176 ϩ 44 mers: the (1R, 2R) and (1S, 2S). To transform milnacipran to its
Da) from the protonated molecular ions of carbamoyl O-glucuronide trans-isomer would require changes at only one chiral center of
metabolites has been frequently reported in the literature (Straub et al., milnacipran: (1S, 2R) to (1R, 2R) or (1S, 2S) and (1R, 2S) to (1R, 2R)
1988; Tremaine et al., 1989; Dow et al., 1994; Schaefer et al., 1998; or (1S, 2S). In this study, the HPLC method provided a good baseline
Beconi et al., 2003). In this study, the fragments of m/z 247 and m/z resolution (Fig. 2) of the trans-isomer of milnacipran (retention time
291 observed in the mass spectra of d- and l-milnacipran carbamoyl ϳ34 min) from milnacipran (retention time ϳ36 min). The trans-
O-glucuronide metabolites support the occurrence of both types of isomer of milnacipran was not observed in the metabolite profile of

EXCRETION AND METABOLISM OF MILNACIPRAN IN HUMANS
1735
plasma or urine by radioactivity analysis (Fig. 1, A and B, respec- scientists and physicians better understand the therapeutic usage of
milnacipran hydrochloride.
tively). In addition, LC-MS/MS analysis of a urine sample (Fig. 8A)
confirmed that milnacipran was not excreted in urine as its trans-
isomer. The absence of milnacipran transformation to its trans-isomer
would imply no chirality change at either chiral center of milnacipran.
Furthermore, previous clinical studies of milnacipran have demon-
strated that there is no interconversion between the d-milnacipran (1S,
2R) and l-milnacipran (1R, 2S) enantiomers (data on file, company
study reports). The conversion of d-milnacipran to l-milnacipran, or
vice versa, would require changes at both chiral centers: 1S to 1R and
2R to 2S (or vice versa). Therefore, the absence of the milnacipran
transformation to its trans-isomer in this study (no change in either
chiral center) strengthens the results of previous clinical studies (no
change in both chiral centers).
Acknowledgments
We thank Melanie Ronsheim and Thomas Chou for their assistance with the
analyses of the study. In addition, we thank Larry Cheng for his guidance
during the study, as well as Dr. Morgan Hill and Mildred Bahn at Prescott
Medical Communications Group (Chicago, IL) for editorial and medical writ-
ing assistance.
Authorship Contributions
Participated in research design: Li, Wangsa, and Ho.
Conducted experiments: Li and Chin.
Performed data analysis: Li, Chin, Wangsa, and Ho.
Wrote or contributed to the writing of the manuscript: Li, Wangsa, and Ho.
References
Previously, the metabolism of milnacipran was studied after oral
administration of [14C]milnacipran hydrochloride to mice (100 mg/
kg), rats (100 mg/kg), and monkeys (60 mg/kg) (data on file, company
study reports). After the oral administration, approximately 40% of
the dose (monkeys) to 63% of the dose (rodents) was excreted in urine
as unchanged milnacipran. Less than 2% of the dose was excreted as
the milnacipran carbamoyl O-glucuronide metabolite in mice (1.0% of
the dose), rats (0.1% of the dose), and monkeys (1.6% of the dose).
Similar amounts of d- and l-milnacipran carbamoyl O-glucuronide
metabolites were excreted by each animal species, and both glucuro-
nide metabolites were observed in plasma of all three animal species.
In addition, milnacipran was transformed to N-desethyl milnacipran
metabolite in the animals, with the N-desethyl milnacipran metabolite
being the major metabolite produced in rats and monkeys. The animal
plasma concentrations of N-desethyl milnacipran metabolite and d-
milnacipran carbamoyl O-glucuronide metabolite were higher than
those observed in humans (100-mg oral dose). Moreover, the plasma
concentration of l-milnacipran carbamoyl O-glucuronide metabolite
found in monkeys was similar to that observed in humans, but lower
plasma concentrations of this metabolite were found in mice and rats.
Overall, the metabolism study of milnacipran hydrochloride was
conducted safely in humans. Other than the common side effect of
emesis observed in two healthy subjects, the 100-mg oral dose of
[14C]milnacipran hydrochloride was well tolerated by the remaining
healthy subjects. In addition to measuring all major plasma metabo-
lites of milnacipran in this study, the excretion of milnacipran and its
major metabolites was measured. We hope that the results of this
comprehensive study of milnacipran metabolism in humans will help
Beconi MG, Mao A, Liu DQ, Kochansky C, Pereira T, Raab C, Pearson P, and Lee Chiu SH
(2003) Metabolism and pharmacokinetics of a dipeptidyl peptidase IV inhibitor in rats, dogs,
and monkeys with selective carbamoyl glucuronidation of the primary amine in dogs. Drug
Metab Dispos 31:1269–1277.
Delbressine LP, Funke CW, van Tilborg M, and Kaspersen FM (1990) On the formation of
carbamate glucuronides. Xenobiotica 20:133–134.
Dow J, Piriou F, Wolf E, Dulery BD, and Haegele KD (1994) Novel carbamate metabolites of
mofegiline, a primary amine monoamine oxidase B inhibitor, in dogs and humans. Drug Metab
Dispos 22:738–749.
Forest Laboratories, Inc. (2009) Savella (milnacipran hydrochloride) full prescribing informa-
tion. Forest Laboratories, Inc., New York.
Gibaldi M and Perrier D (1975) Pharmacokinetics, 2nd ed., Drugs and the Pharmaceutical
Sciences, vol 1, Marcel Dekker Inc., New York.
Kwok DW, Pillai G, Vaughan R, Axelson JE, and McErlane KM (1990) Preparative high-
performance liquid chromatography and preparative thin-layer chromatography isolation of
tocainide carbamoyl-O-␤-D-glucuronide: structural characterization by gas chromatography-
mass spectrometry and fast atom bombardment-mass spectrometry. J Pharm Sci 79:857–861.
Puech A, Montgomery SA, Prost JF, Solles A, and Briley M (1997) Milnacipran, a new serotonin
and noradrenaline reuptake inhibitor: an overview of its antidepressant activity and clinical
tolerability. Int Clin Psychopharmacol 12:99–108.
Puozzo C, Albin H, Vinc¸on G, Deprez D, Raymond JM, and Amouretti M (1998) Pharmaco-
kinetics of milnacipran in liver impairment. Eur J Drug Metab Pharmacokinet 23:273–279.
Puozzo C and Leonard BE (1996) Pharmacokinetics of milnacipran in comparison with other
antidepressants. Int J Clin Psychopharmacol 11 (Suppl 4):S15–S27.
Puozzo C, Panconi A, and Depez D (2002) Pharmacology and pharmacokinetics of milnacipran.
Int Clin Psychopharmacol 17 (Suppl 1):S25–S35.
Schaefer WH (1992) Formation of a carbamoyl glucuronide conjugate of carvedilol in vitro using
dog and rat liver microsomes. Drug Metab Dispos 20:130–133.
Schaefer WH, Politowski J, Hwang B, Dixon F Jr, Goalwin A, Gutzait L, Anderson K, DeBrosse
C, Bean M, and Rhodes GR (1998) Metabolism of carvedilol in dogs, rats, and mice. Drug
Metab Dispos 26:958–969.
Straub K, Davis M, and Hwang B (1988) Benzazepine metabolism revisited. Evidence for the
formation of novel amine conjugates. Drug Metab Dispos 16:359–366.
Tremaine LM, Stroh JG, and Ronfeld RA (1989) Characterization of a carbamic acid ester
glucuronide of the secondary amine sertraline. Drug Metab Dispos 17:58–63.
Address correspondence to: Dr. John Ho, Forest Research Institute, 220 Sea
Lane, Farmingdale, NY 11735. E-mail: john.ho@frx.com