Ion mobility spectrometry-mass spectrometry analysis for the site of aromatic hydroxy

Article abstract of DOI:10.1124/dmd.113.051953

Hydroxylated metabolites often retain the pharmacological activity of parent compound, and the position of hydroxylation determines the formation of chemically reactive intermediates, such as quinones and analogs, from para- and/or ortho-hydroxylation of phenols or arylamines. Therefore, the identification of exact position of hydroxylation is often required at the early development stage of new drug candidates. In many cases, liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides identical MS/MS spectra among isomeric hydroxylated metabolites, and therefore, it alone cannot unequivocally identify the exact position(s) of hydroxylation. Ion mobility spectrometry (IMS), integrated with LC-MS/MS, recently showed the capability of separating isomeric species based on differences in their drift times from IMS, which are linearly proportional to the collision cross-section (CCS) reflecting physical size and shape. In the present study, a chemical derivatization of isomeric hydroxylated metabolites with 2-fluoro-N-methyl pyridinium p-toluenesulfonate was found to confer distinct theoretical CCS value on each isomer by forming corresponding N-methyl pyridine (NMP) derivative. The regression lines established by the comparison between theoretical CCS values and observed drift times from IMS for each set of parent compound (labetalol, ezetimibe, atorvastatin, and warfarin) and its MS/MS product ions accurately and selectively projected the actual drift times of NMP derivatives of corresponding aromatic or isomeric hydroxylated metabolites. The established method was used for the accurate assignment of predominant formation of 2-hydroxylated metabolite from imipramine in NADPH- fortified human liver microsomes. The present application expands the versatility of LC-IMS-MS technique to the structure identification of isomeric hydroxylated metabolites at the early stage for drug development.

Full text of DOI:10.1124/dmd.113.051953

1
521-009X/41/7/1295–1299$25.00
http://dx.doi.org/10.1124/dmd.113.051953
DRUG
M
ETABOLISM AND
DISPOSITION
Drug Metab Dispos 41:1295–1299, July 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Short Communication
Ion Mobility Spectrometry–Mass Spectrometry Analysis for
the Site of Aromatic Hydroxylation
Received March 14, 2013; accepted April 8, 2013
ABSTRACT
Hydroxylated metabolites often retain the pharmacological activity isomeric hydroxylated metabolites with 2-fluoro-N-methyl pyridinium
of parent compound, and the position of hydroxylation determines p-toluenesulfonate was found to confer distinct theoretical CCS
the formation of chemically reactive intermediates, such as quinones value on each isomer by forming corresponding N-methyl pyridine
and analogs, from para- and/or ortho-hydroxylation of phenols or (NMP) derivative. The regression lines established by the compar-
arylamines. Therefore, the identification of exact position of hy- ison between theoretical CCS values and observed drift times
droxylation is often required at the early development stage of new from IMS for each set of parent compound (labetalol, ezetimibe,
drug candidates. In many cases, liquid chromatography–tandem
atorvastatin, and warfarin) and its MS/MS product ions accurately
mass spectrometry (LC-MS/MS) provides identical MS/MS spectra and selectively projected the actual drift times of NMP derivatives
among isomeric hydroxylated metabolites, and therefore, it alone of corresponding aromatic or isomeric hydroxylated metabolites.
cannot unequivocally identify the exact position(s) of hydroxylation. The established method was used for the accurate assignment of
Ion mobility spectrometry (IMS), integrated with LC-MS/MS, recently predominant formation of 2-hydroxylated metabolite from imipra-
showed the capability of separating isomeric species based on mine in NADPH- fortified human liver microsomes. The present
differences in their drift times from IMS, which are linearly pro- application expands the versatility of LC-IMS-MS technique to the
portional to the collision cross-section (CCS) reflecting physical structure identification of isomeric hydroxylated metabolites at the
size and shape. In the present study, a chemical derivatization of early stage for drug development.
Introduction
Recently, we developed a novel method to identify the site of
isomeric glucuronide conjugation by ion mobility spectrometry (IMS)
integrated with LC-MS/MS: the most likely isomer of glucuronide
conjugate was identified from the comparison between actual IMS
drift time of target conjugate and the theoretical value of collision
cross-section (CCS) calculated by MOBCAL on the generated re-
gression line from the relationship between drift times and CCS values
for the set of parent compound and its MS/MS fragments (Shimizu
et al., 2012). The regression line–based approach eliminated potential
variation in the calculation of three-dimensional (3D) structure under
vacuum phase among adopted 2D-3D conversion methods and
inherent error ratios in the theoretical CCS values by the modeling,
both precluding IMS technology from the routine analysis without the
calibration by authentic standards at the early development stage. The
theoretical drift time from IMS is linearly proportional to the value of
CCS when the masses of ions are much larger than the buffer gas
under the same IMS condition, and the value of CCS depends on the
physical shape and size of 3D structure of analyte (Smith et al.,
The fragment analysis using liquid chromatography–tandem mass
spectrometry (LC-MS/MS) is an effective way of metabolite identifica-
tion, especially for the rapid identification of metabolites during the early
period of drug discovery and development (for review of fragment
analyses, Prakash et al., 2007). Although the tandem mass spectrometry
(
MS/MS) analysis provides structure information on the metabolites by
comparing the increase or decrease of the molecular weight of afforded
product ions, it does not always provide unambiguous information on the
unequivocal characterization for the isomeric substituted molecules, such
as hydroxylated metabolites, which often afford identical MS/MS frag-
ments. The recent meta-analysis for the metabolic reactions of over 1000
different drugs and other xenobiotics revealed that aromatic hydroxyl-
ation plays a predominant role in generating pharmacologically active
metabolite(s) (;30% of all active metabolites), suggesting that the
hydroxylated metabolite often retains the target affinity and pharmaco-
logical activity of the parent compound (Testa et al., 2012). In addition,
the same analysis indicated that the formation of quinones or analogs that
is generated from para- and/or ortho-hydroxylation of phenols or
arylamines accounts for over 40% of all toxic and/or reactive metabolites.
Therefore, the identification of the exact position of hydroxylation is
often required at discovery stage from efficacy and safety points of view.
2
009). During the course of application of IMS analysis to the
identification of isomeric hydroxylated metabolites, it was found that
the theoretical values of CCS had little difference among isomers,
likely because of the subtle difference in the physical shape and/or
size of 3D structures among hydroxyl isomers, compared with that
among isomers of glucuronide conjugate. Consistently, the isomer
separation of hydroxylated metabolites of ondansetron required
dx.doi.org/10.1124/dmd.113.051953.
ABBREVIATIONS: CCS, collision cross section; FMPTS, 2-fluoro-1-methyl pyridinium p-toluenesulfonate; IMS, ion mobility spectrometry; IMS-
MS, ion mobility spectrometry–mass spectrometry; LC-IMS-MS, liquid chromatography–ion mobility spectrometry–mass spectrometry; LC-MS,
liquid chromatography–mass spectrometry; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MS/MS, tandem mass spectrometry;
m/z, mass-to-charge ratio; NMP, N-methylpyridyl; TM, trajectory method; UPLC, ultraperformance liquid chromatography.
1295

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Shimizu and Chiba
calibration with authentic standard for each isomer to improve precision analyses were performed on a Synapt G2 high definition mass spectrometer
under the conditions described as follows: capillary voltage, 3 kV; cone voltage,
of determined drift times by ion mobility spectrometry–mass spectrom-
etry (IMS-MS) method (Dear et al., 2010).
Chemical derivatization in combination with LC-MS/MS has been
well established for the enhancement of detection sensitivity by
introducing an easily ionizable function or a constantly charged group
into the intact analyte (Lampinen-Salomonsson et al., 2006). Lin et al.
3
0
0 V; trap collision energy, 0 V or 10-40 V ramping; transfer collision energy,
V; trap/transfer gas, argon; IMS gas, nitrogen; IMS T-wave speed, 900 m/s;
IMS T-wave height, 40 V; IMS gas flow, 20 or 35 mL/min; IMS-MS acquisition
time, 7 minutes after samples injected onto column.
Calculations of CCS. Theoretical values of CCS were calculated using the
method suggested by Dear et al. (2010). In brief, the conformation for each
molecule was energy minimized with the MMFF94 force field, followed by the
(2007) used 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS)
to chemically derivatize phenolic group of estrogens and improved extraction of 3D coordinate sets of each atom. Theoretical CCS values were
sensitivity by LC-MS for 17b-estradiol, estrone, and 17a-ethinylestra- then calculated by the open source software MOBCAL (Jarrold Group, Indiana
diol. FMPTS reacts with aromatic hydroxyl group, forming N- University - Bloomington) (Mesleh et al., 1996) with 3D coordinate data sets.
+
The MOBCAL could be downloaded from http://www.indiana.edu/~nano/
methylpyridyl (NMP) ether ion of m/z (mass-to-charge ratio) [M+92]
software.html freely and operates on Microsoft Windows PC. The MOBCAL
by nucleophilic substitution (Quirke et al., 1994; Lin et al., 2007). NMP
output is based on three different models/algorithms, including projection
moiety possesses similar size to glucuronic acid by 3D modeling, and
approximation, exact hard sphere scattering, and the trajectory method (TM).
Among three outputs, the MOBCAL TM has been demonstrated to provide
most accurate projections of CCS values with percentage differences of ,4.5%
for isomeric organoruthenium anticancer complexes (Williams et al., 2009) and
it also enhances the sensitivity of analyte by LC-MS because of the
presence of cationic quarternary amine. Therefore, the chemical deriv-
atization with FMPTS likely confers distinct physical shape and CCS
value on each isomer of hydroxylated metabolites as seen in the iso-
of ,1% for ondansetron and its hydroxylated metabolites (Dear et al., 2010).
meric glucuronide conjugates, which enables regression-based liquid Therefore, the outputs of MOBCAL TM were used for the projections in this
chromatography–ion mobility spectrometry–mass spectrometry (LC- study.
IMS-MS) analysis to identify the exact position of hydroxylation on the
aromatic moiety without authentic standards.
In this communication, we present an approach for the IMS-MS
technology to the identification of exact site(s) of aromatic hydroxyl-
ation: the chemical derivatization with FMPTS was found to selectively
Regression Line for CCS and IMS Drift Time. Although the TM is
generally accepted as the most accurate method of calculating the theoretical
CCS values among multiple MOBCAL outputs, the relationship between CCS
and IMS drift times cannot serve as an absolute basis for the exact identification
of unknown structure of target analyte. Therefore, in addition to the TM-based
CCS value for the parent compound, those values for its MS/MS fragments
were plotted against corresponding actual IMS drift times to generate the
regression line. Thus, generated regression line facilitates accurate projection
of IMS drift time of unknown analyte (NMP derivative of hydroxylated metab-
derivatize aromatic hydroxyl group by forming NMP ether ion of m/z
+
[
M+92] , and the drift times of derivatized isomeric hydroxyl-
ated metabolites determined by IMS were selectively predicted from
regression-based LC-IMS-MS analysis (Shimizu et al., 2012) based olite) by the comparison with (multiple) theoretical CCS value(s) of potential
on the corresponding theoretical CCS values. The present method, structure(s).
therefore, expands the versatility of LC-IMS-MS technique to the
structure identification of isomeric hydroxylated metabolites, which is
often required during the early stage for new drug development as
pharmacologically and/or toxicologically important molecules.
Derivatization with FMPTS. The compound solution in acetonitrile
1000 ml, 10 mM) was added to 1 ml of FMPTS solution (10 mM in dimethyl
sulfoxide, 10 equivalents) and 1 ml of triethylamine. The resultant mixture was
shaken for 5 minutes at room temperature. The reaction mixture was then
injected onto the LC-IMS-MS system for analysis.
(
Incubation of Imipramine with Human Liver Microsomes. The reaction
mixture contained 0.1 M potassium phosphate buffer (pH 7.4), 3 mM mag-
nesium chloride, 1.0 mg/ml human liver microsomal protein, and NADPH-
Materials and Methods
Materials. Atorvastatin, ortho-hydroxy atorvastatin calcium salt, and para-
hydroxy atorvastatin calcium salt were purchased from TLC PharmaChem Inc.
+
generating system (1 mM NADP , 1 unit glucose-6-phosphate dehydrogenase,
and 10 mM D-glucose-6-phosphate disodium salt). The reaction was initiated
by the addition of imipramine hydrochloride (1 mM in dimethyl sulfoxide) to
achieve 10 mM at final concentration. The reaction was conducted at 37°C for
(
Vaughan, ON, Canada). Labetalol hydrochloride, imipramine hydrochloride,
and warfarin were purchased from Sigma-Aldrich (St. Louis, MO). Authentic
metabolites of 6-hydroxy warfarin, 7-hydroxy warfarin, and 8-hydroxy warfarin
were purchased from Salford Ultrafine Chemicals & Research Ltd. (Manchester,
UK). Ezetimibe and 2-hydroxy-imipramine were purchased from Toronto Research
Chemicals Inc. (North York, ON, Canada). FMPTS (2-Fluoro-1-methylpyridinium
p-toluenesulfonate) was purchased from Tokyo Chemical Industries CO., LTD.
6
0 minutes and terminated by the addition of two volumes of acetonitrile/
methanol (2:1, v/v). The supernatant was separated by centrifugation (13,800g,
0 minutes, 4°C), followed by evaporation under the stream of nitrogen gas.
1
The residue was dissolved in acetonitrile and dried over magnesium sulfate,
followed by centrifugation to remove powder of magnesium sulfate. The
resultant supernatant was treated with FMPTS for the NMP derivatization as
described in the preceding section.
(Tokyo, Japan). Triethylamine was purchased from Nacalai Tesque (Kyoto, Japan).
+
Glucose-6-phosphate dehydrogenase, NADP , and D-glucose-6-phosphate diso-
dium salt were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Pooled
human liver microsomes from 50 individual donors (Lot No.0910398; 31 males
and 19 females) were purchased from XenoTech LLC (Lenexa, KS). All other
reagents were of analytical grade.
Results and Discussion
Analytical Procedure. The LC-IMS-MS was conducted on an Acquity
A novel approach in the present study to the identification of the site
ultraperformance liquid chromatography (UPLC) system equipped with a binary of isomeric hydroxylation is to combine phenolic hydroxyl group
pump, autosampler, thermostat, and column compartment (Waters Corp., selective derivatization by the simple incubation with a chemical
Milford, MA), coupled with Synapt G2 high definition mass spectrometer
derivatization reagent, FMPTS, NMP ether derivatives and the IMS
(
Waters Corp., Manchester, UK) including ion mobility spectrometer and time-
analysis. The derivatization conferred distinct theoretical CCS value
calculated by MOBCAL on each isomeric hydroxylated metabolite,
otherwise not amenable to the discrimination because of subtle dif-
ference in the CCS values among isomers. The regression-line based
projection method, which had been established by the comparison
between theoretical CCS values and observed drift times in IMS for
of-flight–type high-resolution mass spectrometry. Solvent A was 0.1% formic
acid, and solvent B was 0.1% formic acid in acetonitrile. Chromatographic
separations were performed on an Acquity UPLC BEH C18 (1.7 mm, 2.1Â50
mm; Waters Corp., Dublin, Ireland) using a 10-minute gradient at a flow rate of
0.4 ml/min, starting at 10% solvent B, linearly increasing to 70% solvent B
over 7 minutes, followed by 90% solvent B for 1.5 minutes, and re-equilibrated
for 1.5 minutes. Eluents from UPLC during pre- and postanalysis (0.5 and multiple sets of glucuronide conjugate and its MS/MS fragments
minutes, respectively) were discarded through the diversion valve. IMS (Shimizu et al., 2012), accurately and selectively identified the NMP
7

Ion Mobility Site Identification of Hydroxylated Metabolites
1297
2
derivatives of phenolic metabolites of labetalol and ezetimibe and isomeric line (drift time = 0.0372 CCS + 2.2715; R = 0.9960). Similarly,
hydroxylated metabolites of atorvastatin, warfarin, and imipramine. ezetimibe (10 in Fig. 1C) and its MS/MS fragments, 11, 12, and 13
FMPTS is known to react with the hydroxyl group (Mukaiyama (drift time, 7.88, 7.61, 6.16, and 4.43 minutes, respectively; CCS
2
et al., 1975), resulting in the formation of NMP ether ion of m/z value, 144.09, 140.89, 107.49, and 63.648 Å , respectively) projected
+
[
2
M+92] by nucleophilic substitution (Quirke et al., 1994; Lin et al., actual drift time of NMP derivative of ezetimibe 9 (9.13 minutes) at
2
007). FMPTS reacts with primary/secondary alcohols (forming NMP the theoretical CCS value (172.31 Å ) with predicted drift time of 8.99
ether derivatives) and primary/secondary/tertiary thiols (forming NMP minutes based on the regression line (drift time = 0.0423 CCS +
2
sulfide) (Quirke et al., 1994). In addition, the sensitivity for estrogens, 1.6995; R = 0.9975).
such as 17b-estradiol, estrone, and 17a-ethinylestradiol, by LC-MS
The present chemical derivatization to generate NMP derivatives
was improved by the chemical derivatization technique with FMPTS conferred distinct theoretical CCS value on each isomer of potential
to modify the phenolic group of estrogens so that their ionization hydroxylated metabolites of atorvastatin, warfarin, and imipramine
efficiency was increased (Lin et al., 2007). Under the simple condition (Table 1), otherwise not amenable to the discrimination by LC-IMS-
in the present study that the analyte and FMPTS were incubated MS because of the subtle difference in the CCS value among isomers
in triethylamine/acetonitrile for 5 minutes at room temperature, the of intact hydroxylated metabolites. Figure 2 shows the relationships
derivatization selectively afforded NMP derivatives with phenol and linear regression lines between CSS values and IMS drift times for
groups of labetalol and ezetimibe (Fig. 1), both possessing phenolic the sets of parent drug [atorvastatin (Fig. 2A), warfarin (Fig. 2B), and
and aliphatic alcohols for the potential sites of nucleophilic sub- imipramine (Fig. 2C)], its MS/MS fragments, its authentic hydroxyl-
stitution by FMPTS. On the other hand, either imipramine or warfarin ated metabolites (for atorvastatin and warfarin), and the corresponding
did not react with FMPTS under the same condition. To ensure the NMP derivatives. Atorvastatin (Fig. 2A, 18) is known to afford two
accurate projection of thus formed NMP derivatives of labetalol and hydroxylated metabolites by CYP3A (Riedmaier et al., 2011). By
ezetimibe based on the regression line-based LC-IMS-MS analysis, fragment analyses, these two hydroxylated metabolites were both
the linearity of regression line between CCS values and IMS drift assigned as hydroxylations of terminal benzene, but the position of
times was confirmed for both sets of parent compound, its MS/MS hydroxylation was not determined by LC-IMS-MS analysis. The drift
fragments, and NMP derivative of phenolic hydroxyl group of parent times of atorvastatin (18 in Fig. 2A) and its MS/MS fragments (19–24)
compound [labetalol (Fig. 1B) and ezetimibe (Fig. 1C)]. The drift were 7.24, 6.59, 6.43, 6.10, 5.72, 5.29, and 4.97 minutes, respectively,
times of labetalol 2 (Fig. 1B) and its six MS/MS fragments 3–8 (drift and the corresponding CCS values were 182.25, 156.28, 153.06,
2
time, 7.07, 6.91, 6.75, 5.67, 5.45, 4.86, and 4.70 minutes, respectively; 148.85, 137.52, 127.33, and 113.34 Å , respectively. The relationship
CCS value, 128.60, 124.99, 122.15, 88.350, 84.645, 71.466, and generated a linear regression line (drift time = 0.0350 CCS + 0.9527)
2
2
6
6.066 Å , respectively) accurately projected actual drift time of NMP with high correlation coefficient (R = 0.9802), which allowed for an
derivative of labetalol 1 (8.15 minutes) at the theoretical CCS value accurate identification of three potential isomers of NMP derivatives
2
(
155.90 Å ) with predicted drift time of 8.07 minutes from regression of ortho-, meta-, and para-hydroxylated metabolites with predicted
Fig. 1. Scheme of chemical derivatizations of labetalol and ezetimibe with FMPTS, forming corresponding NMP derivatives (A) and plot of the drift times (minutes) against
2
calculated CCS values (Å ) for the singly charged ions of NMP derivatives of hydroxylated metabolites, intact hydroxylated metabolites, parent ion, and its MS/MS product
2
ions of labetalol (B) and ezetimibe (C). Linear regression for labetalol 2 and its MS/MS fragments (3–8) gave drift time = 0.0372 CCS + 2.2715 (R = 0.9960). NMP
derivative at phenolic alcohol (1) is shown by open circle (B). Linear regression for ezetimibe (10) and its MS/MS fragments (11–13) gave drift time = 0.0423 CCS + 1.6995
2
(R
= 0.9975). NMP derivative at phenolic alcohol (9) is shown by open circle (C).

1
298
Shimizu and Chiba
TABLE 1
TM-based theoretical CCS values for the potential hydroxylated metabolites of parent drugs (atorvastatin, warfarin, and
imipramine) and their corresponding N-methyl pyridine derivatives
TM-Based Calculated CCS
Parent Compound
Potential Metabolites
Intact Metabolites
N-Methyl Pyridine Derivatives
2
Å
Atorvastatin
Warfarin
Ortho-hydroxy atorvastatin
Meta-hydroxy atorvastatin
Para-hydroxy atorvastatin
6-Hydroxy warfarin
182.29
188.36
187.73
110.42
110.23
109.84
113.37
114.09
113.38
111.86
200.26
206.61
214.55
140.62
142.60
135.92
138.85
144.42
133.48
134.87
7
8
-Hydroxy warfarin
-Hydroxy warfarin
Imipramine
1-Hydroxy imipramine
2-Hydroxy imipramine
3-Hydroxy imipramine
4-Hydroxy imipramine
drift times of 7.96, 8.18, and 8.46 minutes, respectively. The re- hydroxylation of 6-, 7-, 8-hydroxy warfarin could not be assigned by
gression line thus generated accurately projected actual drift times of LC-IMS-MS (Table 1; 28, 29, and 30 in Fig. 2B). The drift times of
two NMP derivatives of authentic para- (Fig. 2A, 14) and ortho- (Fig. warfarin (31 in Fig. 2B) and its MS/MS fragments (32, 33, and 34)
2
A, 15) hydroxylated metabolites of atorvastatin at 8.59 and 7.99 were 6.75, 6.05, 4.81, and 4.86 minutes, respectively, and the
minutes, respectively. Warfarin 31 is known to afford many hydro- corresponding CCS values were 106.5, 92.886, 64.436, and 71.559
2
xylated metabolites, including 49-, 6-, 7-, 8-, and 10-hydroxy warfarin Å , respectively. The relationship generated a linear regression line
(
Daly and King, 2003). Both 49-hydroxy and 10-hydroxy warfarin (drift time = 0.0486 CCS + 1.5437) with high correlation coefficient
2
were distinguished from other three hydroxylated metabolites (6-, 7-, (R = 0.9832), which allowed for an accurate identification of three
and 8-hydroxy warfarin) by LC-MS/MS, whereas the positions of potential isomers of NMP derivatives of 6-, 7-, and 8-hydroxylated
2
Fig. 2. Plot of the drift times (minutes) against calculated CCS values (Å ) for the singly charged ions of NMP derivatives of hydroxylated metabolites, intact hydroxylated
metabolites, parent ion, and its MS/MS product ions of atorvastatin (A), warfarin (B) and imipramine (C). LC-MS chromatograms of the incubated sample of imipramine
with NADPH-fortified human liver microsomes before (upper panel) and after (lower panel) chemical derivatization of metabolically formed hydroxylated metabolites by
2
FMPTS (D). Linear regression for atorvastatin (18) and its MS/MS fragments (19–24) gave drift time = 0.0350 CCS + 0.9527 (R = 0.9802). Two NMP derivatives of ortho-
and para-hydroxy atorvastatin (14 and 15) are shown by open circles, and two intact hydroxylated metabolites (16 and 17) are shown by open triangles (A). Linear regression
2
for warfarin (31) and its MS/MS fragments (32, 33, and 34) gave drift time = 0.0486 CCS + 1.5437 (R = 0.9832). Three NMP derivatives of hydroxylated metabolites (25,
2
6, and 27) are shown by open circles, and three intact hydroxylated metabolites (28, 29, and 30) are shown by open triangles (B). Linear regression for imipramine (36) and
2
its MS/MS fragments (37, 38, and 39) gave drift time = 0.0444 CCS + 1.6503 (R = 0.9957). Theoretical CCS values for 3-, 4-, 1-, and 2-hydroxylated imipramine are plotted
by dotted lines at 133.48 (a), 134.87 (b), 138.85 (c), and 144.42 (d) Å , respectively (Table 1). The NMP derivative of metabolically formed hydroxylated metabolite agreed
2
well with that predicted for 2-hydroxyimipramine (35), shown by open circle (C).

Ion Mobility Site Identification of Hydroxylated Metabolites
1299
metabolites with predicted drift times of 8.38, 8.47, and 8.15 minutes, stage, without synthesis of authentic metabolites or isolation of
respectively. The actual drift time of three NMP derivatives of metabolites for NMR spectroscopy. Further studies are warranted to
authentic 6-, 7-, and 8-hydroxylated metabolite were 8.53, 8.53, and evaluate potential and versatility of chemical derivatization in the
8
.10 minutes, respectively, indicating that the regression line projected structural identification of isomeric metabolites by LC-IMS-MS
the drift time for 8-hydroxy warfarin separately from 6- or 7-hydroxylated analysis.
metabolite.
Discovery Drug Metabolism &
ATSUSHI SHIMIZU
MASATO CHIBA
The regression line–based approach was applied to the identifica-
tion of positions of hydroxylated metabolites formed from imipramine
in NADPH-fortified human liver microsomes. Figure 2D shows rep-
resentative LC-MS chromatograms of the incubation sample before
and after NMP derivatization. The peak at retention time of 3.68
Pharmacokinetics,
Pharmacokinetics Research
Laboratories, Tsukuba Research
Center, Taiho Pharmaceutical Co.,
Ltd., Ibaraki, Japan
+
minutes on both panels gave molecular ions [M+H] at m/z 281 and
2
67, which corresponded to the intact imipramine and N-desmethyl
Authorship Contributions
Participated in research design: Shimizu, Chiba.
Conducted experiments: Shimizu.
imipramine, respectively. The secondary peak at 3.19 minutes gave
molecular ions [M+H] at m/z 297, which corresponded to the intact
+
hydroxylated metabolite(s) of imiparamine on benzene ring by LC-MS
analyses, whereas the structural information by MS/MS did not afford
unequivocal identification of exact position of hydroxylation among
four potential isomers of hydroxylated imipramine (i.e., 1-, 2-, 3-, and
Contributed new reagents or analytic tools: Shimizu.
Performed data analysis: Shimizu.
Wrote or contributed to the writing of the manuscript: Shimizu, Chiba.
References
4
-position in Table 1). The peak at 2.55 minutes on the lower panel
+
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+
metabolically formed hydroxylated metabolite of imipramine in
NADPH-fortified human liver microsomes. As shown in Fig. 2C,
imipramine 36 and its MS/MS fragments (37, 38, and 39) generated
2
a regression line (drift time = 0.0444 CCS + 1.6503; R = 0.9957)
based on the relationship between drift times (6.48, 5.89, 5.35, and
5
.08 minutes, respectively) and CCS values (109.51, 94.507, 82.984,
2
and 78.197 Å , respectively). The theoretical CCS values of 1-, 2-, 3-,
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and 4-hydroxylated imipramine calculated by MOBCAL were 138.85
2
(
Fig. 2C), 144.42, 133.48, and 134.87 Å , respectively, and predicted
Mesleh MF, Hunter JM, Schvartsburg AA, Schatz GC, and Jarrold MF (1996) Structural in-
IMS drift times were 7.82, 8.06, 7.58, and 7.63 munutes, respectively,
by the regression line. The actual drift time of the peak for the NMP
derivative at 2.55 minutes (Fig. 2D) was 8.21 minutes, suggesting that
the major hydroxylated metabolite of imipramine in the incubated
sample with human liver microsomes was the 2-hydroxy imipramine
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previously established regression-based LC-IMS-MS method (Shimizu
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opportunity for the isomeric aromatic hydroxylated metabolites by one-
step simple derivatization for LC-IMS-MS analysis and has proven to
be a powerful tool for preliminary elucidation of the hydroxylated
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1
Address correspondence to: Dr. Atsushi Shimizu, Discovery Drug Metabolism &
Pharmacokinetics, Pharmacokinetics Research Laboratories, Tsukuba Research
Center, Taiho Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaraki 300-2611,
Japan. Email: a-shimizu@taiho.co.jp