Drug Metabolism and Pharmacokinetics, Novartis Biomedical Research
Institute, East Hanover, New Jersey
Methanol was widely used as a substrate-delivering solvent in in
vitro metabolic stability screenings. Its interaction with enzyme
activities, particularly those of cytochrome P450s, has been
investigated extensively in the past. Little was known about the
interaction of methanol, whether direct or indirect, with substrates.
The present study provided data for the first time to show that use of
methanol may result in the formation of artifacts, which could mislead
the metabolic stability information. The disappearance of LAQ094,
metaraminol, and (
)-isoproterenol following 1-h incubation with human
liver microsomes was 73, 85, and 66%, respectively, in the presence of
1% methanol, but was only 3, 15, and 24%, respectively, in the
absence of organic solvent. The dramatically increased instability in
the presence of methanol of these three compounds, each with
1,2-diamino or 1,2-amino hydroxy functional groups, was due to the
formation of [M + 12] products resulting from condensation reaction
of the substrates with formaldehyde. Formaldehyde was formed from
methanol by human liver microsomal enzymes with an apparent
Km of 35 mM and a
Vmax of 7.9 nmol/min/mg of protein. The
concentration of formaldehyde reached as high as 600 µM following a
60-min incubation. The [M + 12] products were characterized as
five-membered heterocycles by liquid chromatography and tandem mass
spectrometry analysis. Inclusion of 10 mM glutathione prevented the formation of such artifacts and is therefore suggested for future
in vitro screenings. Our study also documented the novel finding of
enzyme-dependent conversion of NADPH to nicotinamide in microsomal incubations.
 |
Introduction |
In vitro metabolic
stability screening of compounds using liver microsomes or
postmitochondrial S9 fractions are commonly used in the pharmaceutical
industry in the early selection of drug candidates, in an effort to
better the chance of finding drug leads with desirable pharmacokinetic
and metabolic properties. Fast chromatography coupled with mass
spectrometric detection (LC/MS1) is typically
used to monitor the disappearance of compounds as a result of
incubation (Van Breemen et al., 1998
; Korfmacher et al., 1999) without
detailed investigation of the structure of metabolites in screening mode.
One of the variables in the in vitro incubations is the selection of
organic solvent, which is used to dissolve compounds and deliver them
to the incubation systems. Commonly used organic solvents are methanol,
ethanol, dimethyl sulfoxide (DMSO), acetonitrile, and acetone. The
selection of organic solvents are governed by their ability to
solubilize the compounds and their effect on the activity of various
cytochrome P450 isoforms (Kawalek and Andrews, 1980; Chauret et
al., 1998; Hickman et al., 1998; Busby et al., 1999), either inhibitory
(Chauret et al., 1998; Busby et al., 1999; Tang et al., 2000) or
stimulatory (Palamanda et al., 2000; Tang et al., 2000). Methanol and
acetonitrile are considered as the better alternatives in light of
their less severe inhibition effect against various P450 isoforms
(Chauret et al., 1998; Hickman et al., 1998; Busby et al., 1999) when
present at low levels.
In the present study, we report that the apparent in vitro metabolic
instability of a certain class of compounds could be dramatically
augmented by the presence of methanol solvent. LAQ094, metaraminol, and
()-isoproterenol (Fig. 1), each with
1,2-diamino or 1,2-amino hydroxy functional groups, were selected to
illustrate those effects. The structures of the unusual products from
these three compounds were characterized and the nature of the methanol solvent effect on metabolic stability was investigated. The kinetics of
formaldehyde formation from methanol in human liver microsomes was
characterized.
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Experimental Procedures |
Materials.
Metaraminol, ()-isoproterenol hydrochloride salt,
d4-methanol, formaldehyde, glutathione,
nicotinamide, and NADPH were purchased from Sigma (St. Louis, MO).
LAQ094 was synthesized by Novartis Pharmaceuticals Corporation (Summit,
NJ). 4-Amino-3-penten-2-one (Fluoral-P) was obtained from Acros
Organics (Fairlawn, NJ). Human liver microsomes (pool of 10 male
subjects) were purchased from Xenotech (Kansas City, KS). All other
reagents are of the highest grade commercially available.
Human Liver Microsomal Incubations.
Incubation mixtures contained a final concentration of 0.1 M potassium
phosphate buffer (pH 7.4), 5 mM MgCl2, 1 mM EDTA,
1.0 mg of human liver microsomal protein per milliliter, 20 µM
substrate, and 1 mM NADPH in a total volume of 0.5 ml. The substrate
was delivered with 1% (final concentration, v/v) methanol, DMSO, or d4-MeOH, in some cases with
subsequent evaporation of the organic solvent, or addition of
glutathione (10 mM). The reactions were initiated by the addition of
NADPH after a 5-min preincubation at 37°C, and terminated by mixing
with an equal volume of cold acetonitrile after a 60-min incubation at
37°C. The zero time point was prepared by mixing the incubates with
an equal volume of cold acetonitrile before the addition of NADPH. The
incubates were mixed by vortexing and centrifuged to precipitate the
proteins. The supernatants were analyzed for disappearance of
substrates and structural characterization of metabolites.
Reaction with Formaldehyde.
Incubation mixture containing 0.1 M potassium phosphate buffer (pH
7.4), 20 µM substrate, and 1% formaldehyde (v/v) were kept at room
temperature for 2 h with gentle shaking and then analyzed by HPLC
with UV and mass spectrometric detection immediately.
Measurement of Substrate Disappearance by HPLC with UV or Mass
Spectrometric Detection.
Disappearance of LAQ094 following human liver microsomal incubations
was carried out by HPLC analysis with UV detection at 247 nm. The HPLC
system consisted of a Waters Alliance 2690 Separations Module equipped
with a 996 photodiode array detector. A Metachem Metasil Basic
C18 column (5 µm, 4.6 × 150 mm) preceded
by a 0.5-µm frit and the corresponding guard column was used.
Separation was carried out with solvent A (10 mM ammonium acetate with
0.05% formic acid, pH 3.5) and solvent B (acetonitrile) with a linear gradient at a flow rate of 1 ml/min. The mobile phase was initially kept at 100% A for 5 min, ramped to 30% B over 15 min, and then to
95% B over 1 min and kept at that condition for 3 min. The total run
time was 30 min. The disappearance of LAQ094 was assessed by comparing
the peak areas from the zero time point and the 60-min incubations.
Disappearance of metaraminol and ()-isoproterenol following
microsomal incubations was carried out by LC/MS/MS analysis. The HPLC
system consisted of Shimadzu LC-10AD pumps (Columbia, MA), a Lee Visco
mixer with 10-µl dead space, and a Leap Technologies HTS-PAL
autosampler (Carrboro, NC) with a 25-µl sample loop. Separation was
achieved with a Keystone betasil C18 column (5 µm, 2.0 × 50 mm) with a flow rate of 0.3 ml/min and a linear
gradient with 10 mM ammonium acetate containing 0.1% formic acid, pH
3.5 (solvent A), and methanol (solvent B). The mobile phase was
initially kept at 5% B for 1 min, ramped to 25% B over 2.5 min, and
then to 5% B over 0.5 min. The total run time was 7 min. A PE Sciex
API3000 mass spectrometer (Concord, Ontario, Canada) with TurboIonSpray interface in positive ionization mode was used for detection. The
instrument was operated at a probe temperature of 375°C, ion spray
voltage of 4500 V, orifice voltage of 30 V, and ring voltage of 200 V. A collision energy of 25 eV was used for MS/MS experiments with a
nitrogen collision gas setting of 4. ()-Isoproterenol and metaraminol
were monitored as the transitions of m/z 212.2 to 152.2 and
m/z 168.2 to 150.2, respectively, with a dwell time of
0.2 s each. The disappearance of substrates was assessed by comparing peak areas from the zero time point and the 60-min incubations.
Structural Characterization of Metabolites by LC/MS/MS Analysis.
LAQ094 and its metabolites were characterized by LC/MS/MS analysis. The
HPLC system used was as described above. The HPLC effluent was diverted
to waste during the first 4 min of each run to protect the mass
spectrometer from nonvolatile salts. Thereafter, the effluent was split
to deliver ~200 µl/min to the mass spectrometer for optimum
sensitivity. Mass spectrometric conditions were as described above.
Full scan spectra were typically obtained from 100 to 650 m/z using a 0.2-amu step size.
Characterization of metabolites of metaraminol and ()-isoproterenol
was carried out on the same LC/MS system used for the assessment of
their disappearance, but with modified HPLC conditions. A YMC ODS-AQ
C18 column (5 µm, 2.1 × 150 mm) with a
flow rate of 0.3 ml/min and a linear gradient with 10 mM ammonium
acetate containing 0.1% formic acid, pH 3.5 (solvent A), and methanol (solvent B) were used. The mobile phase was initially kept at 100% A
for 4 min, ramped to 22% B over 7 min, and then to 95% B over 5 min.
The total run time was 34 min.
Measurement of Formaldehyde Formation in Human Liver Microsomal
Incubations.
Formaldehyde formation from methanol in human liver microsomal
incubations was measured according to literature (Wojciechowski and
Fall, 1996) with modifications. Fluoral-P (2.5 mg/ml) was added to the
incubation mixture to react with formaldehyde to form a stable,
fluorescent compound, 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine, which was quantitated fluorometrically (excitation at 410 nm and emission at 510 nm) on a Spectra Max Plus fluorescence plate reader (Molecular Devices, Sunnyvale, CA). This assay method had a limit of
quantitation of 10 µM formaldehyde. Initial human liver microsomal incubations with 1% methanol (245 mM) were carried out for 0, 5, 10, 15, 30, and 60 min, under the conditions specified previously. Subsequent 60-min incubations with methanol concentrations of 2.45, 6.13, 24.5, 61.3, 245, and 735 mM were carried out to characterize the
formaldehyde formation kinetics.
Kinetic Data Analysis.
Michaelis-Menten parameters (Km,
Vmax) were estimated by nonlinear curve
fitting using the Scientist program (Micromath Scientific Software,
Salt Lake City, UT) to the following equation: v = Vmax*[S]/(Km + [S]), where v is the initial rate of formaldehyde
formation and [S] is the methanol concentration.
| |
Results |
Dependence of Metabolic Stability of LAQ094, Metaraminol, and
()-Isoproterenol on Methanol in Liver Microsomal Incubations.
Following a 60-min incubation of LAQ094 with human liver microsomes,
when methanol (1% v/v) was used as the substrate-delivering solvent,
the concentration of LAQ094 decreased by 73% with the concurrent
formation of a product eluting at 18.1 min (Fig.
2A). The disappearance of LAQ094 was
however minimal (~3%) when methanol was avoided in the incubation
(Fig. 2B), or when DMSO was used as the alternative
substrate-delivering solvent (Fig. 2C). Incubation with
heat-inactivated liver microsomes did not lead to significant substrate
depletion or product formation (Fig. 2D). Inclusion of 10 mM
glutathione effectively prevented the instability of LAQ094 in the
presence of methanol solvent (Fig. 2E). Reaction of 1% (v/v)
formaldehyde with LAQ094 for 2 h at room temperature led to the
complete disappearance of LAQ094, and the concurrent formation of a
product with the same retention time (18.1 min, Fig. 2F) as that from
the enzymatic incubation in the presence of methanol.
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Fig. 2.
HPLC chromatograms of 60-min LAQ094
incubations with human liver microsomes in the presence of 1% methanol
(245 mM) (A); in the absence of organic solvent (B); in the presence of
1% DMSO (C); in the presence of 1% methanol (245 mM) with
heat-inactivated enzymes (D); in the presence of 1% methanol (245 mM)
and 10 mM glutathione (E); and in the absence of enzymes but presence
of 1% formaldehyde for 2 h at room temperature (F).
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|
Incubation of metaraminol and ()-isoproterenol with human liver
microsomes for 60 min led to 85 and 66% substrate disappearance (Table
1), respectively, when methanol (1% v/v)
was used in the incubations. The disappearance in the absence of any
organic solvent was, however, only 15 and 24%, respectively, for
metaraminol and ()-isoproterenol (Table 1).
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TABLE 1
Percentage of disappearance of substrates following a 60-min incubation
with hepatic microsomal fractions in the presence and absence of 1%
methanol (245 mM)
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Characterization of the Metabolites of LAQ094, Metaraminol,
and ()-Isoproterenol.
The products formed from LAQ094, metaraminol, and ()-isoproterenol
were characterized by LC/MS analysis. Incubation of LAQ094 with human
liver microsomes in the presence of 1% methanol led to the formation
of only one major metabolite eluting at 18.1 min (Fig. 2A). The
protonated molecular ion of this metabolite was observed at
m/z 212, 12 amu higher than that of LAQ094 (m/z 200, 17.5 min). Comparison of their MS/MS product ion spectra (Fig.
3) showed that the product ion
(m/z 129) corresponding to the amino chloropyridyl moiety in
LAQ094 is retained in the metabolite. The major product ion
(m/z 183) from LAQ094 was consistent with the loss of
NH3 molecule, whereas that from the metabolite
was consistent with the loss of NH=CH2 molecule,
suggesting the existence in the metabolite molecule a methylene group
connecting the terminal amino group. Based on this information, the
metabolite was proposed as a 1,3-tetrahydroimidazole derivative (Fig.
3). When d4-MeOH was used as the solvent,
the molecular ion of the metabolite was observed at m/z 214 (data not shown). The major product ion (m/z 183) from the
d2-labeled metabolite resulted from the
loss of NH=CD2 molecule (data not shown),
indicating that the methylene group connecting the two nitrogen atoms
originated from methanol. The reaction of LAQ094 with formaldehyde
formed a product (Fig. 2E) with retention time, molecular ion, and
MS/MS product ion spectrum identical to those of the metabolite, thus
further supporting the structural assignment of the unusual metabolite
from LAQ094.
Similar results were obtained following microsomal incubations of
metaraminol. Total ion chromatogram (TIC) (Fig.
4A) of the incubates showed three peaks.
The extracted ion chromatogram (XIC) of metaraminol (Fig. 4B) showed a
weak peak at 7.5 min, which does not correspond to any of the three
peaks in TIC trace. The two peaks at 8.1 and 10.3 min in TIC trace
appeared to be metabolites each with 12 amu mass addition (see XIC in
Fig. 4C). These two metabolites had MS/MS product ion spectra (Fig.
5, bottom) identical to each other and
their major fragments m/z 162, 147, 145,127,121, and 117 are
each 12 amu higher than the fragments m/z 150, 135, 133, 115, 109, and 105 from metaraminol (Fig. 5, top). Based on the mass
spectral information, its formation dependence on methanol solvent, and
analogy to LAQ094 findings, the [M + 12] metabolites of metaraminol
were proposed as isomeric tetrahydrooxazole derivatives (Fig. 5). The
reaction of metaraminol with 1% formaldehyde produced the same two
products with MS/MS product ion spectra (data not shown) and retention
times identical to those of the metabolites, thus further supporting
the structural assignments. The third peak at 8.5 min in the TIC trace
had a molecular ion of m/z 123. Its product ion mass spectra
showed fragments at m/z 123 (100, protonated parent ion),
m/z 106 (6.3, loss of NH3), 96 (6.5, loss of HCN), 80 (63.4, loss of CO=NH) and 78 (12.8, loss of
HCO-NH2) and was, therefore, proposed as
nicotinamide resulting from enzymatic breakdown of NADPH. The product
ion mass spectra of synthetic nicotinamide were identical to this
metabolite, thus further supporting the structural assignment. The
formation of nicotinamide was enzyme-dependent since a 60-min
incubation with heat-inactivated microsomes did not lead to its
formation (data not shown).
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Fig. 4.
Metabolite profile of metaraminol following
a 60-min incubation with human liver microsomes by LC/MS analysis:
total ion chromatogram (A); extracted ion chromatogram for unchanged
metaraminol (B); and extracted ion chromatogram for [M + 12]
metabolites from metaraminol (C).
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Incubation of ()-isoproterenol (m/z 212) with liver
microsomes in the presence of methanol also produced two [M + 12]
metabolites (m/z 224). TIC (Fig.
6A) of the incubates showed three peaks, two of them eluting at 9.5 and 10.2 min correspond to two metabolites each with 12 amu mass addition (see XIC in Fig. 6C), and the third one
eluting at 8.6 min was nicotinamide. The XIC of ()-isoproterenol (Fig. 6B) showed a weak peak at 9.7 min. The two metabolites had molecular ion as well as MS/MS product ion spectra (Fig.
7, bottom) identical to each other. The
two major fragments m/z 164 and 206 are each 12 amu higher
than the fragments m/z 152 and 194 from ()-isoproterenol
(Fig. 7, top). Based on the mass spectral information, its formation
dependence on methanol solvent, and analogy to LAQ094 findings, the two
[M + 12] metabolites of ()-isoproterenol were also proposed as
tetrahydrooxazole derivatives (Fig. 7). The reaction of
()-isoproterenol with 1% formaldehyde produced the same two products
with identical MS/MS product ion spectra (data not shown) and retention
times to that of the metabolites, thus further supporting the
structural assignments.
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Fig. 6.
Metabolite profile of ()-isoproterenol
following a 60-min incubation with human liver microsomes by LC/MS
analysis: total ion chromatogram (A); extracted ion chromatogram for
unchanged ()-isoproterenol (B); and extracted ion chromatogram for
[M + 12] metabolites from ()-isoproterenol (C).
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Fig. 7.
Positive product ion mass spectra of
()-isoproterenol (top) and its [M + 12] metabolites (bottom).
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Kinetics of Formaldehyde Formation by Human Liver Microsomes.
The conversion of methanol to formaldehyde by human liver microsomes
was NADPH-dependent (data not shown), and was linear for at least 60 min at 245 mM methanol and 1 mg/ml protein (data not shown).
Formaldehyde was formed up to 600 µM. The reaction followed a typical
Michaelis-Menten kinetics (Fig. 8), with
an apparent Km of 35 mM and a
Vmax of 7.9 nmol/min/mg of protein.
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Fig. 8.
Enzyme kinetics of formaldehyde formation
from methanol by human liver microsomes.
Concentration of formaldehyde formed in 1 h at 1 mg of protein/ml
was plotted against methanol concentration.
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| |
Discussion |
The current study showed that the apparent in vitro metabolic
instability of compounds with 1,2-diamino or 1,2-amino hydroxy moieties
could be artificially augmented by the presence of methanol, a common
solvent used to dissolve and deliver compounds into the incubation
systems. Investigation of the mechanism of instability of a few new
chemical entities revealed unusual metabolites with 12 amu mass
addition. Three compounds, LAQ094, metaraminol, and ()-isoproterenol,
were chosen in the present study to illustrate such a methanol solvent effect.
Mechanism of [M + 12] Metabolites Formation.
Methanol or other organic solvents are typically used at 1% (v/v) or
less in microsomal or S9 incubations, as a balance of compound
solubility and the solvent inhibition of P450 activity considerations.
Because of the small molecular weight of methanol, 1% (v/v)
corresponds to 245 mM in concentration. It was well known that methanol
can be oxidized to formaldehyde by liver enzymes (Dawidek-Pietryka et
al., 1998). The current study showed that formaldehyde formed by human
liver microsomes in 1 h reached a concentration as high as 600 µM. It was shown previously (Yin et al., 1996) that formaldehyde
readily reacts with ethylene 1,2-diamino and ethanolamine to
yield tetrahydroimidazole and tetrahydrooxazole derivatives,
respectively. By analogy, LAQ094, metaraminol, and ()-isoproterenol,
each containing 1,2-diamino or 1,2-amino hydroxy functional groups, can
react with formaldehyde to form heterocyclic products (Fig.
9) that have molecular weights 12 amu
higher than their respective reactants. Previous work (Yin et al.,
1996) showed glutathione could readily react with formaldehyde to form
a thiohemiacetal intermediate that is further converted to a stable
six-membered heterocyclic ring. When 10 mM glutathione was supplemented
in the microsomal incubations, it appeared all the formaldehyde formed from methanol was effectively trapped and no [M + 12] metabolite from
LAQ094 was produced (Fig. 2E). It is, therefore, suggested to include
glutathione in the incubation system for future in vitro screening to
avoid misleading results, when methanol is used as the
substrate-delivering solvent. Previous work (Koppel et al., 1991; Yin
et al., 1996) showed that primary amines could also react with
formaldehyde to form imine products with 12 amu mass addition. Further
investigation is necessary to examine whether other structural moieties
can also lead to the formation of [M + 12] products in the presence
of formaldehyde.
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Fig. 9.
Proposed mechanism of formation of [M + 12] metabolites from LAQ094, metaraminol, and ()-isoproterenol in
the presence of methanol and liver subcellular fractions.
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Metabolism of LAQ094, Metaraminol, and ()-Isoproterenol by Human
Liver Microsomes.
Based on HPLC chromatography and MS analysis, the [M + 12] metabolite
was the only detectable metabolite from LAQ094 in the presence of
methanol. This compound is very stable during the 60-min incubation in
the absence of methanol. The trace amount of [M + 12] metabolite peak
observed in the incubations with DMSO or no solvent might be due to the
incomplete evaporation of methanol solvent, which was used for the
preparation of stock solution.
Previous work (Fuller et al., 1981; Wollenberg and Rummel, 1984; Causon
et al., 1985) showed O-methylation and
O-sulfation as the major metabolic pathways for metaraminol
and ()-isoproterenol in vivo. The present study with liver microsomes
did not produce these metabolites (data not shown) since the cofactors
required for methyl transferase and sulfur transferase activity were
not supplemented. The two [M + 12] metabolites from metaraminol (Fig. 4C) eluting at 8.1 and 10.3 min well separated from each other and had
comparable abundance and identical product ion mass spectra. These two
metabolites are likely cis- and trans-isomers
(two sets of diastereomers), since a diastereomeric mixture of
metaraminol was used in the study. The two [M + 12] metabolites from
()-isoproterenol (Fig. 6C) eluting at 9.5 and 10.1 min also separated
from each other and had comparable abundance and identical product ion
mass spectra to each other. The mass spectral data suggest that they are not regioisomers, i.e., none of them is the acetal formed from the
reaction of the ortho dihydroxyl functional groups with formaldehyde.
The most likely reason for their chromatographic separation is the
protonation of the nitrogen group resulting in cis- and
trans-isomers at the mobile phase pH of 3.5.
Enzymatic Hydrolysis of NADPH.
Enzyme-dependent conversion of NADPH to nicotinamide was observed in
human liver microsomal incubations in the present study. Such an
enzymatic reaction was not previously documented and the reaction
mechanism warrants further investigation. Since the formation of
nicotinamide is enzyme-dependent and NADPH-dependent, there is a
potential that nicotinamide could be mistakenly treated as a metabolite
in HPLC-UV analysis, and consequently mislead the metabolic instability
information when assessed by relative peak areas.
Other Implications of Methanol Solvent Effect.
In addition to the potential interference in metabolic stability
assays, methanol solvent could impact the in vitro toxicological screening. Previous study (Cunningham et al., 1990a,b) showed that a
mutagenic product, bis-5,5'-(2,4,2',4'-tetraaminotolyl)methane, was
produced in the Ames/Salmonella assay from the reaction of 2,4-diaminotoluene with formaldehyde, which was produced from methanol
in the presence of S9. Koppel et al. (1991) showed the formation of
formaldehyde adducts from various drugs, such as amphetamine,
propafenone, flecainide, 
-blockers, and prilocaine, by use of
methanol in the toxicological screening. They observed [M + 12]
adducts resulting from reactions with formaldehyde and proposed that
formaldehyde was formed by thermal dehydrogenation of methanol in the
injection port of the gas chromatography.
In summary, this article demonstrated that methanol is oxidized to
formaldehyde by human liver microsomes with an apparent Km of 35 mM and a
Vmax of 7.9 nmol/min/mg of protein. The
formaldehyde thus formed may undergo condensation reactions with
compounds with 1,2-diamino or 1,2-amino hydroxy functional
groups. When microsomal incubations are used to assess metabolic
instability by measuring the amount of parent compound remaining, this
reaction can lead to an erroneous conclusion about metabolic
instability. The compound may appear to be unstable, although the
reason for the instability is not a direct consequence of the
metabolism of the compound in question, but rather an artifact of the
methanolic content of the incubation.
We thank Dr. Edwin Villhauer (Novartis Pharmaceuticals Corporation,
Summit, NJ) for providing LAQ094 and Drs. Alban Allentoff, Heidi
Einolf, and James Mangold (Novartis Pharmaceuticals Corporation, East
Hanover, NJ) for helpful discussions.
Abbreviations used are:
LC/MS, liquid
chromatography and mass spectrometry;
DMSO, dimethyl sulfoxide;
Fluoral-P, 4-amino-3-penten-2-one;
HPLC, high-performance liquid
chromatography;
LC/MS/MS, liquid chromatography and tandem mass
spectrometry;
MS/MS, mass spectrometry/mass spectrometry;
amu, atomic
mass unit;
TIC, total ion chromatogram;
XIC, extracted ion
chromatogram;
LAQ094, 2-[(5-chloro-2-pyridinyl)amino]-1,1-dimethyl-ethylamine.
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Abstract
Introduction
