Rats were treated with radiolabeled L-deprenyl to study its metabolites. Urinary samples were analyzed using HPLC and HPLC-MS. Nε-monomethyl-lysine was identified as a metabolite and its structure was elucidated. The urine samples were derivatized with Fmoc chloride and separated by HPLC. Key fractions were analyzed using HPLC-MS which showed that the radiolabeled fragment was present in Nε-monomethyl-lysine, demonstrating transmethylation of the drug's methyl group. This metabolic pathway may help explain the positive effects of certain drugs.

1. HPLC and HPLC–MS
Analysis of Urinary
Nε-Monomethyl-Lysine
Submitted To:DR.NIGHAT FATIMA
Submitted By: FAZAL RABBI
MS:CIIT/FA22-RPY-030/ATD
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2. CONTENTS
INTRODUCTION &
BACKGROUND
01
INSTRUMENTATION &
RESULTS
02
.
DISCUSSION &
CONCLUSION
03
.
SUMMARY
04

3. HPLC &HPLC–MS Analysis
of Urinary
Nε-Monomethyl-Lysine

4. Principle of HPLC-MS
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Mass Spectrometry (LC-MS) The LC-MS
technology involves use of an HPLC,
wherein the individual components in
a mixture are first separated followed
by ionization and separation of the
ions on the basis of their mass/charge
ratio.
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5. ABSTRACT
Administration of 14C-labelled L-deprenyl to Rats results in the urinary
elimination of a 14C-labelled compound. The 9-fluorenylmethoxycarbonyl
chloride-reacted urine sample is fractionated by (HPLC) on an octadecyl
silica (SiCl8) stationary phase. Nε-Monomethyl-lysine is identified in the
fraction containing the majority of the radioactivity. Structural elucidation is
carried out using HPLC–mass spectrometry in atmospheric pressure
chemical ionization mode. Identification of the 14C-labelled fragment in Nε-
monomethyl-lysine is an experimental proof that an N-methylated amino acid
is generated by TRANSMETHYLATION from a well-known drug. This type of
transmethylation may have basic importance in the positive side effects of
certain drugs.
Selegiline
Fmoc-Cl

6. INTRODUCTION & BACKGROUND
rats were treated with the radiolabelled L-deprenyl
well-known metabolites of L-deprenyl in urine
Using TLC and HPLC–MS
identification of Nε-monomethyl-Llysine as
Metabolites
L-Deprenyl (selegiline hydrochloride) is an
irreversible
Selegiline acts as a monoamine oxidase inhibitor
Monoamine oxidase-B (MAO-B)
modest benefits for Parkinson’s movement
symptoms

7. INSTRUMENTATION & RESULTS HPLC
separate the urine samples
after derivatization with
Fmoc hydrochloride. It
contained a DG-208054
degasser, two PU-1580
pumps, an AS2057 plus
automatic sample injector,
and a UV-1575 detector.
Acetonitrile–water (2:1)
also containing 0.1%
formic acid was used as
the mobile phase.
HPLC-MS
A model HP LC–MS 1100
instrument. It was equipped with a
high-pressure gradient pump and
diode-array detector, and the MS
was used in the positive
atmospheric pressure chemical
ionization mode. Acetonitrile–water
(6:1) also containing 0.1% formic
acid was used as the mobile
phase.
HPLC of Rat Urine Sample reacted with Fomc.The separation
was performed using 6µm Kovasil & acetonitrile–water (2:1, also
containing 0.1% formic acid) as mobile phase.

8. Precolumn derivatization with Fmoc chloride
To each 1-mL urine sample, 0.5 mL potassium borate buffer
(0.8M, pH 10) was added, and the mixture was vigorously
shaken for 1 min using a mixer. One milliliter of 10mM Fmoc
chloride solution in acetonitrile was then added and
immediately vortexed for 1 min. One milliliter of n-hexane
was then added, the mixture was shaken for 1 min, and the
upper and lower phases were separated by centrifugation.
The upper (organic) phase containing the excess Fmoc
reagent was discarded. The n-hexane extraction of the
excess Fmoc was repeated twice. Finally, 100 µL of acetic
acid (10%, v/v) was added to the samples, mixed, and 100
µL of sample was subjected to HPLC separation. A similar
derivatization was performed with the standard compounds.
METHODS
Male Wistar rats (200–250 g) were
treated per os with radiolabeled L-
deprenyl. Urine samples were coll
ected for 6 h.
A 1:1 mixture of acetonitrile–water also
containing formic acid is an adequate
mobile phase when using octadecyl silica
stationary phase. Detection at 265 nm
gives certain specificity. The fractions
were collected for 1 min each, and their
radioactivity was determined. show the
presence of Nε-monomethyl-lysine in the
fraction containing the majority of
radioactivity. The mass spectrum of
standard Nε-monomethyl-lysine and that
of the radioactive fraction
Results

10. Conclusion
Experimental results support the formation of
Nεmonomethyl-lysine based on the N-methyl group
of L-deprenyl. The process of methyl transfer was
traced with the help of the 14Cmethyl group of L-
deprenyl, and the detection was facilitated by
forming the Fmoc derivative. Preparative
separation was carried out using HPLC, but the
final structural elucidation was successful
with the help of HPLC–MS

11. SUMMARY
Parkinson Disease
MAO Inhibitors
MAO A –Serotonin
MAO-B Dopamine
INTRODUCTION &
BACKGROUND
TLC Usage
HPLC –MS Usage
Rates used as
experimental
Intend to Test. Urine
sample
Methods and Results
The process of methyl transfer was
traced with the help of the
14Cmethyl group of L-
deprenyl, and the
detection was facilitated
by forming the Fmoc
derivative. Preparative
separation was carried
out using HPLC, but the
final structural
elucidation was
successful with the help
of HPLC–MS.
Conclusion
individual components
in a mixture are first
separated followed by
ionization and
separation of the ions
on the basis of their
mass/charge ratio.
Principle of HPLC-MS

12. Q&A
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