This document discusses various bioanalytical techniques for metabolite identification and quantification. It describes drug metabolic pathways involving phase I and phase II reactions. Several in vitro models for drug metabolism evaluation are discussed, including human liver microsomes and hepatocytes. The key techniques covered are NMR, GC-MS, and LC-MS. NMR provides structural information but has low sensitivity, while GC-MS and LC-MS/MS are more sensitive and commonly used in metabolomics studies. Different modes of LC-MS/MS such as full scan, precursor ion scan, and multiple reaction monitoring are described for metabolite detection.

1. Bioanalytical Techniques
By
Muhammad Rashad
PhD Scholar
(Pharmaceutical Chemistry)
Biochemistry of Drug Metabolizing Enzyme

2. Drug metabolism involves a series of
biological and chemical processes through
which endogenous substances are converted
into more water-soluble substituents resultantly
excreted out from the body.
Drug metabolic pathways accomplished by
phase I and phase II biotransformation
reactions.
Drug Metabolism

3. • Phase I reactions comprise oxidation,
reduction, and hydrolysis.
• Phase II biotransformation reactions also
called conjugation reaction includes
glucuronidation, acetylation and sulfation
reaction.

4. Models for drug metabolism
evaluation
Various in vitro models are developed for the
drug metabolomics analysis, some of them are
described below.

5. Models
Human liver
microsomes
Human liver
cytosol fractions
S9 fractions
Cell lines
Transgenic cell
line
Isolated perfused
liver
Recombinant
human CYP and
UGT enzymes
Hepatocytes
Liver slices

6. Approaches for metabolite quantification
Quantification of the metabolite is essential in
such circumstances when metabolite
concentration just reaches or crosses the limit of
plasma drug concentration or when the drug
metabolites are either pharmacologically active
or toxic.

7. 1. Direct quantification
• Drug metabolites are much more hydrophilic as
compared to the parent drug, especially in case
of glucuronides.
• for precise and definitive quantification
of biological samples, appropriate authentic
standards are required.
• Chemical synthesis is generally appropriate
for making phase I metabolites, including
O-demethylation, N-oxidation, N-
demethylation, carbonyl reduction and
others.

8. 2. Indirect quantification
• This technique has been employed for estimation of
phase II metabolites especially glucuronide
metabolites. These metabolites are detected by
splitting the conjugates to produce original drug,
which is then detected.
• In the case of morphine which is metabolized into two
metabolite isomers, including morphine-3-glucuronide
and morphine-6-glucuronide, former is an inactive
metabolite while later possess more pharmacological
activity even more than the parent drug.
• NMR based metabolomics mostly used for the indirect
quantification of drug metabolites.

9. 3. Qualitative evaluation
• Mostly every type of mass spectrometer can be
employed for the determination and recognition of
glucuronide metabolites. However, those with
tandem or high-resolution MS capacities are
preferred.
• For the detection and validation of metabolites,
LC/QTOF or LC/TOF instruments with tandem MS
are commonly used. Currently, the use of Fourier
Transform Ion Cyclotron Resonance Mass
Spectrometer (FTICR / MS) and Ion Trap-Orbitrap
(LTQ-Orbitrap) become more preferable tools in the
metabolomics analysis as these have more
compatibility with multiple LC methods.

10. Bioanalytic techniques for metabolites
identification
• For the analysis of hundreds of metabolites, several
analytical techniques are used.
• But none of the bioanalytical technique can fully
analyze all metabolites in the sample due to their
physicochemical diversity, including amino and non-
amino organic acids, volatile ketones, alcohols, lipids,
and carbohydrates, etc.
• Hence, different analytical techniques are used in
combination with each other to fully measure the
whole metabolomics.

11. Bioanalytical
Techniques
NMR
GC-MS
LC-MS/MS
LC-MS
However, NMR as compared to GC-MS and LC-MS less
sensitive and more than 80% of metabolomics studies are carried
out by using these two methods.

12. 1. NMR based metabolite
identification and quantification
• NMR coupled with MS provides optimum outcomes,
but this technique is not commonly used because of
its high cost.
• For the structural elucidation of organic compounds,
NMR spectroscopy is frequently used.
• H1 NMR is a more useful and sensitive technique for
the metabolites analytical study than C-NMR
spectroscopy.
• The working principle behind NMR spectroscopy is
the nuclei spinning of the atom, as metabolic
components are made up of atoms and each atom has
its nuclei.

13. H1 NMR spectrum provide following pieces of
information about the metabolites:
• The number of signals reflects the number of
magnetically and chemically different protons. Such as
the methanol molecule is comprised of methyl and
hydroxyl (two sets of) protons, therefore in H1 NMR
spectrum produce two signals.
• The area under the NMR peak refers to the intensity,
which can be measured through the integrator.
• The relative position of NMR signals which is also
known as the chemical shift, determined by special
arrangement of adjacent electrons and atoms.
• The splitting of NMR signals determines the coupling
constants.

14. • The first step in NMR spectroscopy working is the
sample preparation. Biological fluids including
plasma require sample preparation for NMR but
some biological fluids such as cerebrospinal fluids
need no sample preparation.
• Tagging is the second step. Hydrogen atom attached
to N-15 is used as an active label.
• The last step is quantification; a reference sample of
known concentration must be added to determine the
absolute concentration.
Principle

15. • One dimensional H1 NMR spectroscopy is a fast, reliable, and
reproducible technique.
• 1D H1 NMR spectroscopy can identify and quantify 50-100
metabolites at a time.
• Moreover, 2D H1 NMR spectroscopy as compared to 1D H1 NMR
can identify and quantify more metabolites.
• 13C NMR has advantageous resolution results than H1 NMR but
due to the low natural abundance of 13C (∼1.1%) has less
sensitivity towards the 13C nucleus, which significantly reduced
13C NMR applications in metabolomics.
• 15N NMR spectroscopy has wide applications in the
structure elucidation and identification of DNA, RNA and proteins
but owing to the low natural abundance of 15N (0.37%), not
readily used in metabolomics studies. 31P nucleus has about 100
% relative abundance and a hundred times more sensitivity than
H1 NMR spectroscopy.

16. • Liquid chromatography combined with the
NMR spectroscopy (LCNMR) can be used
for simplifying the biofluids' complexity
such as urine and fecal water extracts, that
are not detected and quantified via 1D or 2D
NMR.
• Similarly, LC-NMR-MS is used for the
structural elucidation and detection of novel
metabolites

17. • Fluoxetine drug metabolized to norfluoxetine via
dealkylation reaction, to ensure the drug metabolism
NMR spectroscopy can be used for detection or
quantification of the drug metabolites without further
confirmation tests.
• Irbesartan drug after metabolic reaction converts into
hydroxyl irbesartan, which can be confirmed by NMR
spectroscopy.
• Metabolism of pyragallol drug which is metabolized
via sulfation reaction and converted into the
dihydroxyphenyl-2-O-sulfate metabolite.
• For the metabolism of the benzoic acid, an amino acid
conjugation reaction takes place and transformed the
drug into hippuric acid, this metabolic process can be
confirmed via NMR.

18. 2. GC-MS based metabolite identification
and quantification
• In GC-MS the mass spectrometer is coupled with
the gas chromatography. GC with great
resolution separated the volatile compounds but
cannot appropriately identify them. In MS, ions
moved through the vacuum, and by their mass to
charge ratio ionized atoms or molecules are
separated.
• GC-MS spectroscopy are preferable for detection and
quantification of the small molecular weight
metabolites (<650 daltons) such as small acids,
hydroxyl acids, alcohols, sugars, amino acids, fatty
acids, hormones, sterols, drugs, toxins, catecholamine,
aromatic and several intermediate products of primary

19. • In all GC–MS based metabolomics studies, capillary
columns are used because of their great efficiency.
• GC-MS efficiency, selectivity and analysis time
increase directly with the length of the column.
• Longer columns are used for complex samples and
require more time for analyses vice versa.
• In untargeted GC–MS based metabolomics studies,
generally 30 m columns and non-polar stationary
phases (5% phenylarylene 95% dimethyl polysiloxane
or 5% diphenyl 95% dimethylpolysiloxane) are used.

20. • Assche et al. used GC–MS/MS in scan mode for the
investigation of metabolomics profiles of transgenic
Caenorhabditis elegans and have identified a total of
76 unique metabolites and 8 out of them were
considerably different.
• In 2016, Karimpour et al. have studied the postprandial
effect on human plasma metabolome by using the GC–
TOF–MS technique and identified 200 unique mass
features in the sample.
• Lu et.al identified 2482 mass features in human serum
by using the GC–qTOF–MS.

21. 3. LC-MS/MS-based metabolite identification
and quantification
• LC coupled with MS is composed of three main parts, including
the ion source which converts molecules into ions, the mass
analyzer which separates these ions on bases of m/z ratio and
passed them towards detector and the final element of LC/MS is
the detector which detects or identifies the separated components.
• Hypothetical spectra of unknown ions can be predicted through
in silico splitting techniques. For the estimation of given
molecule’s fragment ions, two main methods are employed. First
is the rule-based predictor which can be applied to the
fragmentation mechanisms obtained from the literature. The
second one is the in silico fragmentation such as Fragment
Identification

22. • Computational techniques can provide great help in
the identification of metabolites thus they are not
much efficient to replace the experimental validation
process employed for the identification of these
metabolites.
• For nonpolar and weakly polar compounds, reverse
phase liquid chromatography with C18 columns is
used which provides high resolution. For the
separation of polar, ionic and hydrophilic molecules,
normal phase (NP) liquid chromatography can be
used.

23. Hydrophilic interaction liquid
chromatography (HILIC) is a novel mode of
separation having high compatibility with
mass spectrometer and improved resolution
for the detection of polar analytes. HILIC
combines the mobile phase used in reverse
phase separation and the stationary phase used
on NP mode.

24. Drug
Metabolic
enzyme
Metabolites Biofluid
Analytical
method
Omeprazole CYP2C19
S-
hydroxyomeprazole
and
omeprazole sulfone
plasma LC–MS/MS
Amodiaquine CYP2C8
N-
desethylamodiaquine
Plasma
and urine
HILIC–
MS/MS
Buspirone CYP3A4
1-[2-pyrimidyl]-
piperazine
plasma LC–MS/MS
Tolbutamide CYP2C9
5-
hydroxysulfadiazine
Plasma
and urine
LC–MS/MS
Caffeine CYP1A2 Paraxanthine
Plasma
and urine
LC–MS/MS
Clarithromycin
CYP3A4 and
ABCB1
14-
hydroxyclarithromyci
n
plasma LC–MS–MS
Tolbutamide
Dehydrogen
ase
Carboxytolbutamide
Plasma
and urine
LC–MS/MS
Debrisoquine CYP2D6
4-
hydroxydebrisoquine
Plasma
and urine
LC–MS/MS
Midazolam CYP3A 1-hydroxymidazolam
Plasma
and urine
LC–MS/MS

25. 4. Other suitable approaches for metabolite
identification
LC-MS instrumentation is critical for both screening,
investigation, and structural interpretation. However, the
non-MS approaches can also play a crucial role in such
situations where just the MS data is not satisfactory.
1. Full scan
Full mass scan technique permits the identification of
almost all ionizable metabolites and provides
comprehensive details regarding the molecular mass of
drug metabolites because of its nonselective nature.
Triple quadrupole mass analyzers have been used
which drastically reduces the sensitivity. This problem
may resolve by employing IT analyzers.

26. 2. Precursor ion and constant neutral loss
scan
The precursor ion and constant neutral loss scan mode are
only used for tandem mass spectrometers and employed for
the determination and identification of unknown drug
metabolites.
Numerous metabolites of phase II during the atomization,
have loosed a definite group that can be used for precise
scanning and isolation of these conjugates.
3. Multiple reaction monitoring
The multiple reaction monitoring mode has more selectivity
for the detection of the metabolite. In this approach, only a
specific precursor ion or metabolite has allowed to pass
through the first quadrupole which afterward has split in the
collision cell and then the product ion separated from the
second quadrupole.

27. 4. Single ion monitoring
To surpass the low sensitivity problem of triple quadrupole
mass spectrometer, the alternative is single ion monitoring.
This offer low specificity and sensitivity as against multiple
reaction monitoring, but it has been provided many
advantages when the exact pattern of the analytes is not
accurately forecasted.
5. Hydrogen/deuterium exchange LC-MS
Hydrogen/deuterium exchange LC-MS can be employed to
obtain information related to drug metabolic pathways, MS
fragment product ion formation and for studying and
differentiation of the structures of the isomeric metabolites.

28. 6. High resolution mass spectrometry
For the identification and determination of metabolites, the
more accurate analyzer in mass prediction is TOF
instrument. It has been declared that by using the accurate
mass TOF, various drugs can be detected 5-25 times better
as compared to nominal mass TOF.
7. Product ion scan
Product scan mode is employed for the structural
determination and elucidation of scanned drug metabolites.
In this mode, in the first quadrupole, a metabolite has been
chosen which split in the collision cell and then the product
ions are examined in the second quadrupole.
To acquire more particular structural data, the multistage
scan by utilizing ion trap instruments can be employed.

29. 8. Ion mobility time-of-flight mass spectrometry
The Ion mobility time-of-flight mass spectrometry is suitable
for segregating ions based on their mass-to-charge ratios. The
major benefit of this technique is its ability to segregate the
metabolite isomers which makes it a stronger bioanalytical
technique for the analysis of complicated samples.
9. Chemical agents for metabolite identification
Particular chemical entities are capable to interact with few
kinds of the metabolites as a result of respective signal
frequencies are altered in both MS and NMR spectra. This
feature has been frequently employed for the determination
and detection of metabolites. In this approach, two 15N
labeled agents, one is aminooxy probe and the second one is
cholamine tag is used. These labels have high specificity and
can covalently bound with metabolites having carbonyl and
carboxyl groups correspondingly. This procedure permits
direct visibility of the metabolite’s signals in 2D 15N-1H-
HSQC-NMR spectra and mass spectra.

30. Conclusion
• Various enzymes catalyze the biotransformation of drugs and are
mainly classified into four categories based on the reactions
catalyzed by these enzymes. Targeted and untargeted
metabolomics strategies are used for the identification, detection,
and quantification of endogenous and exogenous metabolites.
• Metabolite quantification is very crucial when drug metabolite is
toxic or pharmacologically active.
• Mainly three bioanalytical techniques have been used in
metabolomics analysis, including NMR, GC-MS and LC/MS/MS.
• Currently, LC-MS based metabolomics analysis has been
frequently used because of the several advantages over the other
two techniques.
• LC/MS/MS computational framework for the untargeted
metabolomics, significantly helpful in the identification of known
and unknown metabolites.