SEMINAR ON

QUANTITATIVE BIOANALYSIS OF
DRUGS & DRUG
METABOLITES USING HILIC MS

Submitted by: Submitted to: Head of Department

HARSHADA B. KULKARNI Mrs. Sarika H. Alhat Dr. Sonali P. Mahaparale

Roll no. 524
M. Pharm-I (QAT)
 CONTENTS:
■ INTRODUCTION
■ HILIC-MS
■ ADVANTAGES
■ DISADVANTAGES
■ APPLICATIONS
■ CASE STUDIES:
a) Bioanalysis of Monoamine Neurotransmitters
b) Analysis of polar analytes in urine.
■ CONCLUSION
■ FUTURE PRESPECTIVE
 INTRODUCTION:
■ The study of the metabolic fate of drugs is an essential and important
part of the drug development process.
■ The application of untargeted metabolic phenotyping
(metabolomics/metabonomic) to large scale preclinical
metabolism/toxicological and clinical or epidemiological investigations
has delivered new insights into the underlying biology of health,
toxicity, and disease.
■ Liquid chromatography coupled with mass spectrometry (LC-MS) has
become the most powerful analytical tool for screening and
identification of drug metabolites in biological matrices.
 HILIC-MS

■ HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY

■ The term HILIC was proposed by Alpert in 1990 to describe the increased retention of
hydrophilic solutes with a hydrophilic stationary phase and an aqueous organic mobile
phase.
■ Adsorption and partitioning are the two main retention mechanisms of HILIC.
■ HILIC uses a polar stationary phase and a non-polar mobile phase.

Stationary phases
Underivatized Zwitterionic-
diol-, amide-, amino-
(sulfoalkylbetaine)
silica bonded phases
bonded phases
 HILIC-MS

Mobile Phase
■ The mobile phases used for HILIC are
typically a mixture of organic-aqueous
solvents.
■ It consists of a minimum of 3%water, which
enhances MS sensitivity.
■ Acetonitrile is commonly used as the organic
component. Substitution of different solvents
affects the retention and selectivity of analytes
due to changes in the retention mechanisms.
HILIC-MS
 ADVANTAGES OF HILIC-MS
■ Sufficient retention of the most hydrophilic compound that does not possess sufficient
retention under reversed-phase LC condition.
■ Faster or high-resolution analysis.
■ Important advantage related to MS sensitivity reflected in three main points:

Increased Electro
Spray Ionization More efficient Alteration of ion
(ESI) stability: solvent evaporation suppression

• Acetonitrile has a • Due to higher organic • Polar compounds in

surface tension lower content of the eluent
than water, requiring Quicker solvent
lower voltages for the evaporation, which
generation of stable allows efficient release
sprays. of gas phase ions.
• Improves signal
strength & sensitivity.
RPLC are more prone
to ion suppression
because they elute in
the first region of the
chromatogram, where
matrix is mainly
present.
 DISADVANTAGES OF HILIC-MS
■ The incompatibility of UV detection systems at low wavelengths
with acetone-based separations.
■ An understanding of the physicochemical properties of a substance
is essential for selecting the appropriate HILIC phase to work with.
■ Fragmentation patterns are poorly reproducible, databases are
therefore problematic.
■ Not all the compounds will ionise under same conditions so, the
technique is more suited for targeted analysis.
 APPPLICATIONS OF HILIC-MS
HILIC has a wide range of biomedical applications, including:
■ Chemical engineering
■ Biochemistry – To separate polar peptides following digestion of proteins to determine
protein function and stability.
■ Pharmacology – To analyse polar drugs and metabolites, including proteins, peptides,
and antibodies.
■ Agricultural – To analyse plant extract polar components, such as those in phenolic
acids, carbohydrates, peptides, and flavonoids.
■ Food Industry – To detect food contaminants, marine biotoxins, small polar
compounds and biogenic amines (this produced by organisms).
■ Characterization of Intact or Large Fragment Protein Biopharmaceuticals.
Case Study 1 : Bioanalysis of Monoamine Neurotransmitters

■ One set of polar analytes that poses particular challenges are the
monoamine neurotransmitters, dopamine (DA), serotonin (5-HT),
epinephrine (EP) and norepinephrine (NE).
■ The application of HILIC for the analysis of monoamine
neurotransmitters using a 2.5 μm hybrid particle bonded with an
amide moiety. Retention is substantially improved when compared
to reversed-phase analysis, especially for EP and NE
■ Separation and chromatographic resolution are also improved,
allowing unequivocal identification of these closely related
compounds in a short analysis time.
Continue…
■ Sample preparation:

Initial Working
stock stock
10 μg/ml 5-HT, DA,
solutions 1.0 mg/ml for all solution EP, NE and 1 μg/ml
target analytes
NMS

Prepared in Prepared in methanol

methanol containing 0.2%
containing 5% HCl ascorbic acid.
Continue…
■ HILIC-MS:

Columns : Waters XBridge™ BEH Amide XP and XBridge

BEH HILIC XP columns (2.5 μm, 2.1 × 75 mm)
• Flow rate : 0.5 ml/min

Mobile phase A (MPA) consisted of 95:5 water: acetonitrile containing

either 10,20,50 or 100 mM ammonium formate buffered to pH 3.0

• Mobile phase B (MPB) consisted of varying combinations of

acetonitrile, water, and ammonium formate (pH 3.0)
MS detection : Performed using a Waters XevoTM TQ-S triple-quadrupole
MS system
Continue…
■ Result
Analysis time : 4 min.
■ Conclusion
The intermediate length of the
column (75 mm) combined with
the relatively low back pressures
characteristic of HILIC analysis
can allow future investigators to
improve separation, reduce
analysis time, or, if desired, both.
 Case Study 2 : Analysis of polar analytes in urine

■ Urine analysis became so popular that every disease even

the fortune of an individual was being sought in the
matula, a glass vessel in which a physician assessed urine,
examining its colour, turbidity, and transparency.

■ The analysis of polar metabolites in urine using HILIC–

MS, either alone or coupled to IMS-enabled MS data
acquisition (HILIC–IMS–MS).
Continue…
Sample preparation: HILIC-MS::

Waters ACQUITY I-
Urine samples were Class
obtained from male chromatography
Sprague–Dawley rats.
system
Mobile phases :
These urine samples deionised water &
were then diluted 1:9 acetonitrile
v/v with LCMS grade containing 0.1% (v/v)
water.
formic acid.
Flow rate: 0.2
Centrifuged at mL/min
13,000g to remove
Column temp:
any particulates
of 50 °C

900 μL of acetonitrile
Injection volume 0.2
was added to 100 μL of
μL.
the supernatant.
Continue…
■ Result
The conventional HILIC UPLC analysis : 10 minutes
The Rapid HILC/MS method: 3.33 minutes

■ Conclusion
HILIC based microbore UPLC–MS performed using short columns, with rapid
gradients and elevated mobile phase linear velocities, provides a suitable analytical
platform with which to perform high-throughput endogenous metabolic profiling of polar
metabolites in urine. Compared to a conventional UPLC–HILIC/MS method, the
developed Rapid HILIC method provided fourfold reduction in analysis time, a 75%
reduction in solvent use, and an 18-fold reduction in sample consumption over a
conventional HILIC UPLC method.
 FUTURE PERSPECTIVE

■ The choice of HILIC for the analysis of polar compounds is

becoming increasingly popular in the bioanalytical laboratory. The
data provided here demonstrates that HILIC can be an important
choice for the most polar compounds, providing an important
complement to reversed-phase analysis.
■ As more particle chemistries and column options become
available, the potential of HILIC to solve more of these analytical
problems should also increase, providing a tool to fill this
analytical space.
 CONCLUSION
■ Current drug discovery efforts have been focused on identification of
drug metabolism and pharmacokinetic issues at the earliest possible stage
in order to reduce the attrition rate of drug candidates during the
developmental phase. Metabolic fate of drugs can be responsible for
problems associated with their bioavailability, interindividual variability,
drug-drug interactions, pharmacologic activity or the toxicity.
■ From the above case studies, it can be concluded that the ‘Hydrophilic
interaction chromatography’ (HILIC) coupled with ‘Mass spectroscopy’
(MS) is a promising alternative to RPLC, especially for highly polar/ionic
compounds & their metabolites.
 REFERENCES
1. RobertRoškar, Tina Trdan Lušin, Analytical Methods for Quantification of Drug Metabolites in Biological
Samples, Chapter 4, 2012.
2. Adam M. King, Lauren G. Mullin, Ian D. Wilson4, Muireann Coen, Paul D. Rainville, Robert S. Plumb,
Lee A. Gethings, Garth Maker, Robert Trengove, Development of a rapid profiling method for the analysis of
polar analytes in urine using HILIC–MS and ion mobility enabled HILIC–MS, 2019.
3. Jonathan P Danaceau*, Erin E Chambers, Kenneth J Fountain, Hydrophilic interaction chromatography
(HILIC) for LC–MS/MS analysis of monoamine neurotransmitters, Bioanalysis, 2012, 4(7), 783–794.
4. Elisavet Tsakelidou, Christina Virgiliou, Lemonia Valianou 2, Helen G. Gika, Nikolaos Raikos, Georgios
Theodoridis, Sample Preparation Strategies for the Effective Quantitation of Hydrophilic Metabolites in
Serum by Multi-Targeted HILIC-MS/MS, Metabolites, 2017, 7(13).
5. Essam Ezzeldin, Muzaar Iqbal, Yousif A. Asiri, Azza A Ali, Prawez Alam, Toqa El-Nahhas, A Hydrophilic
Interaction Liquid Chromatography Tandem Mass Spectrometry Quantitative Method for Determination of
Baricitinib in Plasma, and Its Application in a Pharmacokinetic Study in Rats, Molecules 2020, 25, 1600.
6. Raquel Pérez-Míguez, María Castro-Puyana, Elena Sánchez-López, Merichel Plaza, María Luisa Marina,
Untargeted HILIC-MS-Based Metabolomics Approach to Evaluate Coffee Roasting Process: Contributing to
an Integrated Metabolomics Multiplatform, Molecules 2020, 25, 887.
7. Regina Roemling, Seiji Itoh, HILIC-MS-High Resolution and Sensitivity for the Analysis of Very Polar
Compounds, jsb, 2007.
8. Tatsunari Yoshida, Kazuo Yamanaka, Hiroki Kumagai, Hydrophilic Interaction Chromatography
(HILIC) Separation of Basic Drugs using MS/MS Detection, © Agilent Technologies, Inc. 2005.
9. Zhan-Zhang Wang, Hao-Yang Lu1, De-Wei Shang, Xiao-Jia Ni, Ming Zhang, Yu-Guan Wen,
Development and validation of an HILIC–MS/MS method by one-step precipitation for chloroquine in
miniature pig plasma, Bioanalysis, 2016, 8(11), 1159–1171.
10. https://www.chromatographyonline.com/view/hilic-ms-high-resolution-and-sensitivity-analysis-very-
polar-compounds
11. https://blog.sepscience.com/liquidchromatography/hilic-ms
12. https://scholars.unh.edu/cgi/viewcontent.cgi?article=3297&context=dissertation
13. https://www.sigmaaldrich.com/technical-documents/articles/reporter-us/minimizing-phospholipid.html
14.https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/jssc.200700451
15. https://pubmed.ncbi.nlm.nih.gov/18428187/
16. https://www.chromatographyonline.com/view/hilic-critical-evaluation
17. https://www.news-medical.net/life-sciences/Hydrophilic-Interaction-Chromatography-
Applications.aspx
THANK YOU