Introduction to Analytical Methods In Invitro Techniques, Analytical Methods, In Vitro Techniques, Spectroscopic Techniques, Chromatography, Electrochemical methods, ELISA, RIA, Bioassay, Electrophoresis Presented by K. Thanmaya Divya Department of Pharmacology

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
ANALYTICAL METHODS IN
INVITRO TECHNIQUES
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
K. Thanmaya Divya
(Reg. No. 20L81S0114)
Under the guidance/Mentorship of
Dr. P. Ramalingam, M.Pharm, Ph.D,
Research Director& Professor of Pharmaceutical and Medicinal Chemistry

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Analytical Methods
• In Vitro Techniques
• Spectroscopic Techniques
• Chromatography
• Electrochemical methods
• ELISA
• RIA
• Bioassay
• Electrophoresis
• References
Contents

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
Analytical Methods
 Analytical methods entailed how chemical components in natural or
synthetic materials are identified, separated and then quantified.
 It involves the use of classical methods along with modern methods involving
the use of scientific instruments.
EXAMPLES
 Spectroscopy, Chromatography, Potentiometry, ELISA….ETC.

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
In vitro Techniques
 In vitro (Latin for within the glass) refers to the technique to study the effect of
compound under standard conditions in a controlled environment outside of a
living organism.
 In Vitro studies include the isolation, growth and identification of cells.
Examples
In Vitro diagnosis
In Vitro fertilization
Cell Culture techniques

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• Enzyme Evaluation in Food Industry.
• Spectroscopic-In-vitro Analysis of Hydrazones as Potential Antituberculosis
Agents.
UV Spectroscopy
 UV Spectophotometric method for In-vitro dissolution studies in Phosphate
buffer pH 7.4 .
 UV for Characterization of curcumin loaded nanostructured lipid nanocarriers
in simulated conditions.
 UV methods for estimation of blood glucose.
Spectroscopic Techniques

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
IR Spectroscopy
 FT-IR analysis and In Vitro anti inflammatory activity.
 FTIR microspectroscopy of tissues for in-vitro cancer diagnostics.
 Near infrared spectroscopy for non-invasive assessment of intracranial
haemoglobin oxygenation in an In Vitro model.
Mass Spectroscopy
 Utility of mass spectrometry for in-vitro ADME assays.
 Liquid chromatography–mass spectrometry in In Vitro drug metabolite
screening
 Mass Spectrometry - The Future of In Vitro Diagnostics (IVDs)

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
NMR Spectroscopy
 In-Vitro Diagnostic NMR Spectroscopy for Lipoprotein and Metabolite
Measurements in Plasma and Serum.
 P NMR Spectroscopy for in vitro Viability Testing of Porcine Pancreatic
Islets.
FLUORIMETRY
 In Vitro Inhibitory Activity of Cystatin a Thermodynamic Study Using UV
and Fluorescence Techniques.
 Spectrofluorometry for evaluation of dry powder inhalers in vitro.

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
 Use of paper chromatography for in vitro studies of lactose metabolism in
mammary-gland preparations.
 Bioautography detection in thin-layer chromatography.
 TLC to evaluate the presence of various phytochemicals in the different
medicinal plant extracts used for the evaluation of an in vitro antioxidant
activity.
 HPTLC method development and validation for the In Vitro interaction study
of mebendazole with atorvastatin.
 In Vitro distribution of labeled amino acids in soluble protein fractions of rat
liver as shown by column chromatography.
Chromatography

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
GC-MS was used to screen the phytochemicals of leaf extracts.
 GC-Mass Spectrometry to in vitro Studies of Nitrogen Metabolism.
Development and Validation of a New HPLC Method for In-vitro Studies of
Haloperidol in Solid Lipid Nanoparticles.
Method development and validation of RP- HPLC in invitro dissolution study
of Lamivudine in bulk drug and tablet formulation.
Ion exchange chromatography for the study of RecA-DNA interaction.

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
Electrochemical methods
Potentiometry
 In-vitro study on the competitive binding of diflunisal and uraemic toxins
to serum albumin and human plasma using a potentiometric ion-probe
technique.
 Contribution of potentiometry in the presence of lipoic acid to the study
and evaluation of the in vitro antibacterial effects of betalactams.
 Automated Potentiometric Procedure for Studying Dissolution Kinetics of
Acidic Drugs under Sink Conditions.
 Study of the in vitro degradation of poly(ethyl glyoxylate) was
investigated using potentiometry.

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
Conductometry
 Conductometry, to analyse "in vitro" the releasing of
potassium chloride from an inert matrix.
Voltametry
 Use of voltammetry for in vitro equilibrium and transport studies of
ionisable drugs.
 Differential Pulse Polarography to the Analysis of Capecitabine and
Investigation of its Electroreduction Mechanism.
 In Vitro Continuous Amperometry with a Diamond Microelectrode
Monitoring Endogenous Norepinephrine and its Effect on the
Contractile Response of a Rat Mesenteric Artery.

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
ELISA
 ELISA an immunosorbent assay, for in vitro oncology studies.
 The spot-ELISA a sensitive in vitro method to study the immune
response to hepatitis B surface antigen.
RIA
 RIA in testing the potency of the naturally occuring isoforms of
intrapitutary FSH.
 RIA is an in vitro assay that measures the presence of an antigen.

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Bioassay
An In Vitrobioassay for evaluating the effect of inhaled bronchodilators on
airway smooth muscle.
Brine shrimp lethality bioassay used for testing the efficacy of phytochemical
present in the plant extracts to know its antioxidant activity.
ELECTROPHORESIS
 It is used to seperate DNA,RNA or protein molecules based on their size and
electric charge.

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
References
 Gbylik, M, Posyniak, A, Mitrowska, K, Bladek, T, Zmudzki, J.
Principles of liquid chromatography-tandem mass spectroscopy.
2013;4:940-948.
 Smith, S.B., Aldridge, P.K., & Callis, J.B. Observation of individual
DNA molecules undergoing gel electrophoresis. Science. 1989;243:
203-206.
 Aldrich, C J, Antona, D, Wyatt, J S, Spencer, J A D, Peebles, D M.
& Reynolds, E 0 R. Fetal cerebral oxygenation measured by near
infrared spectroscopy shortly before birth and acid-base status at
birth. Obsrertics and Gynecology.194;84:861-866.

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15