In this slide contains analytical methods, invitro techniques, chromatography, electrochemical methods, ELISA, RIA, bioassay and electrophoresis. Presented by: THANMAYA DIVYA (Department of pharmacology). RIPER, anantapur

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. 20L812IS00114)
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.
2013,940-948.
 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