UV-visible spectroscopy can be used for clinical and pre-clinical applications in cancer diagnosis and analysis. It allows non-invasive measurement of biomarkers like tumor oxygenation, angiogenesis, redox state, and morphology. Spectroscopy measures how light interacts with tissue via absorption and fluorescence of chromophores and fluorophores. This optical signature contains physiologically meaningful information about cancer progression and treatment response that can provide insights not available from traditional pathology. Quantitative optical techniques have the potential to improve cancer management with personalized, molecularly-informed assessments.

1. APPLICATION OF UV VISIBLE
SPECTROSCOPY IN
PHARMACEUTICAL INDUSTRY

2. SPECTROSCOPY
• It is the branch of science which deals with the interaction of
electromagnetic radiation with matter is called spectroscopy.
• Or
• It is the branch of science which deals with the study of
interaction of matter with light.

3. UV SPECTROSCOPY
• The interaction of electromagnetic radiation with matter when
source is uv is called uv spectroscopy
Spectrophotometer
• Spectrophotometer are spectroscopic instrument that convert
radiant intensities into electrical signal and measure the light
passes through the sample
• Spectrophotometer gives readings in transmittance (T) and
absorbance (A)

4. ULTRAVIOLET-VISIBLE
SPECTROPHOTOMETRY
• UV-Visible spectrophotometry is one of the most frequently
employed technique in pharmaceutical analysis. It involves
measuring the amount of ultraviolet or visible radiation
absorbed by a substance in solution. Instrument which measure
the ratio, or function of ratio, of the intensity of two beams of
light in the U.V-Visible region are called Ultraviolet-Visible
spectrophotometers.

5. APPLICATIONS OF UV SPECTROPHOTOMETRY IN
PHARMACEUTICALS
• Qualitative analysis through spectrophotometric methods
achieves fast and accurate results using only small sample
quantities. This fast and effect instrumentation has become an
essential tool in the pharmaceutical industry thanks to its
adaptability and economic value. Qualitative analysis has
proven highly useful in many major forms of organic
compounds and helps to ensure patient health and safety.

6. QUANTITATIVE ANALYSIS OF
PHARMACEUTICAL SUBSTANCES
• Many drugs are in the form of raw material or in the form of
formulation. They can be assayed by making a suitable solution
of drug in a solvent and measuring the absorbance at specific
wavelength.
• Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at
wavelength 284 nm.

7. UV-VISIBLE SPECTROPHOTOMETRIC METHOD DEVELOPMENT
AND VALIDATION OF ASSAY OF PARACETAMOL TABLET
FORMULATION
Paracetamol
• Paracetamol or acetaminophen is a widely used over-the-counter analgesic
(pain reliever) and antipyretic (fever reducer). It is commonly used for the
relief of headaches and other minor aches and pains and is a major
ingredient in numerous cold and flu remedies. In combination with opioid
analgesics, Paracetamol can also be used in the management of more severe
pain such as post-surgical pain and providing palliative care in advanced
cancer patients. . However, acute overdose of Paracetamol can be
potentially fatal and its toxicity is the leading cause of liver failure.

8. EXPERIMENT
Materials
• Paracetamol standard of was provided by Torque Pharmaceuticals (P) Ltd.
(India). Paracetamol tablets containing 500 mg Paracetamol and the inactive
ingredient used in drug matrix were obtained from market. Analytical grade
methanol and water were obtained from Spectrochem Pvt. Ltd.,
Diluent preparation
• Methanol and water (15:85, v/v) used as a diluent.

9. STANDARD PREPARATION
• 10 mg drug was dissolved in 15 ml methanol and was shaken well. Then 85 ml water was
added to it to adjust the volume up to 100 ml (100 ppm). From that 5 ml was taken and
volume was adjusted up to 50 ml with diluents
Test preparation
• 20 tablets were weighed and powdered. Powdered tablet equivalent to 100 mg of
paracetamol was weighed and taken into 100 ml volumetric flask then 15 ml of methanol
was added and shaken well to dissolve it after that 85 ml of water was added to adjust the
volume up to 100 ml. From that 1 ml of solution was withdrawn and taken in 100 ml
volumetric flask. The volume was adjusted with diluent up to 100 ml.
Instrumentation
• UV-Visible double beam spectrophotometer with matched quartz cells (1 cm)

10. DEVELOPMENT AND OPTIMIZATION OF THE
SPECTROPHOTOMETRIC METHOD
• Proper wave length selection of the methods depends upon the nature of the
sample and its solubility. To develop a rugged and suitable
spectrophotometric method for the quantitative determination of
paracetamol, the analytical condition were selected after testing the
different parameters such as diluents, buffer, buffer concentration, and
other chromatographic conditions.
• Our preliminary trials were by using different compositions of diluents
consisting of water with buffer and methanol. By using diluent consisted of
methanol - water (50:50, v/v) best result was obtained and degassed in an
ultrasonic bath (Enertech Electronics Private Limited). Below figures
represent the spectrums of blank, standard and test preparation
respectively.

11. SELECTION OF WAVELENGTH
• Scan standard solution in UV spectrophotometer between 200 nm to 400
nm on spectrum mode, using diluents as a blank. Paracetamol shows λmax
at 243.
Conclusion
• The present analytical method was validated and it meets to specific
acceptance criteria. It is concluded that the analytical method was specific,
precise, linear, accurate, robust and having stability indicating
characteristics. The present analytical method can be used for its intended
purpose.

12. SPECTROSCOPIC ANALYSIS ON THE BINDING INTERACTION OF
BIOLOGICALLY ACTIVE PYRIMIDINE DERIVATIVE WITH BOVINE
SERUM ALBUMIN
Protein
• Protein, one of the most important bioactive molecules, is related to
alimentation, immunity and metabolism. The content of proteins in body
fluid can be used as a vital index for the clinical diagnosis and health
evaluation; therefore, the direct determination of protein is significant in life
sciences, clinical medicine and chemical investigation. The interaction
between bio-macromolecules and drugs has attracted great interest for
several decades and many researches have been focused on two central
questions about proteins: what are the determinant factors that influence
the protein structures and functions, and how does a factor affect their
biological activity

13.  Serum albumin (SA).
• Serum albumin (SA), the main protein in the blood plasma acting as the transporter and disposition
of many drugs, has been frequently used as a model protein for investigating protein folding
and ligand binding mechanism.
 Bovine serum albumin (BSA).
• BSA is composed of three linearly arranged and structurally homologous sub-domains. It has two
tryptophan residues that possess intrinsic domains (I–III) and each domain in turn is the product of
two fluorescence: Trp-134, which is located on the surface of sub-domain IB, and Trp-212, located
within the hydrophobic binding pocket of sub-domain IIA. The binding sites of BSA for endogenous
and exogenous ligands may be in these domains and the principal regions of drugs binding sites of
albumin are often located in hydrophobic cavities in sub-domains IIA and IIIA. So-called sites I and II
are located in subdomain IIA and IIIA of albumin, respectively.

14. PYRIMIDINE
• Pyrimidine moiety is one of the important classes of N-
containing heterocycles widely used as key building blocks for
pharmaceutical agents. It exhibits a wide spectrum of
pharmacophore such as bactericidal, fungicidal, analgesic,
anti-hypertensive and anti-tumor agents.

15. • Protein–drug interaction plays an important role
in pharmacokinetics and pharmacodynamics. In a series of methods concerning the
interaction of drugs and protein, fluorescence techniques are great aids in the study of
interactions between drugs and serum albumin because of their high sensitivity, rapidity,
and ease of implementation . The aim of the present investigation was to study the
affinity of pyrimidine derivative (AHDMAPPC) for BSA using UV–visible and fluorescence
spectroscopy to understand the carrier role of serum albumin for such compound in the
blood under physiological conditions. Significantly, the determination and understanding
of drug interacting with serum albumin are important for the therapy and design of drug .
Knowledge of the interaction and binding of BSA may open new avenues for the design of
the most suitable pyrimidine derivatives. All the experimental results clarify that
AHDMAPPC can bind to BSA and be effectively transported and eliminated in body, which
can be a useful guideline for further drug design.

16. MATERIALS AND METHODS
• BSA and its molecular weight was assumed to be 66, 463 to calculate the
molar concentrations. All BSA solutions (CBSA=2.0×10−5 M) were prepared in
a pH 7.4 buffer solution and the stock solution was kept in the dark at 4 °C.
Tris–HCl (0.1 M) buffer solution containing NaCl (0.1 M) was used to keep
the pH of the solution at 7.4. A dilution of the BSA stock solution in Tris–HCl
buffer solution was prepared immediately before use. The stock solution of
AHDMAPPC (synthesized) was prepared in (5:95, v/v) ethanol water mixture.
Dissolution of the compound was enhanced by sonication in an ultrasonic
bath (Spectra Lab Model UCB-40). All chemicals were of analytical reagent
grade and were used without further purification. Double distilled water was
used throughout. In order to simulate human body fluid surroundings and
to get the best sensitivity, Tris–HCl solution (pH 7.4) was chosen as the
buffer solution in this work.

17. EQUIPMENT AND SPECTRAL
MEASUREMENTS
• The UV–visible absorption spectra were measured at room
temperature on a Shimadzu UV–3600 UV–vis–NIR
Spectrophotometer equipped with a 1.0 cm quartz cell. The
wavelength range was from 250 to 450 nm. All pH values were
measured by a digital pH-meter with magnetic stirrer (Equip-
Tronics EQ-614A).

18. RESULTS AND DISCUSSIONS
• BSA exhibited a strong fluorescence emission band at 347 nm.
The fluorescence intensities of BSA reduced gradually with
increasing AHDMAPPC concentrations, and a blue shift was also
observed, which suggests that the fluorescence chromophore
of serum albumin is placed in a more hydrophobic environment
after the addition of AHDMAPPC.

19. UV-VISIBLE SPECTROSCOPY FOR CLINICAL AND
PRE-CLINICAL APPLICATION IN CANCER
• Methods of optical spectroscopy which provide quantitative,
physically or physiologically meaningful measures of tissue
properties are an attractive tool for the study, diagnosis,
prognosis, and treatment of various cancers. Recent
development of methodologies to convert measured
reflectance and fluorescence spectra from tissue to cancer-
relevant parameters such as vascular volume, oxygenation,
extracellular matrix extent, metabolic redox states, and
cellular proliferation have significantly advanced the field of
tissue optical spectroscopy

20. INTRODUCTION
• There is a great need to accurately quantify predictive
biomarkers in vivo for the diagnosis, prognosis and treatment
of cancers. Since current approaches in cancer management
are generic across patients and involve empirical routines
there is a growing emphasis toward developing individualized
and personalized approaches which are based on detection of
molecular, metabolic and physiological biomarkers.
Traditional biomarkers include features such as the tumor
grade, size and/or the number of local lymph nodes with
metastasis

21. BIOMARKERS OF CANCER
Vascular and metabolic factors
• Oxygenation and hypoxia
Oxygenation, particularly, the lack of it, is widely recognized as a
crucial factor that influences the growth rate, metabolism, treatment
resistance and metastatic behavior of cancer cells . Hypoxic
microenvironments have routinely been identified in solid tumors of
almost all tissues. Numerous studies have investigated the link
between clinical outcomes and hypoxia using a variety of different
methods to date . All of these studies have demonstrated that
hypoxia is clearly related to clinical outcome, which motivates the
importance of measuring it in vivo.
• Currently, methods to measure tumor hypoxia can be divided into
two classes, indirect and direct. The gold standard of direct tissue

22. ANGIOGENESIS AND BLOOD VOLUME
•
Irregular vasculature has previously been identified has a hallmark
of cancer . Given that tumor cells have a constant need for new
blood vessels to nourish their growth, solid tumors persistently
sprout new segments of vessels to the existing vascular system
leading to a highly irregular, leaky and chaotic network of blood
vessels . The sprouting of new vessels is facilitated by over-
expression of the vascular endothelial growth factor (VEGF), which is
known to be upregulated under hypoxic conditions . This increased
tumor vascularization eventually paves the way for a small, localized
tumor to become an enlarged mass and subsequently metastasize to

23. REDUCTION-OXIDATION STATE OF THE
CELL
•
Cellular respiration occurs via the electron transport chain in all aerobic
cells and in the mitochondrial membranes of these cells, reactive oxygen
species (ROS) are generated during oxidative phosphorylation. There are
several complex cellular biochemical pathways that help cells protect
themselves against low levels of ROS and free radicals by forming a
network of redox buffers (which include the
NAD(P)H/NAD(P)+ species). these mechanisms might be rendered
dysfunctional under abnormally high levels of ROS leading to oxidative
stress in the tumor microenvironment. onset of hypoxia within solid
tumors causes the cells to prefer anaerobic glycolytic pathways over
aerobic oxidative phosphorylation to meet their energy needs, which in
turn influences both the amount of ROS produced, and the amount of
NADH/NAD+ redox buffer available in these cells. There is currently no
accepted clinical gold-standard to estimate the redox status of tumors,
though detection in tumors has been achieved via the use of

24. MORPHOLOGICAL FACTORS
• There are significant changes in cellular morphology and
structure that are associated with the onset and progression
of cancer. Pathologists routinely use microscopic differences
observed in cellular and nuclear features including shape,
size, crowding, chromatin organization and DNA structure in
biopsied tissues to diagnose, prognosticate and stage
disease.

25. OPTICAL SPECTROSCOPY
• Methods of optical science and engineering have been developed for
cancer detection and diagnosis and more recently to assess response
to therapy in a variety of tissue sites for applications in both pre-
clinical and clinical studies . The interaction of light with complex
media such as biological tissues, is characterized by processes that
depend on the physical nature of the light and the specific tissue
morphology and composition . The incident light can be scattered
(elastically or inelastically) multiple times due to microscopic
differences in the index of refraction of cells and subcellular
organelles within the tissues, and may be non-radiatively absorbed by
chromophores present in the medium or by fluorophores, which
release their excess energy by radiative decay, producing
fluorescence. The remitted fluorescent light can, in turn, be multiply
scattered or absorbed. Although complex, these optical responses
can be measured by a variety of spectroscopic techniques and

26. • In optical spectroscopy, the wavelengths of illumination span
the ultraviolet (UV) through the near-infrared (NIR) wavelengths.
In steady-state reflectance spectroscopy, a broadband light
source is used for illumination and a spectrum of the reflected
light is collected , while in steady-state fluorescence
spectroscopy a narrow spectral-band of incident light (obtained
via filtering a broadband source or from a narrowband laser) is
used to excite fluorophores and the emerging fluorescence
spectrum at each excitation wavelength is detected