This document provides an overview of analytical method development for anticancer drugs using UV visible spectroscopy. It discusses cancer and its causes, definition and types of anticancer drugs. It describes the principle, instrumentation and applications of UV visible spectroscopy. The key techniques discussed are single beam and double beam systems. It also covers validation process for analytical methods and documentation requirements. UV visible spectroscopy is a commonly used technique for pharmaceutical analysis due to its simplicity, cost effectiveness and ability to provide accurate results.
3. 1. Introduction:
Cancer is a group of Disease characterized by uncontrolled and
unregulated growth of cells.
Although cancer is often considered a disease of gaining with the
majority of cases diagnosed in those over age 55years, it occurs in
people of all ages.
According to the Indian council of Medical research estimates, by
2020 there would be over 17.3 lakh new cases of cancers with
cancer of breast, lung and cervix are topping on the list
4. 2 Definition
Cancer may be regarded as a group of disease
characterized by an
1) Abnormal growth of cells
2) ability to invade adjacent tissues and even distant organ.
3) the eventual death of the affected patient if the tumour has
progressive beyond that stage when it can be successfully
removed.
5. Etiology
Family history : Cancer is most commonly seen in those person who have a
family history
Genetic factors: Some types of cancer run in certain families but it not clearly
linked to the gene
Tobacco: Consumption of Cigarate and cigar can affect health including risk of
Cancer
Age: Majority of cancer are found in above 50years old People
Chemical & Radiation: some radiation like UV,UVB rays UVA rays can caused
the cancer
Alcohol Consumption :Women & Men who drink > 1 alcoholic beverage per
day may be an increased risk of cancer
Others causes: weight gain and obesity, yearly menarche and late menopause in
case of women hormone use, viral disease.
6. Spreading
Initiation
o It is the first stage
o initiation, involved in mutation, in the cell genetic structure.
o A mutation is any changes in the usual DNA sequence.
Promotion
o Promotion is the second stage in the development of cancer,
o It is characterized by the reversible proliferation of the altered cells.
o The promoting factors include such agents: -Dietary fat, obesity, Cigarate smoking, Alcohol consumption, inactivity.
Progression
o Progression is the final stage in the natural history of cancer.
o The stage is characterized by increase growth rate of the tumour
7. Spreading Rate:
The estimated number of incident cases of cancer in India for the year 2022 was
found to be 14,61,427 (crude rate: 100.4per 100,000).
In India, one in nine people are likely to develop cancer in his/her lifetime
Symptoms
Fever
Weight loss/weight gain.
Anorexia
Nausea
Vomiting
Headache
Prolonged cough
Lump
9. Ratio India/ Another country
Country Number ASR/1,00,000
World 98,94,402 100.1
India 8,46,960 62.7
China 29,92,600 129.0
UK 1,77,628 99.6
US 6,07,636 85.7
Australia 47,452 82.2
Nepal 13,523 54.4
10. Ratio of Men/ Women
0
2
4
6
8
10
12
Men
0
5
10
15
20
25
Women
11. Increase Rate (Last Ten years):-
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
Year 2004 Year 2005 Year 2006 Year 2007 Year 2008 Year 2009 Year 2010 Year 2015 Year 2020
INCREASE RATE OF LAST TEN YEAR
Males Females Total
14. 2.1 Definition Anticancer drug also called anti-Neoplastic drug, any drug that is effect in the malignant disease. A type of anticancer drug that blocks cell growth by interfering
with DNA the genetic material in cell.
2.2 Classification of Anticancer drugs
Alkylating agent:
o Nitrogen mustards- Mechlorethamine, Chlorambucil, Cylophosphamide.
o Ethylenimine-Thio-Tepa
o Alkyl sulphonate-Busulfan
o Triazine-Decarbazine
Antimetabolites:
o Folate antagonist- Methotrexate
o Purine antagonist- 6-mercaptopurine, 6-thioguanine, Azathioprine
o Pyrimidine antagonist- 5 fluorouracil, cytarabine
Plant products:
o vinca alkaloids- vincrstine, vinblastine
Antibiotic
o Anthracyline
o Mintmycin c
o Bleomycin
o Actinomycin d
Hormone based anti-cancer
o Anastrozole
o Bicalutamide
o Letimide
o Letrozole
Miscellaneous
o Mitotane
o Hydroxyur
o Eaetoposide
o Procarbazine
15. Alkylating agents
Binds to DNA
Inhibits transcription
cl-ligands are exchanged
against water
Positively charged ligands
that binds to the DNA
Localized unwinding of
the DNA
Mechanism of action
1 ALKYLATING AGENTS
18. SIDE EFFECTS:
Low blood counts
Tiredness
Mouth sourness
Loss of appetite
Constipation
Hair loss
Skin changes
Nerve changes
Drug interaction: Anytime you take more than one medication, or even mix it with certain foods, beverages, or over-the-
counter medicines, you are at risk of a drug interaction.
Drug-drug interactions - These are the most common type of drug interaction. The more medications you take, the greater the
chance for your drug interacting with another medicine.
Drug-food/beverage interactions - You have probably seen the stickers on your prescription bottle to “avoid grapefruit juice” at one
time or another.
Drug-disease interactions - Drug interactions don't always occur with just other drug or food.
Synergic effectAn interaction between two or more drugs that causes the total effect of the drugs to be greater
than the sum of the individual effects of each drug. A synergistic effect can be beneficial or harmful
Synergistic Effect Examples The skin damage produced by combined tobacco smoke and UV radiation is greater than the skin
damage caused by either tobacco smoke or UV radiation alone. The liver is harmed by both carbon tetrachloride and ethanol (ethyl
liquor). When used together, they cause more serious liver damage than the sum of their separate effects on the liver.
19. Banned drugs: Several popular over the counter painkillers, antibiotics and cough medications have been banned following
a notification of the Union health ministry. As many as 34 drugs -including some anti-infectives like ivermectin, mupirocin and
nicotine replacement therapy - have been added to the NLEM, taking the total drugs under it to 384.
Few of the banned drugs mentioned below
Chlorpheniramine + Vitamin C
Betahistine + Ginkgo Biloba Extract + Vinpocetine + Piracetam
Aceclofenac + Paracetamol + Rabeprazole
Azithromycin + Ambroxol
Ammonium Chloride + Dextromethorphan + Cetirizine + Menthol
Clinical trial conducted till now
Only 16 trials — less than 2 percent — were pregnancy-specific, meaning they aimed to evaluate a treatment’s effects on
foetuses and expectant mothers.
This means that even as researchers learn which treatments work for most people, there will be a gap in the medical
community’s understanding of how these treatments perform during pregnancy.
An analysis of 927 Covid-19 clinical trials across Asia, Europe, and North America published online in May found more than
half explicitly excluded pregnant women.
Pregnant women should have access to drugs that have been vetted for safety, says White. “It’s an issue of justice.”
Anticancer drugs have been found till now: 150 anticancer drugs approved by the US Food and Drug
Administration (FDA). Based on drug mechanism of action, these agents are divided into two groups: 61 cytotoxic-based drugs
and 89 target-based drugs. From above 150 drugs, we built a drug-cancer network, which contained 183 nodes (150 drugs and
33 cancer types) and 248 drug-cancer associations. The network indicated that the cytotoxic drugs tended to be used to treat
more cancer types than targeted drugs.
20. METHOTREXATE: Is a Antineoplastic anti-metabolite. Methotrexate anti-tumour
activity is a result of the inhibition of cellular replication. It is structurally related to
prazosin
22. Definition
Validation is the documented act of proving that any procedure, process, equipment,
material, activity or system actually leads to the expected result.
According to the Food and Drug Administration (FDA), the goal of validation is to:
“Establish documented evidence which provides a high degree of assurance that a
specific process will consistently produce a product meeting its predetermined specifications
and quality attributes.”
23. Types of Process Validation
1. Prospective validation
2. Concurrent validation
3. Retrospective validation
4. Revalidation
24. Scope of Validation
Validation done in a structured way according to documentation including procedures and
protocols.
Validation should be performed for
new premises, equipment, utilities and systems o new processes and procedures; at periodic intervals; o
Whenever major changes have been made.
Validation in accordance with written protocols.
Demonstrate suitability for new manufacturing formula /method Ø A written report on the
outcome to be produced.
Validation over a period of time
E.g.at least three consecutive batches (full production scale) to demonstrate consistency.
(Worst-case situations should be considered.) o Demonstrate suitability for new manufacturing
formula or method
25. Advantages of validation
During the process the knowledge of process increases
Assures the repeatability of the process
Assures the fluency of production
Assures that the product is continuously according to the marketing authorization
Decreases the risk of the manufacturing problems
Decreases the expenses caused by the failures in production
Decreases the risks of failing in GMP
Decreases the expenses of the everyday production even though the validation
itself will create expense
26. Documentation of validation
The validation activity cannot be completed without proper documentation of each
and every minute activity with utmost details.
Documentation of validation is generally different types such as:
1. Validation Master Plan (VMP)
2. Validation Protocols (VP)
3. Validation Reports (VR)
4. Standard operating procedure (SOP)
27. Validation Master Plan (VMP)
A validation Master Plan (VMP) is a comprehensive document describing the
applicable validation requirements for the facility, and providing a plan for the
meeting those requirements.
In short, it is documented evidence that provides a high degree of assurance that a
specific process will consistently produce a product the meets its predetermined
specifications and quality attributes.
Why to perform VMP?
VMP has become an important component an industry standard and regulatory
requirement as well.
It is important to include such a document, as it sets the overall goals and limits that
will be followed during validation, and can be referred to throughout the project. As a
28. Who performs VMP?
Members:
Validation manager,
Quality Assurance dept.
Member from production
Member Engineering (utilities)
Member from Calibration lab
Member from QC lab
Member from Maintenance
Member from HVAC dept.
Member from Product development lab
29. How VMP is prepared?
Protocol:
VMP includes the incorporation of information into formal written
Protocols, which serve as guides for executing the appropriate validation activities.
Protocols should be developed for IQ, OQ, PQ.
The information included in specific protocols are:
Description of the system
Qualification objective Scope
Responsibilities and data collection procedures Test procedures, specific acceptance criteria
Documentation procedures
Summary and deviation report
30. Cleaning validation
Cleaning validation (CLV) is written evidence that determines the a specified
cleaning procedure will lead to reliable and repeatable results in the cleaning of
surfaces with and without contact with the product
32. 4.1 DEFNITION
• Spectroscopy is the branch of science concerned with the
investigation and measurement of electromagnetic
radiation absorbed or emitted when by the matter or
sample
• UV Spectroscopy- It is also known as electronic spectroscopy,
it is a type of absorption spectroscopy in which the amount of
light absorbed at each wavelength of UV region [200-400nm]
of electromagnetic spectrum is absorbed by the molecule is
measure
33. PRINCIPLE-
The principle of UV-spectroscopy is based on the absorption spectrum and it is
based on the principle of Beer`s Lamberts law
Incident Transmittance
Io It
Sample
Absorption = IO - It
34. Beer`s Lamberts law
Beer and Lamberts both are different scientists and laws are
also different but we will study both laws together so this is
called Beer`s Lamberts law
Beer`s law-
According to Beer`s law the absorption of any monochromatic
light is directly proportional to the concentration of the sample
A α C
Lambert law
According to lambert law, the decrease in the intensity of light
is direct proportional to the path length travelled by the light
A α L
Beer`s and Lamberts law
A α C×L
A = €CL
Where , C – Concentration
L - path length and € - absorption coefficient
36. In the single beam system. UV radiation is produced by the source . A convex lens will gathers the beam of
radiation and it will focus it in a inlet slit. Then the inlet slit allows light from the to pass , but it blocks out a
stray radiation . Then the light reaches monochromator , which splits the light according to wavelength to
pass through. Radiation of at all other wavelength is blocked out . Then the selected radiation passes through
the sample cell to the detector , which measures the intensity of the radiation reaching it . The output from
detector is recorded on the graph paper
2] DOUBLE BEAM SYSTEM
37. The radiation from the source is passes from the mirror system to the monochromator unit .
The main function of the monochromator is to allow a narrow range of wavelength to pass
through the exit slits . Then the radiation coming out from the monochromator to the exit slit
received by the rotating sector which divides the single beam into double beams , one passes
through the reference and the other passes through the sample
APPLICATION
1] DNA & RNA analysis
2] Bacterial culture
3] Pharmaceutical analysis
4] Beverage analysis
ADVANTAGES
1] It is simple process and easy to operate
2] It is cost effective instrument
38. 3] Versatile
4] Non-destructive process
5] The main advantage of this technique is it will give accurate result
6] It is easy to handle
7] Covers the entire of ultravoilet and visible
8] Derivative graph can be obtained by this uv spectrophotometer
DISADVANTAGES
1] Only those molecules having chromophores can be analysed
2] It takes times to get ready to use it
3] There are many factors which affects the result of absorption
they are Ph , temperature , contaminants and Impurities
4] The analysis is only done for liquids samples
5] Reading of samples can be affected by cuvette handling
39. MODELS OF UV SPECTROPHOTOMETHER
AVAILABLE IN MARKET
1] UV-1900i
2] UV-2600i
3] UV-1700
4] UV-2700i
5] UV-3600i plus
6] Solid spec-3700i
7] Solid spec-3700i DUV
The UV Spectrophotometer model that is available in our college is
UV-1700
41. METHOD -1
5.1 STANDARD (0.1N NaOH )
Solvent used 0.1N NaoH
PART-A :
Preparation of 0.1N NaOH
Weigh 4gm of sodium hydroxide pellets and transfer it into 1000ml of volumetric flask. Dissolve sodium
hydroxide using distilled water and make the volume up to the mark using distilled water
PART-B
Stock solution -1
Weigh 10mg of pure drug and dissolve in 100ml volumetric flask using 0.1N NaOH and make up the volume up
to the mark with 0.1N NaOH (1000µg/ml)
PART –C
Stock solution -2
Take 10ml of stock solution -1 in 100ml of volumetric flask and make up the volume up to the mark using 0.1N
NaOH(100µg/ml)
PART –D
DILUTIONS
Take 5ml ,6ml,7ml ,8ml and 9ml of stock solution -2 in 10ml of volumetric flask separetly .make up the volume up
to the mark(10ml) using 0.1N NaOH respectively.
50µg/ml,60µg/ml, 70µg/ml,80µg/ml,90µg/ml of series dilutions have been prepared.
42. PART – E
CALIBRATION CURVE
Take 50,60,70,80 and 90µg/ml individually for analysis and the readings have been record and
calculated r2 value and plot the graph between concentration and absorbance.
PART – F
ACCURACY
For the accuracy determination we selected two dilutions one is highest and another one is lowest i.e
50 microgam/ml and 90 microgram /ml .
After that we calculated its absorbance and observed the spectrum between absorbance and
wavelength for three times and determined its reproducibility.
PART –G
PRECISION
For the precision value we took two dilution series i.e 50,60 .70 ,80,90 microgram/ml and observed its
spectrum by using UV probe and this procedure we repeated for two times in same day (intra- day
precision) and next day ( inter-day precision ) .
43. METHOD -2
5.2 Standard (phosphate buffer 6.4)
Solvents used is phosphate buffer 6.4
PART-A
Preparation of phosphate buffer solution pH 6.4
Dissolve 2.5gm of disodium hydrogen phosphate ,2.5 gm of sodium dihydrogen phosphate and 8.2gm of sodium
chloride in 950ml of water adjust the Ph of the solution to 6.4 with 1M sodium hydroxide or1M hydrochloric acid if
necessary. Dilute to 100ml with water.
PART-B
STOCK SOLUTION -1
Weigh 10mg of pure drug and dissolve in 100ml of volumetric flask and make up the volume up to the mark
using phosphate buffer 6.4(1000µg/ml)
PART-C
STOCK SOLUTION -2
Take 10ml of stock solution-1,and dissolve in 100ml of volumetric flask and make up the volume up to the mark
using phosphate buffer 6.4(100µg/ml)
PART –D
DILUTIONS
Take 1ml ,2ml,3ml ,4ml and 5ml of stock solution -2 in 10ml of volumetric flask seapartly .make up the volume up
to the mark(10ml) using 0.1N NaOH respectively.
10µg/ml,20µg/ml, 30µg/ml,40µg/ml,50µg/ml of series dilutions have been prepared.
44. PART –E
CALIBRATION CURVE
Take 10,20,30,40and 50µg/ml individually for analysis and the readings have
been recorded and calculate r2 value and plot the graph between concentration
and absorbance.
PART-F
ACCURACY
For the accuracy determination we selected two dilutions one is highest and
another one is lowest i.e 10 microgam/ml and 50 microgram /ml .
After that we calculated its absorbance and observed the spectrum between
absorbance and wavelength for three times and determined its reproducibility.
PART –G
PRECISION
For the precision value we took two dilution series i.e 10,20 .30 ,40,50 µg/mland
observed its spectrum by using uv probe and this procedure we repeated for two
times in same day (intra- day precision) and next day ( inter-day precision )
45. METHOD -3
5.3 Standard (phosphate buffer 7.4)
Solvents used is phosphate buffer 7.4
PART-A
Preparation of phosphate buffer solution pH 7.4
Dissolve 2.5gm of disodium hydrogen phosphate ,2.5 gm of sodium dihydrogen phosphate and 8.2gm of sodium
chloride in 950ml of water adjust the Ph of the solution to 6.4 with 1M sodium hydroxide or1M hydrochloric acid if
necessary.dilute to 100ml with water.
PART-B
STOCK SOLUTION -1
Weigh 10mg of pure drug and dissolve in 100ml of volumetric flask and make up the volume up to the mark using
phosphate buffer 7.4(1000µg/ml)
PART-C
STOCK SOLUTION -2
Take 10ml of stock solution-1,and dissolve in 100ml of volumetric flask and make up the volume up to the mark
using phosphate buffer 7.4(100µg/ml)
PART –D
DILUTIONS
Take 1ml ,2ml,3ml ,4ml and 5ml of stock solution -2 in 10ml of volumetric flask seapartly .make up the volume up to
the mark(10ml) using phosphate buffer 7.4respectively.
10µg/ml,20µg/ml, 30µg/ml,40µg/ml,50µg/ml of series dilutions have been prepared
46. PART –E
CALIBRATION CURVE
Take 10,20,30,40and 50µg/ml individually for analysis and the readings have
been recorded and calculate r2 value and plot the graph between concentration
and absorbance.
PART-F
ACCURACY
For the accuracy determination we selected two dilutions one is highest and
another one is lowest i.e 10 microgram/ml and 50 microgram /ml .
After that we calculated its absorbance and observed the spectrum between
absorbance and wavelength for three times and determined its reproducibility.
PART –G
PRECISION
For the precision value we took two dilution series i.e 10,20 .30 ,40,50 µg/mland
observed its spectrum by using uv probe and this procedure we repeated for two
times in same day (intra- day precision) and next day ( inter-day precision )
47. METHOD-4
5.4 STANDARD (0.1N NaOH)
Solvent used 0.1N NaOH
PART-A
PREPARATION OF 0.1N NaOH
Weigh 4gm of sodium hydroxide pellets and transfer it into 1000ml of volumetric flask . Dissolve sodium
hydroxide using distilled water and make the volume up to the mark using distilled water
PART-B
STOCK SOLUTION -1
Weigh 50mg of tablet powder of Methotrexate(10mg) and dissolve in 50ml volumetric flask using 0.1N NaOH and
make up the volume up to the mark with 0.1N NaOH (1000µg/ml)
PART-C
STOCK SOLUTION- 2
Take 10ml of stock solution-1 in 100ml of volumetric flask and make up the volume up to the mark using 0.1N
NaOH (100µg/ml)
PART-D
DILUTIONS
Take 5ml , 6ml , 7ml , 8ml and 9ml of stock solution-2 in 10ml of volumetric flask separately make up the volume
up to the mark (10ml) using 0.1N NaOH respectively .
50µg/ml , 60µg/ml , 70µg/ml , 80µg/ml and 90µg/ml of series dilutions have been prepared
48. PART-E
CALIBRATION CURVE
Take 50,60,70,80 and 90µg/ml individually for analysis and the readings have been record and
calculate R² value and plot the graph between Concentration and Absorbance
PART-F
ACCURACY
For the accuracy determination we selected two dilutions one is lowest and another one is lowest
i.e 50µg/ml and 90µg/ml.
After that we calculate its absorbance and observed the spectrum between absorbance and
wavelength for three times and determined its reproducibility
PART-G
PRECISION
For the precision value we took two dilution series i.e 50,60,70,80 and 90µg/ml and observed its
spectrum by using UV probe and this procedure we repeated for two times in same day (intra-day
precision) and next day (inter-day precision)
49. METHOD – 5
5.5 Standard ( Phosphate buffer 6.4 )
Solvents used is phosphate buffer 6.4
PART-A
Preparation of Phosphate buffer solution pH 6.4
Dissolve 2.5gm of disodium hydrogen phosphate , 2.5gm of sodium dihydrogen phosphate and 8.2gm of sodium
chloride in 950ml of water adjust the pH of solution to 6.4 with 1M sodium hydroxide or 1M hydrochloric acid if
necessary dilute to 100ml with water
PART-B
STOCK SOLUTION – 1
Weigh 0.05gm of market formulation of Methotrexate tablet(10mg) and dissolve in 50ml of volumetric flask and make
up the volume up to the mark using phosphate buffer 6.4 (1000µg/ml)
PART-C
STOCK SOLUTION – 2
Take 10ml of stock solution-1 , and dissolve in 100ml of volumetric flask and make up the volume up to the mark
using phosphate buffer 6.4 (100µg/ml)
PART-D
DILUTIONS
Take 5ml , 6ml , 7ml , 8ml and 9ml of stock solution-2 in 10ml of volumetric flask separately make up the volume up to
the mark using phosphate buffer 6.4 respectively.
50µg/ml , 60µg/ml , 70µg/ml , 80µg/ml , 90µg/ml of series dilutions have been prepared
50. PART-E
CALIBRATION CURVE
Take 50,60,70,80 and 90µg/ml individually for analysis and the readings have been recorded and
calculate R² value and plot the graph between Concentration and Absorbance
PART-F
ACCURACY
For the accuracy determination we selected two dilutions one is lowest and another one is highest
i.e 50µg/ml and 90µg/ml
After that we calculate its absorbance and observed the spectrum between absorbance and
wavelength for three times and determined its reproducibility
PART-G
PRECISION
For the precision value we took two dilution series i.e 50,60,70,80 and 90µg/ml and observed its
spectrum by using UV probe and this procedure we repeated for two times in same day (intra-day
precision) and next day ( inter-day precision)
51. METHOD – 6
5.6 Standard (Phosphate buffer 7.4)
Solvents used is phosphate buffer 7.4
PART-A
Preparation of Phosphate buffer solution pH 7.4
Dissolve 2.5gm of disodium hydrogen phosphate, 2.5gm of sodium dihydrogen phosphate and 8.2gm of
sodium chloride in 950ml of water adjust the pH of solution to 6.4 with 1M sodium hydroxide or 1M
hydrochloric acid if necessary dilute to 100ml with water
PART-B
STOCK SOLUTION – 1
Weigh 0.05gm of market formulation of Methotrexate tablet(7.5mg) and dissolve in 50ml of volumetric flask
and make up the volume up to the mark using phosphate buffer 7.4(1000µg/ml)
PART C
STOCK SOLUTION – 2
Take 10ml of stock solution-1 , and dissolve in 100ml of volumetric flask and make up the volume up to the
mark using phosphate buffer 7.4 (100µg/ml)
PART D
DILUTIONS
Take 5ml , 6ml , 7ml , 8ml and 9ml of stock solution-2 in 10ml of volumetric flask separately make up the
volume up to the mark using phosphate buffer 7.4 respectively.
50µg/ml , 60µg/ml , 70µg/ml , 80µg/ml , 90µg/ml of series dilutions have been prepared
52. PART E
CALIBRATION CURVE
Take 50,60,70,80 and 90µg/ml individually for analysis and the readings have been recorded and
calculate R² value and plot the graph between Concentration and Absorbance
PART-F
ACCURACY
For the accuracy determination we selected two dilutions one is lowest and another one is highest
i.e. 50 µg/ml and 90µg/ml
After that we calculate its absorbance, observed the spectrum between absorbance and wavelength
for three times, and determined its reproducibility
PART-G
PRECISION
For the precision value we took two dilution series i.e 50,60,70,80 and 90µg/ml and observed its
spectrum by using UV probe and this procedure we repeated for two times in same day (intra-day
precision) and next day ( inter-day precision)
54. CALIBRATION CURVE OF STANDARD (0.1 N
NaoH )
CALIBRATION CURVE OF STANDARD (0.1 N NaoH )
Calibration curve was plotted between concentration an absorbance by using the
dilution series of 50, 60 , 70 , 80, 90 microgram / ml and observe its r2 value and
slope value .
y = 0.0312x
R² = 0.9768
0
0.5
1
1.5
2
0 10 20 30 40 50 60
Series1
Linear (Series1)