Health Care Rece

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PHARMA INNOVATIONS FOR BETTER PERCEPTIVE IN HEALTH CARE
PREPARATION OF SELECTIVE FAST DISSOLVING TABLETS BY NATURAL POLYMERS
Dr.Satya Bratha Banja
Professor
Malla Reddy college of pharmacy
Date 10/6/2019
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Aim:
Development and evaluation of orodispersible tablets Valsartan by using Natural crosslinking
polymers like Guar gum and Agar.
Chemicals:
Valsatran, Guar gum, Agar, Microcrystalline Cellulose, Mannitol, talc , Magnesium stearate and
Aspartame.
Apparatus:
Digital weighing balance, Tablet punching machine (Remek), and bulk density apparatus.
Principle:
Oral routes of drug administration have wide acceptance up to 50-60% of total dosage
forms. Solid dosage forms are popular because of ease of administration, accurate dosage, self-
medication, pain avoidance and most importantly the patient compliance .The most popular solid
dosage forms are being tablets and capsules; one important drawback of this dosage forms for
some patients, is the difficulty to swallow. Drinking water plays an important role in the
swallowing of oral dosage forms. Often times people experience inconvenience in swallowing
conventional dosage forms such as tablet when water is not available, in the case of the motion
sickness ( kinetosis) and sudden episodes of coughing during the common cold, allergic condition
and bronchitis.In certain people, the use of conventional tablets can give trouble, such as the
elderly(geriatric) who are experiencing difficulties in using conventional dosage forms(solutions,
suspensions, tablets, and capsules) because of hand tremors and dysphagia; and children (pediatric)
who have problems in swallowing drugs because of muscular and nervous system has not fully
developed. Also in patients who have trouble using conventional tablets, such as in mentally ill
patients, patients who are paralyzed, patients who are unable to swallow and patients who have to
avoid much water, as well as in people who experience nausea .ODT is not only indicated for
people who have swallowing difficulties, but also are ideal for active working or travelling people
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For these reasons, tablets that can rapidly dissolve or disintegrate in the oral cavity have attracted a
great deal of attention. Orodispersible tablets are solid dosage form that disintegrates and
dissolves in mouth without water within 60 seconds or less and are also called as fast-dissolving
tablets, melt-in mouth tablets, rapimelts, porous tablets, quick dissolving etc.The faster the drug
into solution, quicker the absorption and onset of clinical effect. Some drugs are absorbed from the
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mouth, pharynx and esophagus as the saliva passes down into the stomach .In such cases,
bioavailability of drug is significantly greater than those observed from conventional tablets dosage
form. The basic approach in development of ODT is the use of natural superdisintegrants like,
Guar gum and agar etc, which provide instantaneous disintegration of tablet after putting on
tongue, there by release the drug in saliva. The bioavailability of some drugs may be increased due
to absorption of drug in oral cavity and also due to pre-gastric absorption of saliva containing
dispersed drugs that pass down into the stomach. More ever, the amount of drug that is subjected
to first pass metabolism is reduced as compared to standard tablet.Valsartan is a potent and
specific competitive angiotensin II type1 receptor (AT1) antagonist. It is used orally for the
treatment of hypertension and has a low bioavailability of 23%, because of its poor absorption in
lower gastro intestinal tract. It undergoes little or no hepatic metabolism, its elimination half life is
6hrs. In the present study it was proposed to formulate an oral delivery device, in the form of
mouth dissolving tablets by using direct compression technology, with the aim of rapid
disintegration and a complete drug release in a short period of time.
Plan of work:
A) Evaluation of powder blend (Precompression parameters)
 Angle of repose
 Bulk density
 Tapped density
 Compressibility index
 Hausner’s ratio
B) Formulation of ODTs
Preparation of Valsartan tablets by direct compression method:
The direct compression technique was used to manufacture the tablets. The drug and the
excipients were passed through #60-sieve.Weighed amount of drug and excipients except
magnesium stearate were mixed in a polybag by geometric addition method for 10 minutes
manually. The blend was then lubricated by further mixing with magnesium stearate (#60-
sieve). All the above ingredients were subjected for drying to remove the moisture content at
40 to 45oC, the mixture was blended with flavor and the powder blend was then compressed on
10-station rotary punching machine (Remik minipress1) using flat faced punches.
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Table No 1: Formulation of Valsartan fast dissoving tablets
Ingredients (mg) F1 F2 F3 F4 F5 F6 F7 F8
Valsartan 40 40 40 40 40 40 40 40
Microcrystalline
56 51 46 41 56 51 46 41
cellulose
Guar gum 1.5 3 6 9 - - - -
Agar - - - - 1.5 3 6 9
Mannitol
90 93.5 95.5 97.5 90 93.5 95.5 97.5
Talc 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Magnesium stearate 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5
Aspartame
7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5
Total wt (mg)
150 150 150 150 150 150 150 150
Preformulation Studies
Bulk Density .
Weighed quantity of Valsartan (25gm) was transferred into 100 ml measuring cylinder without
tapping during transfer. The volume occupied by the drug was measured. Bulk density was
measured by using formula b = m / Vb. The values obtained are reported in the table

b= m / Vb
where

b = bulk density
m = mass of the blend
Vb = untapped volume
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Tapped density:
25 gm of Valsartan was taken in 100 ml measuring cylinder that was placed in Electro lab tapped
density apparatus (method USP-I). Initial volume (V0) of the cylinder was noted and then the
cylinder was tapped 500 times and volume was measured. Then further an additional 750 tapings
were repeated. No difference was noted between the volumes of the two tapings (500 and 750).
The final volume (V) was considered after completion of 750 taps. Tapped density was measured
by using formula t = m / Vt.
Compressibility Index:
Weighed amount of Valsartan (25gm) was transferred to 100ml-graduated cylinder and subjected
to 500,750 &1250 taps in tap density tester (Electro lab). The difference between two taps should
be less than 2%. The % of compressibility index calculated using formula
Compressibility Index = 100 * (tapped -bulk) / tapped
Hausner’s ratio:
It is measurement of frictional resistance of the drug .The ideal range should be 1.2 –1.5.it is the
determined by the ratio of tapped density and bulk density.
Hausners ratio = tapped / bulk
Angle of repose
It is defined as the maximum angle that can be obtained between the free standing of powder heap
and horizontal plane, which is given by the equation:
 = tan-1 h / r
where  = Angle of repose

h = Height of the pile
r = Radius of the base of the conical pile

Procedure: Weighed quantity of the drug was passed through a funnel kept at a height 2 cm from
the base. The powder is passed till it forms a heap and touches the tip of the funnel. The radius was
measured and angle of repose was calculated by using the above formula.
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Table No 02: Angle of repose ,Bulkdensity,Tappeddensity,Carr’s index and Haunser
ratio.
For Angle of Bulk density Tapped Carr’s index Haunser

mula repose (gm/cc) density (%) ratio
tion (degree) (gm/cc)
code
F1 26.96±1.42 0.568±0.005 0.810±0.01 15.69±1.51 1.31±0.02
F2 25.50±1.32 0.612±0.005 0.762±0.01 14.72±1.20 1.31±0.03
F3 27.30±1.50 0.625±0.005 0.745±0.02 13.45±1.57 1.14±0.02
F4 29.10±1.25 0.639±0.005 0.736±0.02 14.47±1.37 1.19±0.04
F5 27.10±1.36 0.600±0.005 0.794±0.01 16.64±1.44 1.26±0.02
F6 24.79±1.48 0.622±0.005 0.745±0.02 12.46±1.29 1.24±0.04
F7 26.22±1.67 0.620±0.005 0.736±0.01 12.40±2.02 1.15±0.03
F8 24.64±1.36 0.635±0.005 0.721±0.02 12.54±1.51 1.21±0.03
Report: Valsartan fast dissolving tablets can be efficiently and successfully formulated by using
natural polymers employing direct Compression method. Pre-compression parameters were
conducted for all formulations blend and were within I.P limits and mention the table 2. Bulk
density was found in the range 0.56 8 to 0.635 g/sqcm and tapped density in the range of 0.81 to
0.721 g/sqcm using these two density factors hausner`s ratio and compressibility index were
calculated. The powder blend of all formulations hausner`s ratio less than 1.31 which indicates
better flow property and compressibility index between 15.69 to 12.54 which indicates fair
flowability property. The fair flowability property of the powder blend was also evidenced with
angle of repose between 29.10 and 24.46 which is below 40.0.
References:
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EVALUATION OF PREPARED FAST DISSOLVING TABLETS USING NATURAL

POLYMERS
Mrs. D.Nirmala
Associate Professor
Malla Reddy College of Pharmacy
Date 11/6/2019
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Aim: Evaluation of prepared Valsatran Fast dissolving tablets using natural polymers.
Chemicals: Valsatran Fast dissolving tablets, Distilled water, pH 6.8 phosphate buffer, and
Amaranth
Apparatus: Monsanto hardness tester, Petri plates, tissue paper, disintegration test apparatus,
Roches friabilator, verniar callipers, stop watch, digital balance and UV spectrophotometer.
Evaluation of tablets (Post compression parameters)
 Hardness
 Thickness
 Weight variation
 Friability
 Drug content
 Wetting time and water absorption ratio (R)
 In vitro disintegration time
 In-vitro dispersion time
Hardness:
This test gives the indication for the tablets ability to with stand its integrity of drug with
the drug release can be optimized. It was determined by placing the tablet between the anvils only
one of which is movable, driven by electricity. It presses the tablet at constant load till the tablet
breaks. It was recorded in KP (1kP = 1 kg). Hardness of 10 tablets determined and average
hardness and range was calculated.
Thickness:
The thickness in millimeters (mm) was measured individually for 10 preweighed tablets by
using a Mitutoyo portable dial hand micrometer. The average weight, standard deviation and
relative standard variation were reported.
Friability :
Friability is related to tablets ability to withstand both shocks and abrasion without
crumbling during manufacturing, packing, transportation and consumer handling. Friability can be
evaluated by means of friability test apparatus. Compressed tablets that loose less than 0.5% to
1.0% in weight are generally considered acceptable. Friability of the formulated tablets was
determined in Roche friabilator. Ten tablets were weighed accurately and then initial weight was
note down. There are introduced in the apparatus and subjected to 100 revolutions at a speed of 25
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rpm for 4 min. When the drum stopped, tablets were taken and dedusted and final weight was
taken. % Friability was calculated by the formula.
Initial weight (gm) – Final weight (gm)
% Friability = ---------------------------------------------------- × 100
Initial weight (gm)
Acceptance criteria: the friability value should be less than 1.0%.
Weight variation:
Weight variation test was performed according to USP. Average weight of twenty tablets was
calculated and individual weight of each tablet was taken. % deviation was calculated with respect
to average weight. The maximum % deviation allowed is 5% as the tablet weight is more than 324
mg. the tablets meet the USP test if no more than two tablets are outside the% limit and if no tablet
differs by more than two times the % limit.
Content uniformity :
Ten tablets of each batch were weighed and powdered. Aliquot of this powder containing
Valsartan equivalent to 40 mg was accurately weighed, suspended in approximately 50 ml of Ph
6.8 Phosphate buffer and shaken for 15 minutes. Final volume was adjusted to 100 ml with Ph 6.8
Phosphate buffer and filtered (Whatman No.1 filter paper). From this 10 ml was diluted to 100 ml.
The final volume was made by taking 2 ml of above solution and diluted to 10 ml with Ph 6.8
Phosphate buffer. Absorbance of this solution was recorded at 250 nm using UV/Vis
spectrophotometer. The mean percentage drug content was calculated.
Wetting Time :
Five circular tissue papers were placed in a petridish of 10cm diameter. Ten milliliters of
water was added to the petridish. A tablet was carefully placed on the surface of the tissue paper in
the petridish at 250C. The time required for water to reach the upper surface of the tablets and to
completely wet them was noted as the wetting time. These measurements were carried out in
replicates of six. Wetting time was recorded using a stopwatch.
Water absorption Ratio :

The weight of the tablet prior to placement in the petridish was noted (W b) using a Shimadzu
digital balance. The wetted tablet was removed and reweighed (W a). Water absorption ratio, R,
was then determined according to the following equation.
R = 100 * (Wa - Wb) / Wb
where Wb and Wa were tablet weights before and after water absorption, respectively
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In-Vitro Dispersion time :
In vitro dispersion time was measured by dropping a tablet in spoonful of water or in 20ml of
water in a beaker. The time for the tablet to completely disintegrate into fine particles was noted.
Three tablets from each batch were randomly selected and in vitro dispersion time was performed.
In-Vitro disintegration test :
In-vitro disintegration time is measured by dropping a tablet in a beaker containing 50 ml of
buffer pH 6.8.Three tablets from each formulation are randomly selected and in-vitro dispersion
time is carried out.
Table No 01: Hardness, Thickness, Friability and Weight variation
Formulation Hardness(kg/cm2) Thickness(mm) Friability (%) Weight variation(mg)

code
F1 2.6±0.02 3.6±0.07 0.30 151±0.4
F2 2.8±0.10 3.7±0.06 0.22 148±0.6
F3 2.1±0.04 3.8±0.03 0.57 149±0.4
F4 2.6±0.06 3.8±0.05 0.41 152±0.6
F5 2.5±0.06 3.8±0.08 0.26 148±0.2
F6 2.8±0.05 3.7±0.06 0.422 149±0.8
F7 2.4±0.07 3.5±0.06 0.38 150±0.2
F8 2.6±0.05 3.2±0.01 0.35 152±0.8
Table No 02: Wetting time, Water absorption ratio, In-vitro disintegration time, In-vitro
dispersion time and drug content uniformity of valsatran prepared fast dissolving tablet
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Formu Wetting time Water In-vitro In-vitro Drug content
lation (sec) absorption Disintegration Dispersion uniformity (%)
Code ratio (%) time (sec) Time (sec)
F1 77±0.25 10±0.48 117 100±1.12 99
F2 62±0.87 8±0.95 112 109±1.54 101
F3 71±0.99 10±0.35 90 168±1.25 98
F4 57±1.25 16±0.85 40 52±1.14 102
F5 73±1.35 14±0.92 197 79±1.04 100
F6 66±1.15 14±0.95 92 127±0.89 98
F7 68±0.98 9±0.58 60 73±0.54 97
F8 75±0.88 10±0.54 80 82±1.08 99
Report :
The results of evaluation parameters of prepared fast dissolving tablets were shown in
Table 1 and 2. Hardness (2.8±0.10 kg/cm2), friability (0.76%), weight variation (152±0.8) and
drug content (102%) indicate that values were within permissible limit for all formulations. The
wetting time (57±1.25sec), water absorption ratio (16±0.85 %), in-vitro disintegration time (40
sec), and in-vitro dispersion time (52±1.14 sec), revealed that a successful formulation of
Valsartan as fast dissolving tablets using natural polymers.
References:
Dasari Nirmala, Vidyavathi Marvajhala. Design and Evaluation of Fast Dissolving Tablets
of Domperidone using Cationic Exchange Resin. International Journal of Pharma Research and
Health Sciences. Volume 4 (1), 2016, Page-991-997
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PROTOCOL FOR SANDWICH ELISA

Dr.Girish K Radha krishna
National Animal Biotechnology Institute
Scientist-E
Date 12/6/2019
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Aim: To estimate the amount TNF-α by performing sandwich elisa
Introduction:
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for

detecting and quantifying peptides, proteins, antibodies and hormones. In an ELISA, an antigen
must be immobilized to a solid surface and then complexed with an antibody that is linked to an
enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation
with a substrate to produce a measureable product. The most crucial element of the detection
strategy is a highly specific antibody-antigen interaction. ELISAs are typically performed in 96-
well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this
binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having
the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound
from non-bound material during the assay. This ability to wash away nonspecifically bound
materials makes the ELISA a powerful tool for measuring specific analytes within a crude
preparation.
Materials required
Plate sealers, substrate solution, stop solution, plate coating buffer (PBS),wash buffer, and
Reagent Diluent Concentrate 296 well microplates: (R&D Systems, Catalog # DY990).
Plate Sealers: (R&D Systems, Catalog # DY992).
PBS: 137 mMNaCl, 2.7 mMKCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2-7.4, 0.2 μm
filtered (R&D Systems, Catalog # DY006).
Wash Buffer: 0.05% Tween® 20 in PBS, pH 7.2-7.
Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 μm filtered(R&D Systems, Catalog #

DY995).
Quality of BSA is critical • The use of high quality Bovine Serum Albumin (BSA) for the
Reagent Diluent is crucial for the optimum performance of the DuoSet ELISA Development kit.
Impurities such as proteases, binding proteins, soluble receptors or other interfering substances
can be found to varying degrees in virtually all BSA preparations and can inhibit or interfere with
the detection of certain analytes.
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If the standard curve appears suppressed, consider evaluating a different preparation of BSA
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color
Reagent B (Tetramethylbenzidine) (R&D Systems, Catalog # DY999).
Stop Solution: 2 N H2SO4(R&D Systems, Catalog # DY994).
REAGENT PREPARATION
Bring all reagents to room temperature before use. Allow all components to sit for a minimum of
15 minutes with gentle agitation after initial reconstitution. Working dilutions should be prepared
and used immediately.
Streptavidin-HRP: Each vial contains 2.0 mL of streptavidin conjugated to horseradish-
peroxidase. Dilute to the working concentration specified on the vial label using Reagent
Diluent.
Goat Anti-Mouse TNF-α Capture Antibody: Refer to the lot-specific C of A for amount
supplied. Reconstitute each vial with 0.5 mL of PBS. Dilute in PBS, without carrier protein to the
working concentration indicated on the C of A.
Biotinylated Goat Anti-Mouse TNF-α Detection Antibody: Refer to the lot-specific C of A for
amount supplied. Reconstitute each vial with
1.0 mL of Reagent Diluent. Dilute in Reagent Diluent to the working concentration indicated on
the C of A.
Recombinant Mouse TNF-α Standard: Refer to the lot-specific C of A for amount supplied.
Reconstitute each vial with 0.5 mL of Reagent Diluent. A seven point standard curve using 2-fold
serial dilutions in Reagent Diluent is recommended. Prepare 1000 μL of high standard per plate
assayed at the concentration indicated on the C of A.
PRECAUTIONS
It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS
supplemented with 10-50% animal serum. Do not use buffers with animal serum to reconstitute or
dilute the Detection Antibody or Streptavidin-HRP.
It is important that the Reagent Diluent selected for reconstitution and dilution of the standard
reflects the environment of the samples being measured.
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Avoid microbial contamination of reagents and buffers.
• A thorough and consistent wash technique is essential for proper assay performance. Wash
Buffer should be dispensed forcefully and removed completely from the wells by aspiration
or decanting. Remove any remaining Wash Buffer by inverting the plate and blotting it against
clean paper towels.
• Individual results may vary due to differences in technique, plasticware and water sources.
• It is recommended that all standards and samples be assayed in duplicate.
• The use of PBS from tablets may interfere in this assay.
Assay Procedure
 Add 100 μL of sample or standards I Reagent Diluent, or an appropriate diluent, per
well. Cover with an adhesive strip and incubate 2 hours at room temperature.
 Repeat the aspiration/wash as in step 2 of Plate Preparation.
 Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover
with a new adhesive strip and incubate 2 hours at room temperature.
 Repeat the aspiration/wash as in step 2 of Plate Preparation.
 Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and
incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
 Repeat the aspiration/wash as in step 2.
 Add 100 μL of Substrate Solution to each well. Incubate for
 20 minutes at room temperature. Avoid placing the plate in direct light.
 Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
 Determine the optical density of each well immediately, using a microplate reader set to
450 nm. If wavelength correction is available, set to 540nm or 570nm. If wavelength
correction is not available, subtract readings at 540nm or 570nm from the readings at
450nm. This subtraction will correct for optimal imperfections in the plate. Reading made
directly at 45nm with correction may be higher and less accurate.
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WESTERN BLOT PROTOCOL
DR.ANAND SRIVASTAVA
National Animal Biotechnology Institute
Scientist-E
12/06/2019
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INTRODUCTION
Western blotting is used to identify individual proteins within a cell or tissue lysate using
antibodies. The antibodies bind to specific epitopes (highly specific sequences of amino acids).
Because epitopes in proteins vary from protein to protein, western blotting analysis can be used to
identify and quantify a single protein in a lysate that contains thousands of different proteins. In
order to do so, firstly, proteins are separated from each other based on their size by SDS-PAGE gel
electrophoresis. Then, the proteins are transferred from the gel to a membrane by application of an
electrical current/voltage. The membrane can then be processed with primary antibodies specific
for target proteins of interest. Next, secondary antibodies bound to enzymes are applied and finally
a substrate that reacts with the secondary antibody-bound enzyme is added for detection of the
antibody/protein complex.
CHEMICLAS:
Buffer and reagent require
Lysis Buffer
 NP-40
 150 mMNaCl
 1% NP-40 or Triton X-100
 50 mMTris pH 8.0
RIPA
 150 mMNaCl
 1% NP-40 or Triton X-100
 0.5% sodium deoxycholate
 0.1% SDS
 50 mMTris, pH 8.0
 Tris-HCl
 20 mMTris-Hcl, pH 7.5
Loading Buffer (2X Laemmli buffer)

 4% SDS
 5 % 2-mercaptoethanol
 20% glycerol
 0.004% bromophenol blue
 0.125 M TrisHCl, pH 6.8
Running Buffer (1X running buffer)

 25 mMTris base
 192 mM glycine
 0.1% SDS
 Adjust to pH 8.3
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Transfer Buffer (1X transfer buffer)
 25 mMTris base
 192 mM glycine
 20 % methanol
 Adjust to pH to 8.3
Blocking Buffer
 1X TBST
 5% non-fat dry milk OR 5% BSA
LYSATE PREPARATION
Choice of starting material: It depends on the source of proteins to be used for analysis such as
whole cell lysate, nucleus, mitochondria, cytoplasm, membrane bound proteins etc. Based on the
choice of starting material protocol may be modified. Here we describe method for whole cell
lysate.
Cells from the cell culture:Aspirate PBS and add ice-cold lysis buffer (1 mL per confluent 10 7
cells/100mm dish/150 cm2 flask). Wash cell culture dish on ice with ice-cold PBS.Aspirate PBS
and add ice-cold lysis buffer (1 mL per confluent 107 cells/100mm dish/150 cm2 flask). Using a
cell scraper, scrape adherent cells off the dish and transfer the cell suspension into a
microcentrifuge tube. If required, the cells can be trypsinized and washed with PBS prior to
resuspension in lysis buffer.Agitate cells for 30 minutes at 4˚C.Centrifuge cell lysate mixture at
4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20
minutes at 12,000 rpm.Transfer the supernatant (lysate) to a fresh tube on ice.
SAMPLE PREPARATION
 Determine the protein concentration of each cell lysate.
 Determine how much protein to load (Recommended: 10-50 μg/lane) and add an equal
volume 2X Laemmli buffer.
 Reduce and denature the samples by boiling the lysates in sample buffer at 95-100˚C for 5
minutes.
SDS-PAGE
 Prepare gel of appropriate polyacrylamide percentage to best resolve protein of interest
based on molecular weight.
 Load samples containing equal amounts of protein (10-50 μg/lane protein from cell lysate
or 10-100 ng/lane purified protein) prepared in sample buffer into SDS-PAGE wells.
Include a molecular weight marker in one of the lanes.
 Fill the electrophoresis apparatus with 1X running buffer as instructed by the manufacturer.
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 Run the gel as recommended by the manufacturer; 1-2 hours at 100 V is standard, but time
and voltage may require optimization.
PROTEIN TRANSFER
 Prepare PVDF membrane by wetting it in methanol for 30 seconds and then soaking it
briefly in distilled water followed by 1X transfer buffer. Handle the membrane carefully,
ideally with rounded tweezers to avoid scratching or puncturing the surface.
 Soak filter papers and sponges in the transfer buffer for 10 minutes prior to assembly of the
transfer “sandwich”.
 After electrophoresis, remove the gel from the electrophoresis apparatus and equilibrate it
by soaking in transfer buffer for 10 minutes.
 Prepare the sandwich and sequentially assemble the layers of the sandwich. Gently remove
any air bubbles with a roller or pipette. Bubbles between the gel and the membrane will
inhibit the transfer of proteins to the membrane.
 Place the sandwich into a transfer cassette and perform semi-dry or wet transfer according
to the manufacturer’s instructions of the blotting apparatus.
IMMUNOBLOTTING
 After transfer, rinse the membrane briefly in 1X TBST.
 Incubate membrane in blocking solution for 1 hour at room temperature or overnight at
4˚C with constant rocking.
 Optional step: Rinse the membrane for 5 minutes in 1X TBST.
 Dilute the primary antibody to working concentration in 1X TBST with 1% milk or BSA
(use whatever was chosen for blocking).
 Incubate the membrane in primary antibody solution for 1 hour at room temperature or
overnight at 4˚C with gentle rocking. This time may require optimization. In most cases,
overnight incubation at 4˚C increases signal strength and reduces background signal
relative to 1 hour incubation at room temperature.
 Wash the membrane with 1X TBST three times for 10 minutes each with gentle rocking.
 Incubate the membrane in the appropriate diluted secondary antibody (in 1X TBST and
may include 1% milk or BSA) for 1 hour at room temperature with gentle rocking. HRP
conjugated secondary antibodies are most common for western blot analysis.
 Wash the membrane in 1X TBST three times for 10 minutes each with gentle rocking.
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DETECTION
 Prepare the ECL substrate just prior to use according to the manufacturer’s instructions.
 Incubate the membrane in the substrate according to manufacturer’s directions. Typical
incubation times are 1-5 minutes.
 Carefully remove the membrane from the detection reagent and sandwich it between layers
of plastic (i.e. a sheet protector or plastic wrap).
 Expose the membrane to autoradiography film in a dark room or image with a
chemiluminescent imaging system, such as a ChemiDoc.
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Handling of HPLC, FT-IR and drug estimation by HPLC and FT-IR
Dr.C.Parthiban
Professor
Date 13/6/2019
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HPLC
Make : Agilent HPLC
Model : 1220
Column: C18 (100 mm × 4.5 mm, 3.5μm)
EZ Chrome software
UV detector
Handling of HPLC:
1. Switch on the instrument and computer.
2. Open EZ CHROME ELITE soft ware.
3. Go to control and check instrumental status.
4. Switch on the pump and detector.
5. Do purging for 5minutes with the selected mobile phase.
6. Set new method for analyzing sample. i.e. select detection wave length and flow rate,
run time, mobile phase etc.
7. Save new method.
8. Download method in control option.
9. Do base line monitoring for 10 min. with mobile phase.
10. Load the sample in HPLC injector.
11. Go for single run.
12. When the instrument displays waiting for trigger turn the injection port to inject
position.
13. Get the spectra click on Area % option
14. Displays area and run time of the sample.
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Exercise Done:
Determination of Bicalutamide bulk and pharmaceutical dosage forms.
Method:
Reverse-phase chromatography was performed on Agilent HPLC using C18 (100 mm × 4.5 mm,
3.5μm) column with EZ Chrome software and UV detector. Methanol : phosphate buffer pH 3
(80:20) (v/v) was used as mobile phase at a flow rate of 1 mL min -1 with UV detection at 270 nm.
Materials and Reagents
Bicalutamide was gift sample from shilpa medicare. Acetonitrile HPLC Grade was
purchased from Merck Chemical Company. HPLC Grade water was purchased from Avantor
performance materials limited. The 0.45 μm pump Nylon filter was obtained from Advanced
Micro Devices (Ambala Cantt, India) & whatman no 5 filter paper was obtained from Modern
Science lab, (Nashik, India). Glass wares used were Class A grade.
HPLC instrumentation and conditions
Chromatographic separation was achieved by using maintained at 25 ºC. Isocratic elution
was performed using methanol: phosphate buffer pH 3 (80:20) (v/v) at a flow rate 1 mL min -1with
UV detection at 270 nm. The overall run time was 5 min and 25μL of sample was injected into the
HPLC system.
Preparation of stock solution
Bicalutamide stock solution (1000 μg mL-1) was prepared by accurately weighing 50mg of
Bicalutamide in a 50 mL volumetric flask and making up to volume with mobile phase. Working
solutions for HPLC injections were prepared on a daily basis from the stock solution in a solvent
mixture of methanol: phosphate buffer pH 3 (80:20) (mobile phase). Solutions were filtered
through a 0.45 μm membrane filter prior to injection.
Preparation of sample solution
Twenty tablets were weighed accurately and finely powdered. A powder equivalent to
25mg of Bicalutamide was transferred carefully to 25mL volumetric flask and about 15mL diluent
was added. The mixture was sonicated for 10 minutes. The volume was made up to 25mL with
diluent, filtered through whatman no. 5 filter paper. From the filtrate further dilutions were made
to obtain 25μg/mL. The final solution was injected in HPLC, chromatogram was recorded and area
was measured.
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Figure : Chromatogram of Bicalutamide Standard
Figure: Chromatogram of Bicalutamide Formulation

Table : Assay of Bicalutamide in Tablet Formulation
Formulation Label claim Amount found % Purity %RSD
Casodex (50mg) 50mg 49.97mg 99.9% 0.771
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FT-IR
Make : BRUKER
Model : ALPHA
Soft Ware : OPUS
Handling of IR:
1. Switch on the instrument and computer.
2. Open OPUS software select ATR sampling technique.
3. Type the sample name and nature of the sample.
4. Clean the germanium crystal compartment and do base line
correction.
5. Place the sample in the crystal and start sample scan.
6. Get the spectra and smooth the spectra.
7. Integrate the spectra to get wave numbers for the peak.
8. Click GO to save the spectra with a file name.
9. Do interpretation for the spectra to find the unknown sample.
Exercise Done:
INTERPRETATION OF UNKNOWN SAMPLE-1 BY FT-IR
AIM: To identify unknown sample.
SAMPLING TECHNIQUE: Attenuated total reflectance
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IR GRAPH:
INTERACTIONS:
N-H = stretching = 3348 cm-1
C-H = bending = 2980 cm-1
C=O = stretching = 1740 cm-1
C=O = stretching = 1368 cm-1
O-H = stretching = 1214 cm-1
C-H = bending = 1430 cm-1
REPORT: The interpretation of given unknown sample was found to be PARACETAMOL.
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INTERPRETATION OF UNKNOWN SAMPLE-2 BY FT-IR
AIM: To identify unknown sample.
SAMPLING TECHNIQUE: Attenuated total reflectance
IR GRAPH:
INTERACTIONS:
C-H = stretching = 2977 cm-1
C=O = stretching = 1703.83 cm-1
C-H = bending = 1360.41 cm-1
C=O = bending = 1092.50 cm-1
REPORT: The interpretation of given unknown sample was found to be ACETONE.
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PREPARATION OF SELECTIVE DRUG LOADED NANOPARTICLES
Dr.K.Vijaya Sri
Professor
Date 14/6/2019
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Introduction:
Nanoparticles (NPs) are at the forefront of rapid development in nanotechnology. Their

exclusive size-dependent properties make these materials indispensable and superior in many areas
of human activities.1Being the most current transition metal in the Earth’s crust, iron stands as the
backbone of current infrastructure.2 However, in comparison to group elements such as cobalt,
nickel, gold, and platinum, iron oxides are somewhat neglected.2
Nanoparticles (NPs) are at the forefront of rapid development in nanotechnology. Their
exclusive size-dependent properties make these materials indispensable and superior in many areas
of human activities [1].Magnetic Iron oxide Nano Particles are inexpensive to produce, physically
and chemically stable, biocompatible, and environmentally safe. Magnetic nanoparticles are a class
of nanoparticle that can be manipulated using magnetic fields. Such particles commonly consist of
two components, a magnetic material, often iron, nickel and cobalt, and a chemical component that
has functionality. While nanoparticles are smaller than 1 micro meter in diameter (typically 1–100
nanometres), the larger microbeads are 0.5–500 micro meter in diameter.
There are two types of nanoparticles i.e.; organic and in organic/metal oxide nanoparticles.
Metal oxides play a very important role in many areas of chemistry, physics andmaterials science.
There are various types of metal oxide nanoparticles like gold nanoparticles silver nanoparticles
zinc oxide nanoparticles and iron oxide nanoparticles
GNPs occur in various size ranges from 2-100 nm; however, 20-50 nm particle size ranges
showed the most efficient cellular uptake. In contrast, a larger particle, i.e., 80-100 nm does not
diffuse into the tumor and stay near the blood vessels there are several advantages such as Gold
nanoparticles has unique physical and chemical properties which enhance the efficiency of drugs,
drug loading, biocompatible, easily reach to the targeted site with blood flow, non-cytotoxic to the
normal cells
Silver oxide nanoparticles are the particles of silver or silver oxide, with particle size
between 1 and 100 nm in size. Silver nanoparticles are used into a wide range of medical devices,
including bone cement, surgical instruments, surgical masks, etc. Moreover, it has also been shown
that ionic silver, in the right quantities, is suitable in treating wounds. In fact, silver nanoparticles
are now replacing silver sulfadiazine as an effective agent in the treatment of wounds. Due to their
attractive physiochemical properties these nanomaterials have received considerable attention in
biomedical imaging using SERS(Surface-enhanced Raman spectroscopy).
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Zinc oxide nanoparticles (ZnO NPs), as one of the most important metal oxide
nanoparticles, are popularly employed in various fields due to their peculiar physical and chemical
properties.ZnO NPs have superior antibacterial, antimicrobial, and excellent UV-blocking
propertiesTreatment using zinc was approved by the FDA and nowadays Zn is available as a food
additive Their white color, UV-blocking activity, and ability to prevent biofilm formation makes
them suitable for fabric and glass industries as coating materials designated for medical and other
devices[2].
Magnetic nanoparticles: Iron (III) oxide (Fe3O3) is a reddish brown, inorganic compound
which is paramagnetic in nature and also one of the three main oxides of iron, while other two
being FeO and Fe3O4. Fe3O4 is naturally a mineral magnetite and is super paramagnetic in
nature.Magnetic iron oxide nanoparticles have been functionalized with antibodies, nucleosides,
proteins and enzymes for directing them to diseased tissues such as tumors.
Magnetic nanoparticles have several applications such as Super paramagnetic iron oxide
nanoparticles (SPIONs) have emerged as promising candidates for various biomedical
applications, such as enhanced resolution contrast agents and targeted drug delivery, hyperthermia,
gene therapy, stem cell tracking, molecular/cellular tracking, magnetic separation technologies for
early cancer, diabetes, and atherosclerosis.
Magnetic nanoparticle clusters that are composed of a number of individual magnetic
nanoparticles are known as magnetic nanobeads with a diameter of 50–200 nanometres [3].The
magnetic nanoparticles have been the focus of much research recently because they possess
attractive properties which could see potential use in catalysis including nanomaterial-based
catalysts,biomedicineand tissue specific targeting,microfluidics, magnetic resonance imaging,
magnetic particle imaging, data storage, environmental remediation, nanofluids, optical filters,
defect sensor, magnetic cooling and cation sensors.
1.1. Iron oxides: Eight iron oxides are known, among these iron oxides, hematite (α-
Fe2O3), magnetite (Fe3O4) and maghemite (γ-Fe2O3) are very promising and popular candidates
due to their polymorphism involving temperature-induced phase transition. Each of these three
iron oxides has unique biochemical, magnetic, catalytic, and other properties which provide
suitability for specific technical and biomedical applications.
1.1.1. Hematite (α-Fe2O3): It is the most stable iron oxide and n-type semiconductor
under ambient conditions, hematite (α-Fe2O3) is widely used in catalysts, pigments and gas
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sensors due to its low cost and high resistance to corrosion. It can also be used as a starting
material for the synthesis of magnetite (Fe3O4) and maghemite (γ-Fe2O3), which have been
intensively pursued for both fundamental scientific interests and technological applications.α-
Fe2O3 has weak ferromagnetism at room temperature, while the saturation magnetization is often
smaller than 1 emu g−1.Hematite is an n-type semiconductor with a band gap of 2.3 eV, where the
conduction band (CB) is composed of empty d-orbitals of Fe3+ and the valence band (VB)
consists of occupied 3d crystal field orbitals of Fe3+with some admixture from the O 2p non-
bonding orbitals.Fe3+ions occupy two-thirds of the octahedral sites that are confined by the nearly
ideal hexagonal close-packed O latticeas shown in figure 1(a).
1.1.2. Magnetite (Fe3O4):Fe3O4 has the face centered cubic spinel structure, based on 32
O2− ions and close-packed along the direction as shown in figure 1(b). Fe3O4 differs from most
other iron oxides in that it contains both divalent and trivalent iron. Fe3O4 has a cubic inverse
spinel structure that consists of a cubic close packed array of oxide ions, where all of the Fe2+ ions
occupy half of the octahedral sites and the Fe3+are split evenly across the remaining octahedral
sites and the tetrahedral sites. Thus, Fe3O4 can be both an n- and p-type semiconductor.
However,Fe3O4 has the lowest resistivity among iron oxides due to its small bandgap.
1.1.3. Maghemite (γ-Fe2O3): The structure of γ-Fe2O3 is cubic; each unit of maghemite
contains 32 O2− ions, 21⅓ Fe3+ ions and 2⅓ vacancies as shown in figure 1(c). Oxygen anions
give rise to a cubic close-packed array while ferric ions are distributed over tetrahedral sites (eight
Fe ions per unit cell) and octahedral sites (the remaining Fe ions and vacancies). Therefore, the
maghemite can be considered as fully oxidized magnetite, and it is an n-type semiconductor with a
bandgap of 2.0 eV.γ-Fe2O3 and Fe3O4 exhibit ferrimagnetism at room temperature, with the
saturation magnetization reaching to 92 emu g−1[4].
1.2 Morphology of Iron oxide nanoparticles structure:
There are four types of magnetic nanoparticles composite structures. They are core–shell
structure, matrix dispersed structure, Janus-type heterostructures and shell–core–shell structure
Core-Shell Structure:In this structure, the iron oxide core was encapsulated in an inorganic
or an organic coating that renders the whole particle stable and biocompatible, and may serve as a
support for biomolecules. Generally, IONPs are not located at the center of the functional coating
material; this structure is also known as a yolk structure. Indeed, the magnetic composite
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nanomaterials not only provide the material with an improved stability of the nanoparticulate
building blocks, but also further introduce new physical and biological properties, and
multifunctional behaviours. Thus, in the inverse core–shell structure, the magnetic IONPs will coat
the surface of non-magnetic functional materials. Moreover, magnetic IONPs can combine one or
more functional materials and further coat with another functional material on the functionalized
surface.
Matrix dispersed structure:Magnetic IONPs are dispersed in a matrix to prevent the
superparamagnetic NPs from aggregating into large ferromagnetic species. Matrix-dispersed NPs
can be created in a variety of different states, e.g. dispersed in a continuous amorphous matrix,
grafted on larger, mesoscale particles, or well defined, three-dimensional superstructures of NPs.
Janus structure:In Janus structure, one side is magnetic IONPs, and the other side is
functional materials. Anisotropic surface chemical compositions are interesting for applications
even if one is not concerned with self-assembly. This Janus particle can be used in target-specific
platin delivery.
Shell–core–shell structure:In this structure, the location of magnetic IONPs is between the
two functional materials. Most applications require magnetic IONPs to be embedded in the
nonmagnetic layers to avoid aggregation and sedimentation of magnetic IONPs as well as to
endow them with particular surface properties for specific applications[5]
1.3 Surface functionalisation of Iron oxide nanoparticles:
A prerequisite for every possible applied structure is the proper surface protection or
functionalization of such magnetic composite Nanoparticles.
Iron oxides with bare surface tend to agglomerate due to strong magnetic attraction among
particles, Vanderwaalsforces, and high energy surface. Consequently, the reticuloendothelial
system eliminates the agglomerated iron oxide Nanoparticles.High concentration of local Ferrous
ions is also toxic to organisms from Ferrous dissolution. Magnetic nanoparticles have hydrophobic
surface with large surface -area -to- volume ratio. These particles agglomerate and form large
clusters and increase the particle size and also increases the saturation magnetisation.
These can be avoided by coating a shell on the iron oxide Nanoparticles surface which
makes them hydrophilic, compatible to bioenvironments, and functionalized.The appropriate
surface coating allows a targetable delivery with particle localization in a specific area and is also
considered nontoxic and biocompatible.There are different types of coating techniques[18].
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1.3.1 Organic Materials:Several approaches have been developed to functionalize iron
oxide nanoparticles, including in situ coatings and post-synthesis coating, which are the common
routes for organic material coating on the iron oxide nanoparticle surface.iron oxide nanoparticles
are functionalised either with small molecules or surfactants and polymers
A. Small molecules and surfactants:For surface modification magnetic nanoparticles
should be functionalised by special groups (e.g. –OH, –COOH, –NH2, –SH), which are suitable
for the attachment of different bioactive molecules for various applications.
A small molecule silicane is often used to modify and endow the functionalized end groups
to the surface of bare iron oxide nanoparticles directly for post-connecting with metal ions,
polymers, biomolecules or other biological entities.However, fabrication of oil-soluble type iron
oxide nanoparticles is very important for obtaining monodisperse iron oxide nanoparticles. The
most common organic compounds are oleic acid and oleyamine are used. oleic acid is widely used
in iron oxide nanoparticle synthesis because it can form a dense protective monolayer, thereby
producing highly uniform iron oxide nanoparticles. Generally, the oleic acid and oleyamine are
often used in the high-temperature thermal decomposition reaction process.
To synthesize water-soluble magnetic iron oxide nanoparticles directly, one way is to use
small molecules (such as amino acid, citric acid, vitamin, cyclodextrin, etc) in the reaction process.
Generally, surface of magnetic nanoparticles is hydrophobic in order to change the polarity
to hydrophilic excess of ligand should be added to the nanoparticulate solution, resulting in the
displacement of the original ligand on the surface of nanoparticles. Examples Nitrosonium
tetrafluoroborate (NOBF4) is used to replace the original organic ligands attached to the
nanoparticles surface, stabilizing the nanopaticles in various polar and hydrophilic media for years,
without aggregation or precipitation[19].
B. Natural and synthetic polymers:Compared with small molecules and surfactants,
polymer functionalization not only provides multifunctional groups and more colloid stability, but
also plays a significant role in pharmacokinetics and biodistribution. Polymers are the most
common surface coating used in iron oxide nanoparticles, since they can prevent oxidation and
confer stability to the nanoparticles and the polymers get attached to the surface of magnetic
nanoparticles by either adsorption by covalent bond linkage or by crosslinker like
glutaldehyde.The polymer nature can be synthetic, encompassing polyethylene glycol,
poly(vinylpyrrolidone), polyvinyl alcohol and poly (lactic-co-glycolic acid), or natural, as in the
case of chitosan[20].
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Poly Ethylene Glycol (PEG):PEG is a hydrophilic, uncharged polyether polymer well
known for its biocompatibility. It has been commonly used as iron oxide nanoparticles-coating due
to non-fouling properties, reduced blood proteins opsonization and, as a result, escapes recognition
by the immune system. Such properties increase its time in blood circulation and the accumulation
in the target cells/organ.
Poly Vinyl Pyrrolidine and Poly Vinyl Alcohol (PVP&PVA) :PVP and PVA are water-
soluble synthetic polymers. Specifically, PVP is derived from the monomer N-vinyl pyrrolidone.
Given its biocompatible, stable and safe properties, PVP is widely used in biomedical and
pharmaceutical applications. In turn, PVA exhibits emulsifying and adhesive properties, forming a
hydrogel structure that involves the iron oxide nanoparticles by means of hydrogen bonds between
the polymer chains. This leads to increases in polymer-surface interactions, preventing the
agglomeration of the particles.
PLGA:PLGA is a copolymer of poly lactic acid and poly glycolic acid (PGA) with great
potential for use in drug delivery and tissue engineering. Besides presenting solubility in most of
common solvents, PLGA can take different shapes and sizes, and encapsulates molecules of all
sizes. Usually, higher rates of PGA (which contains methyl site groups) lead to a higher
hydrophobicity and degradation of the polymer. On the other hand, lactide-rich PLGA are less
hydrophilic and degrade more slowly, since it absorbs less water. Moreover, physical properties of
PLGA are known to vary depending on different factors such as the molecular weight, which plays
an important role in the drug-loading capacity on the polymer surface.
Chitosan: Iron oxide nanoparticles have also been coated with Chitosan. This material is a
natural, long-chain polymer, generated by the combination of 2-amino-2-deoxy -D-glucan with
glycosidic linkages, which can be obtained by chitin deacetylation. Its positive charge drives the
Chitosan carriers to the cell membrane (negatively charged) and its mucoadhesive properties
extend the Chitosan retention in the target sites, making it interesting for application in drug
delivery systems. Furthermore, Chitosan is biocompatible, biodegradable and presents low
toxicity.
Many Chitosan-nanosystems have been developed over the last few years, relying on the
aforementioned advantages and water solubility. Coating of iron oxide nanoparticles with this
polymer does not change the thermal and magnetic properties of the nanoparticles, serving as
support for drug binding. Also, a one-pot synthesis in the presence of Chitosan of low molecular
weight showed that it was capable of protecting iron oxide nanoparticles from aggregation due to
the electrostatic repulsion between the positively charged nanoparticles.This polymer, however,
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presents certain limitations as a coating material, mainly associatedwith the partial protonation of
its amino groups in water at physiological pH, what reduces Chitosansolubility. To overcome such
issues, chemical changes can be performed in order to makeChitosan derivatives more water-
soluble. A practical example is the O-carboxymethyl Chitosan, which useshydrogen bonding
between water and the polymer in combination with carboxyl group to obtainwater solubilization.
Also, a polyelectrolyte complex of carboxymethyl starch-Chitosan can be used as acoating for iron
oxide nanoparticles, producing stable, biocompatible and mucoadhesive nanosystems[21].
C. Biomolecules: Recently, biomolecule functionalized magnetic iron oxide
nanoparticles have become a common and effective strategy in the biological separation, detection,
sensor and other bio-applications due to their higher biocompatibility. The various biomolecules,
including enzymes, antibodies, proteins, biotin, bovine/human serum albumin, avidin and
polypeptides have been bound onto the surface of iron oxide nanoparticles[22]
1.3.2 Inorganic Materials:
a. Silicon:Silica-coated iron oxide nanoparticles(IONP@silica) is a classical and
important composite material for both fundamental study and bio-applications. Silica coating can
enhance the dispersion in solutionand also stabilise the nanoparticles and protect from acidic
environment.Three different approaches have been explored to generate Silica-coated iron oxide
nanoparticles (IONP@silica) nanomaterials. The first method relied on the well-known Stober
process, in whichthe iron oxide nanoparticles were homogeneously dispersed in the alcohol, then
the silane was added, and finally the water or ammonia aqueous solution was dropped into the
mixed solution and Silica-coated iron oxide nanoparticles (IONP@Silica)are formed.
Tetraethoxysilane (TEOS), Vinyltriethoxysilane (VTEOS), Octadecyltrimethoxy silane are the
most common used silanes, which easily bind on the surface of iron oxide nanoparticles through
OH groups.
The second approach was based on microemulsion synthesis, in which micelles or inverse
micelles were used to confine and control the coating of silica on core nanoparticles. It is
noteworthy that this method requires much effort to separate the core–shell nanoparticles from the
large number of surfactants associated with the microemulsion system.
The third approach is aerosol pyrolysis, in which Silica-coated iron oxide
nanoparticles(IONP@SiO2)were prepared by aerosol pyrolysis of a precursor mixture composed
of silicon alkoxides and metal compound in a flame environment.
b. Carbon: Carbon protected iron oxide nanoparticles have good chemical and thermal
stability, and intrinsic high electrical conductivity. The carbon coating provides an effective
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oxidation barrier and prevents corrosion in magnetic core materials. Hydrophilic carbon coating on
iron oxide nanoparticle cores endows better dispersibility and stability than those shown by bare
iron oxide nanoparticles.The common approach is a three-step process: firstly, magnetic iron oxide
nanoparticles are prepared as seeds by various methods, and then the polymer is coated through the
polymerization process, finally forming IONP@C composite materials by annealing treatment.
c. Metals:Surface modification of iron oxide nanoparticles with metallic elements can
provide an inert layer, which typically exhibit a core-shell, core-satellite or dumbbell structure.
Metallic coatings facilitate further functionalization of the iron oxide nanoparticles to improve
stability and compatibility. Gold and silver are the most common noble metal element used for
surface coating.
In general, the core–shell, core–satellites and dumbbell structures of iron oxide
nanoparticles can be formed by microemulsion and thermal decomposition methods and then
direct and indirect method are the two routes used to achieve the gold shell coating on the surface
of the magnetic iron oxide nanoparticles.
In direct method, a gold or silver shell is formed on the surface of Ferrous oxide
nanoparticles by reduction of Au+3 or Ag+2 ions in the presence of reducing agents. Direct gold
coating is carried out in aqueous or organic solutions.In the aqueous phase, the sodium citrate and
sodium borohydride are the frequently used reducing agents. In the organic phase,oleic acid and
oleylamineare used as reducing agents.
In indirect method, Gold/silver coated iron oxide nanoparticles are synthesised by
forming a “glue layer” between the iron oxide nanoparticles core and the gold shell. The “glue”
layer should be capable of enhancing the Fe3O4magnetic nanoparticles stability, and also have
metal binding groups to attach gold seeds to promote the formation of gold shell. Materials used as
the “glue” layer are often polymers, silica and carbon.
d. Metal oxides and sulphides:More and more metal oxides or sulphides have been
used to protect or functionalize iron oxide nanoparticles, mainly because of the fantastic magnetic
properties of iron oxide nanoparticles and other unique physical or chemical properties of metal
oxides and sulphides.
Oxide and sulphide semiconductors are the most common compounds that are used to
functionalize magnetic iron oxide nanoparticles, such as TiO2,ZnO, SnO2, WO3, Cu2O, CdS,
ZnS, PbS, Bi2S3etc[23].
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PREPARATION OF QUERCETIN LOADED FERROUS OXIDE NANOPARTICLES
Aim: To formulate and determine the particle size of quercetin loaded ferrous oxide
nanoparticles.
Chemicals: Ferrous oxide nanoparticles, Quercetin, Ethanol, poloxomer 188,  -cyclodextrin,
Dimethyl Sulphoxide.
Principle:
Magnetic nanoparticle clusters that are composed of a number of individual magnetic nanoparticles
are known as magnetic nanobeads with a diameter of 50–200 nanometres [3]. Iron oxides with bare surface
tend to agglomerate due to strong magnetic attraction among particles, van der Waals forces, and high
energy surface. Magnetic nanoparticles have hydrophobic surface with large surface -area -to- volume ratio.
These particles agglomerate and form large clusters and increase the particle size and also increases the
saturation magnetisation.
Cyclodextrins are cyclic (α-1,4) linked oligosaccharides of α-D-glucopyranose, containing
a relatively hydrophobic central cavity and hydrophilic outer surface. Owing to lack of free
rotation about the bonds connecting the glucopyranose units, the cyclodextrins are not perfectly
cylindrical molecules but are toroidal or cone shaped. Based on this architecture, the primary
hydroxyl groups are located on the narrow side of the torus, while the secondary hydroxyl groups
are located on the wider edge. When cyclodextrins are used to solubilize water insoluble drugs, it
is generally assumed that the solubilization proceeds through inclusion complexes.
Quercetin is mainly present in nature as its glycosides in which one or more sugar groups are bound
to phenolic groups by glycosidic linkage. Quercetin has been reported to exert numerous pharmacological
activities, such as free radical scavenging, TNF alpha inhibition, cardio protective and anticarcinogenic
effects (1-2). The therapeutic value of quercetin is limited by poor absorption from the GIT, low skin
penetration, low solubility, rapid excretion from the body and resulting in low absorption invivo. In this
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present research work to novel formulation of quercetin loaded Ferrous oxide nanoparticles with
cyclodextrins which decrease particle size the enhance the solubility, dissolution of quercetin.
Procedure:
Preparation of quercetin loaded ferrous oxide nanoparticles:
Various quantity of Ferrous oxide nanoparticles is dispersed in distilled water and is

sonicated for 10 minutes. Poloxomer 188 is dissolved in ethanol and is added to the magnetite
solution and isstirred at 600 rpm for 2 hours. To this solution add - cyclodextrin dissolved in
ethanol and isstirred at 8000 rpm for 2hrs. This results in the formation of cyclodextrin coated
magnetitenanoparticles. These nanoparticles were filtered and dried in oven. These nanoparticles
weredispersed in water and quercetin dissolved in dimethyl sulphoxide is injected into the solution
andis stirred at 6000rpm for 1hrs which results in the formation of Quercetin loaded ferrous
oxidenanoparticles and mentioned in table in 1.
Table: 1 Formulation of Quercetin loaded ferrous oxide nanoparticles:
Ingredients F-1 F-2 F-3 F-4 F--5

Iron oxidenanoparticles(mg) 750 750 750 750 500
Quercetin (mg) 1000 1000 1000 1000 1000
PEG (mg) 200 100 150 100 150
-Cyclodextrin (ml) 50 100 75 50 50
DMSO (ml) 1 1 1 1 1
Ethanol (ml) 2 2 2 2 2
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Evaluation of Quercetin loaded ferrous oxide nanoparticles:
Particle size: Particle size growth is mainly responsible for agglomeration. Precise sizing
techniques can give useful information about the particle size distribution in nanosuspensions. The
particle size of nanoparticles are measured using Particle size ananlyser. The average values were
employed for the calculations of the response surfaces.
PARTICLE SIZE , ZETAPOTENTIAL AND POLYDISPERSITY INDEX:

S.NO FORMULATION PARTICLE SIZE PDI ZETA POTENTIAL
1 F-1 383.7 0.474 -16.8
2 F-2 409.1 0.438 -24.6
3 F-3 699.8 0.546 -24.2
4 F-4 687.4 0.618 -10.6
5 F-5 1098 0.635 -24.4
Figure : Particle Size Graphs
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Report: Quercetin loaded iron oxide nanoparticles were successful prepared. The prepared
nanoparticles were further evaluated by particle size.
References:
1. Enteshari Najafaadi R, Kazemipour N, Esmaeili A, Beheshti S, Nazifi S. Using superparamagnetic

iron oxide nanoparticles to enhance bioavailability of quercetin in the intact rat brain.BMC
Pharmacol Toxicol. 2018 ;19(1):59
2. K.Vijaya Sri, A.Kondaiah, J.VijayaRatna and A. Annapurna; Preparation and characterization of
Quercetin and Rutin cyclodextrin inclusion complexes. Drug development and industrial
pharmacy. 2007,33(3): 245- 253
3. Vijaya Sri and J.Vijaya Ratna. Formulation and Invitro evaluation of quercetin –CD Tablets. Asian
journal of chemistry,2011,23:4491-4493.
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PREPARATION OF DRUG NANOPARTICLES ESTIMATION BY HPLC
Dr.K.Vijaya Sri
Professor
Date 17/6/2019
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Aim: Drug loaded nanoparticles estimation by High performance liquid chrmatgraphy.
Chemicals :
Quercetin was purchased from Hetero Laboratories Ltd. Acetetonitrile and methanol was
purchased from Merck Chemical Company. PVP-K30, Sodium lauryl sulphate were procured from
commercial sources. The 0.45 μm pump Nylon filter was obtained from Advanced Micro Devices
(Ambala Cantt, India) & whatman no 5 filter paper was obtained from Modern Science lab,
(Nashik, India). Other chemicals used were analytical or HPLC-grade.
HPLC instrumentation and conditions
Chromatographic separation was achieved by using BDS hypersil (250 mm × 4.6 mm, 6
μm) column of maintained at 25 ºC. Isocratic elution was performed using methanol: water:
methanol: water containing 0.1% phosphoric acid 65:35 at a flow rate 1 mL min -1 with UV
detection at 370 nm. The overall run time was 10 min and 5 μL of sample was injected into the
HPLC system.
Preparation of stock solution
Quercetin stock solution (1000 μg mL-1) was prepared by accurately weighing 50 mg of
quercetin in a 50 mL volumetric flask and making up to volume with ethanol.
Working solutions for HPLC injections were prepared from the stock solution in a solvent
mixture of methanol: water containing 0.1% phosphoric acid 65:35 (v/v) (mobile phase).
Solutions were filtered through a 0.45 μm membrane filter prior to injection.
Development of calibration curve
Aliquots of standard quercetin stock solution were taken in different volumetric flasks and
the resultant solution was diluted up to the mark with the mobile phase to obtain a final
concentration of 2,4,6,8,10 and 12 μg mL-1. Each of the solutions was injected using isocratic
130
mode with UV detection at 370 nm and flow rate of 1mL min -1. From the resultant chromatogram
obtained, peak area was used to plot the calibration curve.
Preparation of quercetin loaded ferrous oxide nanoparticles:
Various quantity of Ferrous oxide nanoparticles is dispersed in distilled water and is
sonicated for 10 minutes. Poloxomer 188 is dissolved in ethanol and is added to the magnetite
solution and is stirred at 600 rpm for 2 hours. To this solution add - cyclodextrin dissolved in
ethanol and is stirred at 8000 rpm for 2hrs. This results in the formation of cyclodextrin coated
magnetite nanoparticles. These nanoparticles were filtered and dried in oven. These nanoparticles
were dispersed in water and quercetin dissolved in dimethyl sulphoxide is injected into the solution
and is stirred at 6000rpm for 1hrs which results in the formation of Quercetin loaded ferrous oxide
nanoparticles.
Entrapment efficiency:
Quercetin ferrous oxide nanoparticles are centrifuged at 10,000 rpm for 30 min using
centrifuge. After centrifugation, supernatant is collected and the amount of untrapped drug is
determined by using HPLC. The percentage entrapment efficiency is calculated by the equation
%entrapment efficiency = 𝑥100
Results
The mobile phase methanol: water containing 0.1% phosphoric acid 77:23 (v/v) showed
good separation and good peak symmetry. By the proposed method, the retention time of was
found to be 3.6 minutes. The resulting chromatogram obtained is shown in Figure 1.
Linearity
Linearity was obeyed in the concentration range of 2-12μg/mL and the correlation 0.9998.
The regression equation of quercetin concentration over its peak area ratio was found to be y
=15116X+906.1, where y is the mean peak area and x is the concentration of quercetin (μg/ml).
The calibration curve and chromatograph obtained is shown in Figure 2 and 3 and Linearity data
are shown in Table 1.
131
Table: 1 Standard graph of Quercetin
S.NO Concentration Peak area

(ug/ml)
1 0 0
2 2 312066
3 4 591584
4 6 906028
5 8 1221365
6 10 1513107
7 12 1811128
Figure: Calibration curve of quercetin
132
Table 2: Data of Entrappment efficiency of quercetin loaded ferrous oxide nanoparticles
S.No Formulation % Drug Entrapped

1 F-1
2 F-2
3 F-3
4 F-4
5 F-5
133
References:
1.K.Vijaya Sri, J.VijayaRatna, A. Annapurna, and B.V.V. Ravi Kumar; Reversed -phase HPLC
method for the determination of Quercetin in human plasma. 2009,21(1):101-104.
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FOOD CONTAIN ADULTERANTS ESTIMATION
Dr.K.Vijaya Sri
Professor
Date 18/6/2019
150
Aim: To perform the qualitative tests for the samples and identify the food adulterants.
Chemicals: Milk, Honey, Tincture iodine solution, conc sulphuric acid, formalin
Principle:
 Food Adulteration refers to the process by which the quality or the nature of a given food is
reduced through addition of adulterants or removal of vital substance.
 Food adulterants refer to the foreign and usually inferior chemical substance present in food
that cause harm or is unwanted in the food.
 Basically, during food adulteration, small quantity of non-nutritious substances are added
intentionally to improve the appearance, texture or storage properties of the food.
Methods of food adulteration:
1. Mixing: Mixing of clay, stones, pebbles, sand, marble chips, etc.

2. Substitution: Cheaper and inferior substances being replaced wholly or partially with good
ones.
3. Concealing quality: Trying to hide the food standard. E.G. adding captions of qualitative
food to low quality for selling.
4. Decomposed food: Mainly in fruits and vegetables. The decomposed ones are mixed with
good ones
5. Misbranding/ False labels: Includes duplicate food stuffs, changing of manufacture and
expiry dates.
6. Addition of toxicants: adding non-edible substances like argemone in mustard oil, low
quality preservatives, colouring agents, etc
151
Methods for Detection of Common adulterants in Milk
S.No Food Adulterant Method for Detection Remarks

Article
1 Milk Water The presence of water can be by putting a There is no
drop of milk on a polished slanting water in milk.
surface. The drop of pure milk either or
flows slowly leaving a white trail behind
it, where as milk adulterated water will
flow immediately without leaving mark.
starch Add a few drops of tincture of Iodine or blue colour
Iodine solution. Formation of blue colour formation.
indicates presence of starch
Formalin Take 10ml of milk in test tube and add There is no
5ml of conc sulphuric acid from the sides preservative.
of the wall without shaking. If a violet or
blue ring appears at the intersection of
two layers then it shows presence of
formalin.
Detergent Shake 5-10ml of sample with an equal Detergent is
amount of water lather indicates the absent.
presence of detergent.
S.NO Food Article Adulterant Method for detection Remarks

2 Iodized salt Common salt Cut a piece of Potato ,add salt and Blue colour is
wait minute and add two drops of observed.so
lemon juice .If iodized salt blue iodized salt is
colour will develop. In case of present.
common salt ,there will be no blue
colour.
3 Honey Sugar A cotton wick dipped in pure The given
solution honey when lighted with a match sample is pure.
stick burns and shows the purity of
honey.If adulterated ,the presence
of water will not allow the honey
to burn,if it does ;it will produce a
cracking sound.
4 Sugar Chalk Dissolve 10gm of sample in a Chalk powder is
powder glass of water,allow settling,Chalk absent.
will settle down at the bottom.
Urea On dissolving in water it gives a There is no
smell of ammonia. ammonia smell.
Yellow Take 5ml in a test tube from the There is no pink
colour solution and add a few drops of colouration.
conc.Hcl. A pink colour in lower
acid layers shows the presence of
non –permitted colour.
152
Report: The Samples for which adulterant tests have done i.e Milk, Sugar, Honey and Iodized salt
are pure.
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HANDLING AND MAINTAINING THE DRUG STORES IN HOSPITAL –MEDICATION

PRESCRIPTION ERRORS, ADR REPORTS
BY
DR.B.V.S.LAKSHMI
19/6/2019
180
A CASE STUDY LOOKING AT ADVERSE DRUG REACTIONS.
ADVERSE DRUG REACTIONS DETECTION, REPORTING AND MONITORING
Introduction
An adverse drug reaction (ADR) is an unwanted, undesirable effect of a medication that occurs
during usual clinical use. Adverse drug reactions occur almost daily in health care institutions and
can adversely affect a patient’s quality of life, often causing considerable morbidity and mortality.
Much attention has been given to identifying the patient populations most at risk, the drugs most
commonly responsible, and the potential causes of ADRs. An increase in the number of drugs on
the market, an aging population, and an upward trend in polypharmacy are contributing factors to
the prevalence of ADRs worldwide. Adverse drug reactions may cause patients to lose confidence
in or have negative emotions toward their physicians and seek self-treatment options, which may
consequently precipitate additional ADRs. Around 5% of all hospital admissions are the result of
an ADR, and around 10%– 20% of inpatients will have at least one ADR duringtheir hospital stay.
The actual incidence of ADRs may be even greater because some ADRs mimic natural disease
states and may thus go undetected and/or unreported.
Although some ADRs present as minor symptoms, others are serious and cause death in as many
as 0.1%–0.3% of hospitalized patients. Adverse drug reactions should be quickly identified and
managed to limit their detrimental effects on the patient.
Pharmacovigilance involves the study of drug-related injuries and making warning or withdrawal
recommendations for pharmaceutical agents; it encompasses the detection, assessment,
understanding, and prevention of ADRs. Pharmacists play a vital role in every step of the
pharmacovigilance process, which can prevent patients from undergoing unnecessary procedures
or taking unwarranted drugs. In addition to preserving the safety and quality of life for the patient,
pharmacovigilance can represent a cost savings to the patient and the health care institution. By
reporting known or suspected ADRs, pharmacists, other health care practitioners, and patients can
assist in identifying patterns and trends, which may lead to increased regulatory scrutiny or even
the withdrawal of drugs that do not have a favorable risk-benefit ratio.
181
Case study-1
A 67 year old woman with an extensive rash is referred urgently to hospital. The rash started on
the backs of her hands and spread very quickly to the arms, trunk, neck, and face. The lesions
consist of concentric rings with frank blistering in some areas. Lesions have also started to appear
on her lips and inside her mouth. Her medications include: ramipril 10mg once daily, simvastatin
40mg at night, aspirin 75mg once daily, metformin 1g twice daily, gliclazide 40mg each morning.
She was started on aspirin 5 years ago following a stroke. At about the same time, she was
diagnosed with type 2 diabetes and has been taking metformin, ramipril, and simvastatin
for over 4 years. She was prescribed gliclazide during her annual diabetes review 2 months ago.
The patient denies taking any over-the-counter medicines or herbal remedies. She has not made
any significant changes to her diet and there is no history of recent infection.
On admission:
Blood pressure = 127/75 mmHg
Body Mass Index = 26kg/m2
HbA1c = 8.0% (64mmol/mol)
Her U + Es, renal, and liver function are normal.
Case study analysis
1. Which drug is most likely to be causing erythema multiforme?

According to the side-effects under the prescribing notes for sulphonylureas, hypersensitivity
reactions can occur, usually in the first 6–8 weeks of therapy. They consist mainly of allergic skin
reactions which progress rarely to erythema multiforme. The monograph for ramipril, also includes
erythema multiforme as a side-effect. The time at which erythema mutiforme occurred in this
patient is closely related to the time from when gliclazide was started and characteristic of
hypersensitivity reactions with sulphonylureas.
2. How should this adverse drug reaction (ADR) be managed?
This patient has suffered a major manifestation of erythema multiforme accompanied by mucosal
involvement after starting gliclazide. Gliclazide should be stopped and the reaction should be
clearly documented in the patient’s medical record and allergy history to prevent recurrence. If the
reaction does not subside after stopping gliclazide, other causes for the reaction should be
182
considered. Treatment to ameliorate the symptoms of erythema multiforme can be provided as
necessary.
3. Should this ADR be reported through the Yellow Card scheme to the Medicines and
Healthcare products Regulatory Agency (MHRA)?
According to Adverse Reactions to Drugs, the MHRA requests that all serious suspected reactions
to established drugs in adults are reported even if the effect is well recognised. Serious reactions
include those that are fatal, lifethreatening, disabling, incapacitating, or which result in
hospitalisation, or prolong hospital stay.
Although erythema multiforme is a recognised side-effect of sulphonylureas that usually resolves
spontaneously when the drug has been discontinued, the ADR should be reported in this case
because it has led to hospitalisation of the patient. If you are not sure whether to report an ADR or
whether someone else has reported it, you should still report the ADR. Only a suspicion of an
ADR is needed to report. ADRs should be reported through the Yellow Card Scheme.
Alternatively, prepaid Yellow The patient’s gliclazide therapy is stopped and the erythema
mutiforme resolves.
She is started on sitagliptin 100mg once daily.

She is readmitted 3 weeks later with nausea, vomiting, and severe, persistent abdominal pain
which radiates to the back. Symptoms started 6 hours ago.
Serum amylase = 610iu/litre (0–140)
Serum lipase = 530iu/litre (0–160)
Urea = 7.4mmol/litre (2.5–6.5)
Calcium = 2.4mmol/litre (2.2–2.6)
Blood glucose = 11mmol/litre (3.5–10)
White blood cell count = 12 x 109/litre (4–11 x 109)
Bilirubin = 10micromol/litre (5–17)
Serum aspartate aminotransferase = 45iu/litre (0–35)
eGFR = 105mL/minute/1.73m2
Blood pressure = 120/70mmHg
X-ray of the abdomen shows no evidence of gallstones or pancreatic calcification.
Abdominal ultrasound shows no evidence of pancreatic necrosis or dilatation of intrahepatic ducts.

A diagnosis of acute pancreatitis is made but causality is not known.
183
The patient is nil by mouth and placed on supportive therapy including parenteral analgesia,
intravenous fluids, and intravenous insulin.
4. Which drug is most likely to be causing acute pancreatitis?
According to the prescribing notes for statins, pancreatitis can occur very rarely. ACE inhibitors
can also cause pancreatitis. The monograph for sitagliptin, includes pancreatitis as a side-effect
that is also reported. This episode of pancreatitis seems to have coincided with initiation of
treatment with sitagliptin.
5. How frequently has pancreatitis been reported with sitagliptin?
Occasionally a rare side-effect might be listed first if it is considered to be particularly important

because of its seriousness. The frequency of side effects is described as:
Side-effects very common (greater than 1 in 10) and common (1in 100 to 1in 10);
Less commonly (1 in 1000 to 1 in 100);
rarely (1 in 10 000 to 1 in 1000);
very rarely (less than 1 in 10 000);
also reported, frequency not known
The frequency of pancreatitis with sitagliptin is not known. Pancreatitis was identified as a side-
effect with sitagliptin from spontaneous reports received post-marketing. It is not possible to
calculate the frequency of side-effects from spontaneous reporting rates alone as neither the
number of patients using the drug nor the extent of under-reporting is known.
6. Why is Januvia® (the proprietary preparation of sitagliptin) assigned the▼symbol?
According to Adverse Reactions to Drugs, the black triangle symbol ▼ identifies newly licensed
medicines that are monitored intensively by the MHRA. Only limited information is available
from clinical trials on the safety of new medicines. Further understanding about the safety of
medicines depends on the availability of information from routine clinical practice.
7. Should this suspected ADR with sitagliptin be reported through the Yellow Card
Scheme?
According to Adverse Reactions to Drugs, for medicines showing the black triangle symbol (e.g.
sitagliptin), all suspected reactions (including those considered not to be serious) should be
reported through the Yellow Card Scheme. An adverse reaction should be reported even if it is not
certain that the drug has caused it, or if the reaction is well recognised, or if other drugs have been
given at the same time.
184
8. The patient makes a good recovery and recommences her usual oral medications;
however sitagliptin is not restarted. How should this patient’s type 2 diabetes be
treated?
This patient is at high risk of complications because she has a long history of type 2 diabetes, she
has a history of stroke, and she seems to have been unable to tolerate two oral antidiabetic drugs.
She should continue to take metformin and she can also be prescribed a long-acting insulin at
bedtime.
Case study-2
D.W. is a 9-year-old boy with osteosarcoma, which is being treated with ifosfamide and etoposide.
He has no history of allergy. After two courses of chemotherapy, D.W. develops an upper arm
deep venous thrombosis because of the chemotherapy. He is admitted to the hospital and initiated
on heparin, omeprazole, and prophylactic antibiotics (piperacillin/tazobactam). On the third day of
treatment, the swelling and pain in his upper arm have decreased significantly.
1. Which one of the following best classifies D.W.’s chemotherapy adverse drug reaction (ADR)?
A. Type A.
B. Type B.
C. Type C.
D Type D.
2. Which one of the following would be best to use to document D.W.’s ADR?
A. Internal hospital quality reporting system.
B. FDA MedWatch.
C. Quantros MEDMARX international reporting system.
D. The Institute for Safe Medication Practices (ISMP) medication error reporting program.
3. On the third hospital day, D.W.’s platelet count has dropped by 50% from baseline, and his
alkaline phosphatase has increased to twice the normal value. He also begins to have some oozing
of blood from his central catheter line site. The team agrees that these events are ADRs and asks
you to narrow the suspected drugs to two agents. Which two-drug option is most likely causing
these ADRs in D.W.?
A. Omeprazole and heparin.
B. Heparin and ifosfamide.
C. Ifosfamide and omeprazole.
D. Omeprazole and piperacillin/tazobactam.
185
Case study-3
F.G. is a 68-year-old man (weight 78 kg) who presents to the emergency department with signs of
an ischemic stroke. Initial examination reveals a patient in atrial fibrillation with poor nutritional
status. Further examination reveals a patient with normal renal function for age but severe carotid
artery stenosis. F.G. is deemed a poor surgical candidate; he is stabilized in the neurologic
intensive care unit and, 3 days later, is transferred to a regular floor. A feeding tube is placed
before discharge. At discharge, the patient is prescribed enoxaparin 80 mg subcutaneously every
12 hours. Four days after discharge, F.G. is readmitted with a severe abdominal hematoma and
significant drop in hemoglobin. Enoxaparin is discontinued with improvement in the hematoma
and the hemoglobin concentration.
1. Which one of the following risk factors is most likely to be a contributing cause when assessing
a possible enoxaparin ADR in F.G.?
A. Atrial fibrillation and poor nutritional status.
B. Incorrect enoxaparin dosage.
C. Feeding tube placement.
D. Recent stroke event.
2. Using the Liverpool adverse drug reaction casualty assessment tool, which one of the following
is the mostly likely probability of an enoxaparin ADR in F.G.?
A. Unlikely.
B. Unassessable.
C. Possible.
D. Probable.
Case study- 4
K.L., a 34-year-old woman with end-stage cancer, is being cared for by the palliative pain
management service. She has received her home doses of hydromorphone 8 mg orally every 4
hours since her admission 3 days ago. On the morning of hospital day 4, K.L. has “pain and
twitching all over.” The resident physician asks you to evaluate K.L. for an ADR from opioid
analgesics.
1. Which one of the following is the best first step to determine whether K.L. experienced an ADR
to opioid analgesics?
A. Replace hydromorphone with other opioids to see whether that changes her symptoms.
B. Add another opiate in equal doses, and see whether her symptoms change.
186
C. Review the medical record to determine any temporal relationships associated with a potential
ADR.
D. Use both the Naranjo algorithm and the Liverpool adverse drug reaction causality assessment
tool to score the potential ADR.
2. The nurse on K.L.’s unit decides to complete an ADR report. The nurse also notes that this is the
third patient she has seen with opioid neurotoxicity in the past week. After completing a reporting
form for review by the hospital’s chief medical officer, this ADR should also be reported to which
reporting system?
A. Internal hospital quality reporting system.
B. FDA MedWatch.
C. Quantros MEDMARX international reporting system.
D. ISMP medication error reporting program.
3. Which one of the following best describes the ADR classification of the neurotoxicity K.L.
experienced from hydromorphone?
A. Augmented.
B. Bizarre.
C. Chronic.
D. Delayed.
187
Drug interactions
Case Study 5
A 39-year-old man was admitted to the hospital with new onset seizures and a 2-week history of
blurred vision. After a work-up, diagnoses included advanced HIV infection, CMV retinitis, and
probable toxoplasmic encephalitis. Sulfadiazine and pyrimethamine were started for treatment of
toxoplasmic encephalitis. Phenytoin was initiated at a dosage of 300 mg daily for seizure. The
patient also received valganciclovir for treatment of CMV retinitis. After experiencing another
seizure while hospitalized, the phenytoin dose was increased to 300 mg twice daily on 19
September. Antiretroviral therapy with efavirenz (EFV), emtricitabine, and tenofovir was started
on 22 September. A plan was made to monitor both phenytoin and EFV plasma concentrations to
ensure adequate safety and therapeutic efficacy.
On 30 September, the first measurement of EFV plasma concentration was obtained, with a result
of 0.34 µg/mL. Target EFV concentrations of 1 – 4 µg/mL or >2.2 µg/mL have been proposed in
the literature, while concentrations <1.1 µg/mL have been associated with treatment failure.
Because EFV has a long half-life (40-55 hours), it was recognized the plasma level could be low
because the sample was obtained prior to steady-state.
1. Other than obtaining the level before achieving steady-state, what are other potential
explanations for the low EFV exposure? Consider the patient’s concurrent medications.
There was concern that phenytoin was the cause of the lower-than-expected EFV concentrations.
As persistently low EFV exposure could put the patient at risk for treatment failure and
development of virologic resistance, it was decided to rapidly taper the phenytoin and commence
therapy with levetiracetam —an anticonvulsant without CYP modulation potential. In the
meantime, the EFV dosage was increased from 600 to 800 mg/day under the assumption that CYP
induction could continue for up to 7-14 days after discontinuation of phenytoin. Prior to
implementation of the EFV dosage increase, a 2nd test result returned an EFV concentration of
0.58 µg/mL.
The patient received his last dose of phenytoin on 22 October. A third measurement of EFV level
was obtained on 9 November, after what was predicted to be sufficient time for reversal of
phenytoin’s enzyme induction. The result was an EFV concentration of 2.5 µg/mL. At that time
the EFV dosage was reduced back to the recommended dose of 600 mg/day. On 16 November, the
188
patient had an HIV RNA of 189 copies/mL and a CD4 cell count of 100 cells/mm3, which
indicated continued virologic response to antiretroviral therapy. The patient’s latest measured
EFV, after 3 weeks at a dosage of 600 mg/day, was 2 µg/mL.
Phenytoin levels measured after EFV therapy initiation showed a gradual increase, despite a stable
phenytoin dosage and no changes in medications.
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Health Camp
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CHILDRENS AWERNESS CAMP
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20/6/19 nacto
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NACTO INDUSTRY VISIT
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1

PREPARATION OF SELECTIVE FAST DISSOLVING TABLETS BY NATURAL POLYMERS

Dr.Satya Bratha Banja

Malla Reddy college of pharmacy

Talc 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5

Hausners ratio = tapped / bulk

where  = Angle of repose

r = Radius of the base of the conical pile

For Angle of Bulk density Tapped Carr’s index Haunser

EVALUATION OF PREPARED FAST DISSOLVING TABLETS USING NATURAL

Evaluation of tablets (Post compression parameters)

Water absorption Ratio :

Formulation Hardness(kg/cm2) Thickness(mm) Friability (%) Weight variation(mg)

F1 2.6±0.02 3.6±0.07 0.30 151±0.4

F2 2.8±0.10 3.7±0.06 0.22 148±0.6

F3 2.1±0.04 3.8±0.03 0.57 149±0.4

F4 2.6±0.06 3.8±0.05 0.41 152±0.6

F5 2.5±0.06 3.8±0.08 0.26 148±0.2

F6 2.8±0.05 3.7±0.06 0.422 149±0.8

F7 2.4±0.07 3.5±0.06 0.38 150±0.2

F8 2.6±0.05 3.2±0.01 0.35 152±0.8

F1 77±0.25 10±0.48 117 100±1.12 99

F2 62±0.87 8±0.95 112 109±1.54 101

F3 71±0.99 10±0.35 90 168±1.25 98

F4 57±1.25 16±0.85 40 52±1.14 102

F5 73±1.35 14±0.92 197 79±1.04 100

F6 66±1.15 14±0.95 92 127±0.89 98

F7 68±0.98 9±0.58 60 73±0.54 97

F8 75±0.88 10±0.54 80 82±1.08 99

PROTOCOL FOR SANDWICH ELISA

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for

Plate Sealers: (R&D Systems, Catalog # DY992).

Wash Buffer: 0.05% Tween® 20 in PBS, pH 7.2-7.

Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 μm filtered(R&D Systems, Catalog #

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color

Reagent B (Tetramethylbenzidine) (R&D Systems, Catalog # DY999).

Stop Solution: 2 N H2SO4(R&D Systems, Catalog # DY994).

WESTERN BLOT PROTOCOL

Loading Buffer (2X Laemmli buffer)

Running Buffer (1X running buffer)

Handling of HPLC, FT-IR and drug estimation by HPLC and FT-IR

Figure: Chromatogram of Bicalutamide Formulation

Formulation Label claim Amount found % Purity %RSD

Casodex (50mg) 50mg 49.97mg 99.9% 0.771

PREPARATION OF SELECTIVE DRUG LOADED NANOPARTICLES

Nanoparticles (NPs) are at the forefront of rapid development in nanotechnology. Their

1.3 Surface functionalisation of Iron oxide nanoparticles:

Chemicals: Ferrous oxide nanoparticles, Quercetin, Ethanol, poloxomer 188,  -cyclodextrin,

Cyclodextrins are cyclic (α-1,4) linked oligosaccharides of α-D-glucopyranose, containing

is generally assumed that the solubilization proceeds through inclusion complexes.

Preparation of quercetin loaded ferrous oxide nanoparticles:

Various quantity of Ferrous oxide nanoparticles is dispersed in distilled water and is

Table: 1 Formulation of Quercetin loaded ferrous oxide nanoparticles:

Ingredients F-1 F-2 F-3 F-4 F--5

PARTICLE SIZE , ZETAPOTENTIAL AND POLYDISPERSITY INDEX:

Figure : Particle Size Graphs

1. Enteshari Najafaadi R, Kazemipour N, Esmaeili A, Beheshti S, Nazifi S. Using superparamagnetic

PREPARATION OF DRUG NANOPARTICLES ESTIMATION BY HPLC