SUBMITTED BY: Benish Baseerat

SUBMITTED TO: Dr. Maqsood ur Rehman

SUBJECT: PQM

TOPIC: Bioassays of official Drugs; Insulin

Heparin
Oxytocin
Antibiotics.

ROLL NO: 513

CLASS NO: 05

SEMESTER: 8TH

DEPARTMENT: PHARMACY

SESSION: 2017 to 2022

DATE: 04/06/2021

University of Malakand chakdara Dir Lower

 Insulin

 Introduction :
Insulin was discovered in 1921, which helped millions suffering from type-1 diabetes.
• It is a hormone made in pancreas, special cells call “beta cell” produce insulin.
• When a person suffer from type-1 diabetes the capability of these cells is lost.
• Most people now a days use human insulin or insulin analogs.
• it is produced by bacteria (lilly) or by yeast (Novo-Nordisk) by using genetic engineering.

 The most prominent manifestation of insulin activity (an abrupt decrease in blood
glucose)was the basis for biologic assay from the the time of its first clinical use.
 The liquid chromatography was a sophisticated physicochemical method( e.g liquid
chromatography) to measure insulin potency quantitatively has resulted in a more
accurate and precise compendial test for insulin and insulin products.
 However, the bioidentity of insulin and insulin products cannot be assessed by these
methods.
 Thus a bioidentity test is developed.

 Mechanism of action:
Every pancreatic islet contains ~1,000 endocrine cells of which 75% are insulin-producing beta-
cells.
• Insulin is synthesised as pro-insulin in the endoplasmic reticulum and is processed to
the biologically active form inside the secretory granules.
• The beta-cell is electrically excitable and uses changes in membrane potential to couple
variations in blood glucose to trigger insulin secretion.
• The beta-cell contains about 20 different ion channels proteins
• Two types of ion channels are particularly important for the initiation of insulin
secretion.
• The KATP-channels are active at low glucose concentrations, because of the high
intracellular ADP levels.

 Bioassay of insulin

Principle:
The principle of insulin injection is estimated by comparing the hypoglycemic effect it produce
with that produced by a standard preparation of insulin under the conditions of a suitable
method of assay.

Requirements :
Test sample solution.
Std solution.
Standard preparation and unit:
It is pure, dry and crystalline insulin. One unit contains 0.04082 mg.
• Preparation of standard solution:
Accurately weigh 20 units of insulin and dissolve it in normal saline. Acidify it with HCl to pH
2.5. Add 0.5% phenol as preservative. Add 1.4% to 1.8% glycerin. Final volume should contain
20 units/ml. Store the solution in a cool place and use it within six months.
• Preparation of test sample solution:
The solution of the test sample is prepared in the same way as the standard solution.

 The Rabbit Method

Selection of rabbits: They should be healthy, weighing about 1800-3000 gms. They should then
be maintained on uniform diet but are fasted for 18 hrs. before assay water is withdrawn
during the experiment.

• Principle
– Potency of a sample of insulin injection is estimated by comparing the
hypoglycemic effect it produces with that produced by a standard preparation of
insulin under conditions of a suitable method of assay
• The Rabbit Blood Sugar Method—Quantitative is used to
– Determine the potency of Insulin Reference Standards, for the validation of the
stability of new insulin preparations,
– Determine the specific activities of insulin analogs.
• Diluent
– Prepare an aqueous solution containing
• 0.1%–0.25% (w/v) of either cresol or phenol
• 1.4%–1.8% (w/v) of glycerin
• Sufficient hydrochloric acid to produce a pH between 2.5 and 3.5, unless
otherwise directed in the individual monograph
• Standard stock solution
– Prepare a solution containing 40 USP insulin units/ml of USP insulin RS of the
appropriate species in Diluent
– For Insulin of mixed Bovine and Porcine species, Prepare a solution in a Diluent,
containing;
• 34.8 USP Insulin units/ml of beef
• 5.2 USP Insulin Units/ml of Pork
– Having PH between 2.5 and 3.5, unless otherwise directed in the individual
monograph
• Store in cold place, avoiding from freezing
• Use within 6 months
• Standard solutions
– Dilute portions of the Standard stock solution with Diluent to make two solutions
 • Standard solution 1
• Contain 1.0 USP Insulin Unit/mL
• Standard solution 2
• Contain 2.0 USP Insulin Units/mL
• Sample stock solution
– Proceed as directed in the Standard stock solution
– Use a suitable quantity of the preparation under test
– The Sample stock solution contains about 40 USP Insulin Units/mL.
• Sample solutions
– Dilute portions of the Sample stock solution with Diluent to make two dilutions
of the preparation under test
• Sample solution 1
• Contain 1.0 USP Insulin Unit/mL
• Sample solution 2
• Contain 2.0 USP Insulin Units/mL
– In the case of neutral insulin injection, adjust to a pH of 2.5–3.5 before making
the dilutions
• Doses of the solutions to be injected
– On the basis of trial or experience, Select the dose of the dilutions to be injected,
– The volume will be between 0.30 and 0.50 mL.
– For each animal, the volume of the Standard solution is the same as that of the
Sample solution.
• Selection of animal
– Select suitable, healthy rabbits each weighing NLT 1.8 kg.
– Keep the rabbits in the laboratory for NLT 1 week before use in the assay,
maintaining them on an adequate uniform diet, with water available at all times.
• Preparation of animal
– Divide the rabbits into four equal groups of preferably 3 rabbits each.
– On the preceding day, approximately 20 h before the assay, provide each rabbit
with an amount of food that will be consumed within 6 h.
– Follow the same feeding schedule before each test day.
– During the assay, withhold all food until after the final blood specimen is taken.
– Handle the rabbits with care to avoid undue excitement.
• Initial Blood Test
– A sample of blood is taken from the marginal ear vein of each rabbit.
– Presence of reducing sugar is estimated per 100 ml of blood by a suitable
chemical method. This concentration is called ‘Initial Blood Sugar Level’.
• Injection of Insulin
• Inject subcutaneously the doses indicated in the following Table
• The second injection being made on the day after the first injection, or
NMT 1 week later.
• The time between the first and second injections is the same for all
rabbits.
 GROUP FIRST INJECTION SECOND INJECTION

1 Standard solution 2 Sample solution 1

2 Standard solution 1 Sample solution 2

3 Sample solution 2 Standard solution 1

4 Sample solution 1 Standard solution 2

 Final Blood Test

– At 1 h ± 5 min and 2½ h ± 5 min after the time of injection, obtain from each
rabbit a suitable blood specimen from a marginal ear vein.
– Blood can also be collected effectively from the central auricular artery
– Blood sugar is determined again. This is known as ‘Final Blood Sugar Level’.
– The potency of a test sample is estimated by comparing the hypoglycemic effect
of the sample with that of the std. preparation of insulin.
 Interpretation
– If the potency value obtained is NLT 15 USP Units/mg, the Bio-identity Test
requirement is met.
– If the potency value is less than 15 USP Units/mg, repeat the test using eight
more rabbits.
– If the average potency of the two sets of tests is NLT 15 USP Units/mg, the
requirement of the test is met.

 Mouse Method
• Mice show characteristic convulsions after s.c. inj. of insulin at elevated temperatures.
• The percentage convulsions produced by the test and standard preparations are
compared.
Experimental procedure
 At least 96 mice weighing between 18-22 gms. of the same strain are used.
 They should be maintained on constant diet.
 Prepared for the Assay by deprivation of food for 2 hours prior to the
experiment.
 Standard and sample dilutions
 Standard solution is prepared by dissolving a quantity of standard preparation
with sterile saline solution and acidified with HCl to PH 2.5 and containing
sufficient of a substance to prevent the growth of moulds
 Should be stored at a temperature between 2°C and 10°C, protected from
freezing
 Further dilutions of standard are prepared as;
 Std dilution I (contain 0.064 units/ml)
  Std. dilution II (contain 0.096 units/ml)
 Similarly, test sample solutions are also prepared.
 Mice are divided into 4 groups each containing 24 mice
 S.C injections, of two dilutions of standard preparations and two dilutions of the sample
being tested, are given
 24 mice
 Standard dilution (0.064 units/ml)
 24 mice
 Standard dilution (0.096 units/ml)
 24 mice
 Test sample dilution
 24 mice
 Test sample dilution

 Mice after injections, are put in an air incubator, with transparent front, at uniform
temperature between 29°C and 35°C and observed for 1½ hr.
 The number of mice, which convulse or die, is recorded.
 These mice usually convulse severely but failure of the animal to upright itself when
placed on its back, should as well be considered as convulsion.

 Interpretation
 The result is calculated by standard statistical methods (Refer PP 1973 for
details)
 The death or convulsion in the test animal is due to the rapid onset of
Hypoglycaemia
 Normal sugar levels are 1000 µg/ml (100 mg)
 Death or convulsion may occur at any time when the blood sugar level falls
below 350 µg/ml.

Heparin

Introduction
• Heparin is a highly-sulfated glycosaminoglycan of natural origin. • It is also one of the oldest
drugs still in widespread use – Heparin, along with vitamin K antagonists, have been the main
anticoagulant drugs for more than 70 years.

Bioassay of Heparin
• Heparin: anticoagulant
• Preparation: Injection
• Appearance: White or almost white, hygroscopic nature
• Solubility: Freely soluble in water
• Identification:
• delays the clotting of recalcified citrated sheep plasma
• Nuclear magnetic resonance spectrometry
 • Assay potency: 90-111% of stable potency
• Unit: I.U., (One mg of heparin = 130 I.U.)
Principle:
•The potency of heparin sodium is determined in vitro by comparing the concentration
necessary to prevent the clotting of sheep or goat or human plasma with the concentration of
the standard preparation of heparin sodium.
•Its is necessary to give the same effect under the same conditions of the method of assay.
Requirements:
•Standard heparin (for assay)
•Plasma (form sheep or goats, human)
•Solution of standard solution
•Test drug/ heparin

Plasma preparation

• The blood is placed into a test-tube with 8 % w/v sodium citrate (ratio of blood
to sodium citrate is 19: 1)
 • The above is immediately mixed by gentle agitation and inversion of the vessel
The mixture is centrifuged and the separated plasma is pooled out. 0.2 ml of 1%
w/v solution of CaCl2 is added to 1ml of the pooled plasma.
• This plasma becomes suitable if clot forms within 5 min. Prepared plasma
Sodium citrate
• The potency of standard heparin is determined in relation to the International
Standard stated by the World Health Organization.
• Test solution:
•Accurately about 25 mg of the test sample is weighed
•Sufficient saline is added to give the concentration of 1 mg/ml

Procedure

• In clean test-tubes, graded amount of the solution of standard preparation is added (the largest
dose not exceed 0.8 ml)
• Sufficient volume of saline is then added to make total volume of 0.8 ml and add 1 ml of
prepared plasma to each test tube
• 0.2 ml of 1 % w/v solution of calcium chloride is added, the time is noted and each tube is
immediately stoppered with a suitable stopper
• The contents are mixed by inverting three times in such a way that the entire inner surface of
the tube is wet
• The sample of heparin to be tested is diluted to the same concentration corresponding to that
of the standard
• The procedures are repeated as mentioned for the standard heparin

NB: The entire process of preparing and mixing the tubes of both the solution of standard preparation
and the test solution must be completed within 20 minutes after the addition of the prepared plasma

• Exactly one hour after the addition of the calcium chloride solution, the extent of clotting is
determined in each tube, recognizing three grades between zero and full clotting.

Results:

S1 = T1

• If the degree of clotting observed in the series of dilutions of the solution of standard
preparation lies between that observed in two of the series of dilutions of the sample being
examined, the potency of the latter is estimated
• If there is no such correspondence between the degrees of clotting produced by the solution of
standard preparation and any of the dilutions of the sample being examined, new dilutions of
the latter are prepared and assay is repeated
Limits of errors:

• The Limit of errors of estimated potency (P=0.99) are in the range of –90-110 with three
determination –92-108 with four determination

Conclusion

• Heparin bioassays are performed to monitor and adjust standard heparin.

• •This is done in order to evaluate the concentration of heparin in blood and helps doctors to
monitor therapy.
• •If concentrations are within an established therapeutic interval and the person is doing well
clinically i.e. there is no clotting, excessive bleeding, or other complications – then the dosage is
considered appropriate.

Antibiotics

“Antibiotic” is from antibiosis, meaning against life.

Substances derived from a microorganism or produced synthetically (Sulfonamides & Quinolones) to kill
or suppress the growth of other microorganisms.

Bioassay of Antibiotics:

principle

The microbiological assay of an antibiotic is based upon a comparison of the inhibition of growth of
micro-organisms by measured concentrations of the antibiotics under examination with that produced
by known concentration of a standard preparation of the antibiotic having a known activity.

Requirenments

• Culture mediaMedia has to be prepared for the specified test organism for the ingredients listed
in IP according to the individual requirement of the test organism.

Preparation of the test

• Standard preparation and units of activityA Standard Preparation is an authentic sample of the
appropriate antibiotic for which the potency has been precisely determined by reference to the
appropriate international standard. The Potency of the standard preparation may be expressed
in International Units or in μgper mg of the pure antibiotic

•Preparation of the standard solution

• The stock solution is prepared by dissolving a quantity of the Standard Preparation of a given
antibiotic as per IP
•Preparation of the sample solution

• From the information available for the substance under examination, a stock solution is
prepared as specified in IP

•Test organism

• The test organism for each antibiotic is listed in Table, together with its identification number in
the American Type Culture Collection (ATCC)

•Temperature control

• Thermostatic control is required at several stages of microbial assay when culturing a micro
organism and preparing its inoculum and during incubation in a plate assay.
• Closer control of the temperature is required during incubation in a tube assay.

Methods

A. Cylinder plate or cup plate method

• A previously liquefied medium with the required quantity of microbial suspension is inoculated
• The suspension is added to the medium at a temperature between 40-50 degree and inoculated
medium is immediately poured
• The solution are applied to the surface of the solider medium in sterile cylinder or in ager
cavities
• They are incubated for about 18 hours at the temperature indicated
• The quantities estimation of antibiotic is done by accurately measuring the diameter or areas
of the circular inhibition zones .
B. Turbidimetric or Tube Assay method •
• Advantage-shorter incubation period for the growth of the test organism(usually 3 to 4
hrs)
• Disadvantage-The presence of solvent residues inhibitory substances affects more.
• Not recommended for cloudy or turbid preparation.
• Five different concentration of the standard solution are prepared for preparing the
standard curve.
• 1mm of each concentration of the standard solution of the sample solution are placed in
each of the tubes in duplicate at 9 ml of nutrients medium previously seeded with the
appropriate test organism at to each other
Three control tubes are prepared

• One containing the inoculated culture medium(culture control)

• Another one treated immediately with 0.5 ml of dilute formaldehyde solution(blank)
• Third containing uninoculatedculture medium.
• All the tubes are placed in an inocubatorand maintain at the specified temperature for 3 to 4
hour.
• The growth of the test organism is measured by determining the absorbance at 530 nm of its
against the blank.
• The standard calibration card is prepared and the absorbance obtained for the sample is plotted
on it to obtain the concentration of the test antibiotic.

Oxytocin

Introduction

• Oxytocin is a peptide hormones and Neuropeptide.

• Oxytocin is normally produced by the paraventicular nucleus of the hypothalamus and released
by the posterior pituitary.
• Oxytocin is a natural hormone that causes the uterus to contract.
• Oxytocin is used to induce labor or strengthen labor contractions during childbirth, and to
control bleeding after childbirth.
• Oxytocin is also used to stimulate uterine contractions in a woman with an incomplete or
threatened miscarriage.
Bioassy of oxytocin
• The potency of oxytocin injection is determined by comparing its activity with that of the
standard preparation of oxytocin under the conditions of the following method of assay.

PRINCIPLE:

Potency is determined by comparing its activity

– Depression of BP
– Contraction of Uterus
– Milk Ejection Pressure
– Vasopressor activity with standard preparation of oxytocin

STANDARD PREPARATION

– Consisting free dried synthetic oxytocin peptide with human albumin citric acid (12.5 units)
METHOD-A (Depression of the BP in chicken) Test Animals: Cockerel (young male chicken), 1.2 - 2.3
Kg, Healthy

Anaesthesised cock-prolonged & constant high B.P

Expose gluteus primus muscle(thigh) & remove politeal artery & crural vein.

Cannulate the popliteal artery & record B.P response

Cannulate the crural or brachial vein.

Prepare std soln with saline. Inject 0.1 - 0.5ml

Inject 2 doses of std soln into cannulate vein is record B.P response

Dose should cause decrease in B.P (reqd. dose between 20-100mUnits)

Interval bween 2 injection, bween 3-10mins depend on rate @ which B.P return normal

Dil. test preparation with saline so as to get same response as standard

The ratio between standard & test should be equal

If animal rapidly becomes insensitive to repeated injection the soln another must used.

Measure all responses are calculated result of the assay by std statistical method.
METHOD—B: (By contraction of the rat uterus):

• Test animals: Female rat 120 – 200g Inject 100ug of oestradiol benzoate IM into female
rat before the assay
• Immediately before assay confirm by vaginal smear that rate in oestrus or pre oestrus.
• Kill rat & suspend one horn of uterus in organ bath containing a solution of following
Nacl,Kcl,Cacl2, NaHco3, Na2Hpo4, NaH2po4, Mgcl2, Dextrose
• Maintain the bath at temp at of 32 c
• Bath liquid required dose between 10-50 units/ml.
• Oxygenate solution with mix of 95% of O2, 5% of CO2 record -contraction of muscle.
• Record contraction produces by addition of two dose of std. ppn (Reqd. Dose 10 &
50munits/ml of bath liquid)
• when maximum contraction has been reached replace - bath liquid by fresh solution.
• Dose should be added at regular interval[3-5minutes]
• Similarly record the contraction of test preparation as standard.
• Ratio between two dose of test & two dose of std should be equal. This ratio kept
constant through out the assay.
• Measure all response & calculate result of assay by standard statistical method.

METHOD C: (Milk ejection pressure in Lactating rat) TEST ANIMALS:

• Lactating rat, 3-21 day after parturition, 300 g Separate from litter & 30-60 minutes later
anaesthetise (IP Pentobarbitone Na).
• Tie rat to an operating table, at 37º, by its hind legs leaving front legs free.
• Cannulate trachea with a short PE tube of i.d. 2.5 mm in such a manner so as to ensure a free
airway; apply artificial respiration only if necessary.
• Cannulate an external jugular or femoral vein with a PE tube of i.d. 0.4 mm filled with saline &
closed with a pin.
• Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat,
preferably the lower inguinal teat.
• Insert a PE tube of i.d. 0.3 mm & e.d. 0.6 mm, to a depth sufficient to obtain appropriate
measurement of pressure (3-10 mm depth), into the primary teat duct which opens onto the cut
surface and tie firmly in place with a ligature.
• Connect this cannula with a suitable strain gauge transducer (such as that used for recording
arterial BP in rat) and fill with a 3.8% w/v of Na citrate /saline contain 50 Units of heparin Na/
ml to prevent clotting of milk.
• After cannulation, inject 0.05 - 0.2 ml of this solution into teat duct through transducer to clear
milk from tip of the cannula. (This procedure may be repeated during the assay should
obstruction arise from milk ejected into the cannula).
• Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is
preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale
deflection for an increase in milk-ejection pressure of 5.3 kPa.
• Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and
wash them in with 0.2 ml of saline .
• Prepare a solution of Std. & Test Ppn in saline solution so that the volume to be injected is
between 0.1 - 0.4 ml.
• Choose two doses of Std Ppn such that the increase in milk-ejection pressure is about 1.35 kPa
for Lr dose and about 2.7 kPa for Hr dose.
• As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of
1.5 to 2 times this amount may be tried.
• Choose two doses of the Test Ppn with the same inter-dose ratio, matching effects of doses of
the Std Ppn as closely as possible.
• Inject four doses (2 doses of Std & 2 doses of Test) at intervals of 3- 5 minutes.
• 2 doses of Std and 2 doses of test should be given according to randomised block or a Latin
square design & at least four responses to each -recorded.
• Measure all responses & calculate result of the assay by std statistical methods.
• Potency - 90% - 111%. Fiducial limits of error are 80% - 125%stated potency.

Vasopressor activity:

• NMT 0.5 Unit /20 Units of oxytocic activity - by biological assay for vasopressor activity-
comparing activity of Test & Std Ppn of arginine vasopressin Freeze-dried syn. arginine
vasopressin peptide acetate with human albumin & citric acid (supplied in ampoules
containing 8.20 Units)
• Inject slowly into tail vein of male albino rat weighing 300g -solution of a suitable a-
adrenoceptor blocking agent, (10 ml/kg body weight of solution prepared by dissolving 5
mg of phenoxybenzamine HCl in 0.1 ml of ethanol (95%) , adding 0.05 ml of 1 M HCl & dil to
5ml with saline .
• After 18 hours, anaesthetise rat - that will maintain -prolonged & uniform BP.
• After 45-60 minutes, tie the rat on its back to the operating table by its hind legs.
• Cannulate trachea with short PE of E.D. 2.5 mm & dissect carotid artery ready for
cannulation.
• Then cannulate the femoral vein close to the inguinal ligament.
• Retract the abdominal muscles to expose the inguinal ligament.
• Retract superficial pudendal vein to one side & dissect femoral vein towards inguinal
ligament from corresponding artery.
• When dissecting, a deep branch reaching femoral vein must be found & tied off to prevent
bleeding during cannulation.
• Tie a short PE cannula of E.D. about 1 mm into femoral vein by two ligatures & join by a
short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing
saline at about 37º.
• Firmly fix wet absorbent cotton swab to thigh so as to cover incision and cannula. At this
stage inject through venous cannula 200 Units of heparin, dissolved in saline /100 g of body
weight.
• Then tie in a carotid cannula of E.D.about 1 mm & connect by a column of saline contain
heparin with a pressure measuring device such as Hg manometer of I.D. about 2-3 mm.
• central & peripheral nervous system including both vagus & associated sympathetic nerves
is left intact.
• No artificial respiration is necessary.
• No air is injected, inject all solutions through venous cannula by means of a 1-ml syringe &
wash in with 0.2 ml of saline from burette.
• Dil extract of Std & Test Ppn with saline so that volume to be injected is between 0.1 & 0.5
ml.
• Choose 2 doses of the Std Ppn such that the elevation of the BP is about 4 kPa for Lr dose &
about 7 kPa but always submaximal for higher, ratio of low to high dose being determined
by response & usually being 3-5. As an initial approximation doses of 3 and 5 MUnits may be
tried.
• Choose 2 doses of Test ppn with same inter-dose ratio, matching effects of dose of Std Ppn.
Inject doses at intervals of 10 - 15 minutes.
• 2 doses of Std & 2 doses of Test Ppn should given in randomised block / Latin square design
& 4-5 responses to each recorded.
• Measure all responses & calculate result of the assay by Std statistical methods.