13054 Parenterals Complete

8/3/2019 13054 Parenterals Complete

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PARENTERAL DRUGDELIVERY

Presented by:

Rahul Bhaskar


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Definitions related to the topic:

Parenteral Products Sterilization & Sterile Product

Pyrogen

SVP

LVP Light Resistant Containers

Well closed containers

Tightly closed containers

Single dose container

Multiple dose container

Hermetically sealed container


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PARENTERALS

para: outside

enteron: intestine (i.e. beside the intestine)

These are the preparations which are given other

than oral routes.Injections:

These are

Sterile,

Pyrogen free preparations intended to be

administered parenterally (outside alimentary

tract).


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Why Parenteral?Parenteral Route Is Used bcoz

1) Rapid action

2) Oral route can not be used

3) Not effective except as injection4) Many new drugs particularly those derived from

new development in biotechnologically can onlybe given by parenteral coz they are inactivated

in GIT if given orally.5) New drugs require to maintain potency &

specificity sodium that they are given byparenteral.


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Advantages:

Quick onset of action

Suitable for the drugs which are not

administered by oral route

Useful for unconscious or vomitingpatients.

Duration of action can be prolonged by

modifying formulation.

Suitable for nutritive like glucose &

electrolyte.

Suitable for the drugs which are inactivated

in GIT or HCl (GI fluid)


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Disadvantages:

Once injected cannot be controlled(retreat)

Injections may cause pain at the site ofinjection

Only trained person is required

If given by wrong route, difficult to controladverse effect

Difficult to save patient if overdose

Sensitivity or allergic reaction at the site ofinjection

Requires strict control of sterility & nonpyrogenicity than other formulation.


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Necessities of Parenteral preparations:

Sterility (must)

Pyrogen free (must)

Free from particulate matter(must)

Clarity (must)

Stability (must)

Isotonicity (should)

Solvents or vehicles used must meet special purity and otherstandards.

Restrictions on buffers, stabilizers, antimicrobialpreservative. Do not use coloring agents.

Must be prepared under aseptic conditions.

Specific and high quality packaging.


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Routes of Parenteral Administration

Intradermal (23)Intramuscular (20)

Intravenous (21)Subcutaneous (21)

Dermis

Intra arterial (20-22)

Vein

Artery

Muscle

Epidermis

Subcutaneoustissue


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Parental Routes of Administration:

Most Common: 1. Subcutaneous (SC;SQ;SubQ)

2. Intramuscular (IM)

3. Intravenous (IV)Others: 4. Intra-arterial (IA)

5. Intrathecal

6. Intraarticular

7. Intrapleural

8. Intracardial

9. Intradermal (Diagnostic)


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Subcutaneous (SC;SQ;SubQ):

The injection is given under the skin

Need to be isotonic

Upto 2 ml is given

Using to 1 inch 23 gauge needle or

smaller needle

Given:

Vaccines

Insulin Scopolamine

Epinephrine


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Intramuscular (IM): Striated muscle fibre

0.5 to 2 ml sometimes upto 4 ml 1 to 1.5 inch & 19 to 22 gauge needle is used

Preferably isotonic

Principle sites:

Gluteal (buttocks) Deltoid (upper arms)

Vastus lateralis (lateral thigh)

Given:

Solutions

Emulsions Oils

Suspension


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Intravenous (IV): Into the vein

1 to 1000 ml

1 inch ,19 to 20 gauge needle with injection rate

1ml/ 10 sec. for volume upto 5 ml & 1 ml/ 20 sec.for volume more than 5 ml.

Given:

Aqueous solutions

Hydro alcoholic solutions Emulsions

Liposome


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IV infusion of large volume fluids (100- 1000 ml)has become increasingly popular. This

technique is called as Venoclysis.

This is used to supply electrolytes & nutrients torestore blood volume & to prevent tissuedehydration.

Combination of parenteral dosage forms foradministration as a unit product is known as anIV admixture. Lactated Ringer Injection USP

NaCl Injection USP (0.9 %) (replenish fluid &electrolyte)

Dextrose Injection USP (fluid & electrolyte)


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Intra-arterial (IA):

Direct into the artery 2 to 20 ml

20 to 22 gauge

Solutions & emulsions can be administered Given:

Radio opaque media

Antineoplastic

Antibiotics


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Intrathecal:

Also called intra-spinal

Directly given into the spinal cord

1 to 4 ml

24 to 28 gauge Must be isotonic

Given:

LA

Analgesics

Neuroleptics


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Intraarticular:

Given directly into the joints

2 to 20 ml

5 inch 22 gauge

Must be isotonic

Given: Morphine

LA

Steroids

NSAIDs

Antibiotics


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Intrapleural:

Given directly into the pleural cavity or lung Used for fluid withdrawal

2 to 30 ml

2 to 5 inch, 16 to 22 gauge needle

Given:

LA

Narcotics

Chemotherapeutic agents


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Intracardial:

Directly given into the heart

0.2 to 1 ml

5 inch , 22 gauge needle

Given:

Cadiotonics

Calcium salts as a calcium channelblockers


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Intradermal:

Also called as diagnostic testing

0.05 ml

inch, 25 to 26 gauge needle

Should be isotonic Given:

Diagnostic agents


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PREFORMULATION FACTORS:

It is study about physical & chemical properties of drugsubstance

prior formulation is called as preformulation.

They are

pH

Solubilty

pka

Dissociation constant

Compatabilty studies- FTIR / DSCStability (Oxidation & reduction)

particle size

Desired type of packaging


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Official Types of Injections:

1. Solutions of Medicinal

Example: Codeine Phosphate Injection

Insulin Injection

2. Dry solids or liquid concentrate does

not contain diluents etc.

Example: Sterile Ampicillin Sodium

3. If diluents present, referred to as.....for

injection

Example: Methicillin Sodium for

injection


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4. Suspensions

"Sterile....Suspension"

Example: Sterile DexamethasoneAcetate Suspension

5.Dry solids, which upon the addition of

suitable vehicles yield preparationscontaining in all respects to therequirements for sterile suspensions.

Title: Sterile....for Suspension

Example: Sterile Ampicillin forSuspension

6. Injectable Emulsions:

Example: Propofol injection


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Formulation of Parenteral:

1. Therapeutic agents2. Vehicles

i. Water

ii. Water miscible vehicles

iii. Non- aqueous vehicles

3. Added substances (Additives)

i. Antimicrobials

ii. Antioxidants

iii. Buffers

iv. Bulking agents

v. Chelating agents

vi. Protectantsvii. Solubilizing agents

viii.Surfactants

ix. Tonicity- adjusting agents


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General steps involved

1. Cleaning

2. Preparation of bulk products

3. Filtration

4. Filling of solution in or product in ampoule or vial

7. Tests forQuality control

5.Sealing

6. Sterilization


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Formulation of Parenteral

1.Therapeutic ingredients: Insulin

Antibiotics

Anticancer Steroids

Vaccines

Antipyretic

Analgesics

Anti- inflammatory

LVPs like Dextrose, NaCl or combination etc.


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2.Solvents:

o Water

o Should meet compendial requirements

o Water miscible vehicles

o Ethyl alcohol

o PEG

o PG

o Non aqueous vehicles

o Fixed oils



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SolventsSolvents used must be:

Non-irritating Non-toxic

Non-sensitizing

No pharmacological activity of its own Not affect activity of medicinal


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3. Added substances (Additives)

Antimicrobials: Added for fungistatic or bacteriostat action or

concentration

Used to prevent the multiplication of micro-organisms

Ex.. Benzyl alcohol ------ 0.5 10 %

Benzethonium chloride -- 0.01 %

Methyl paraben ---- 0.01 0.18 %

Propyl paraben --- 0.005 0.035 %

Phenol --- 0.065 0.5 %



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Preservatives: Multidose containers

must have preservatives unless

prohibited by monograph.

Large volume parenteral must not

contain preservative becoz it may be

dangerous to human body if it contain in

high doses.


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Antioxidants:

Used to protect product from oxidation Acts as reducing agent or prevents oxidation

Ex:

A) Reducing agent:

Ascorbic acid --0.02 0.1 %

Sodium bisulphite-- 0.1 0.15 % Sodium metabisulphite-- 0.1 0.15 %

Thiourea - 0.005 %

B) Blocking agents:

Ascorbic acid esters- 0.01 0.015%

BHT- 0.005 0.02 %

C) Synergistic: Ascorbic acid , Citric acid , Tartaric acid

D) Chelating agent:

EDTA- 0.01- 0.075 %


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Buffers:

Added to maintain pH, Change in pH may causes degradation of the products

Acetates, citrates, phosphates are generally used.

Factors affecting selection of buffers: Effective range,

Concentration

Chemical effect on the total product

EXAMPLES:

Acetic acid ,adipic acid, benzoic acid, citric acid,

lactic acid

Used in the conc. of 0.1 to 5.0 %


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Chelating agents:

Used to form the complex with themetallic ions present in the formulation

so that the ions will not interfere during

mfg. of formulation.

They form a complex which getsdissolved in the solvents.

Examples:

Disodium edetate 0.00368 - .05 % Disodium calcium edetate - 0.04 %

Tetrasodium edetate 0.01 %


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Stabilizers:

As parenterals are available in solutionform they are most prone to unstabilize

Used to stabilize the formulation

Maintain stable

Examples: Creatinine 0.5- 0.8 %

Glycerin 1.5 2.25 %

Niacinamide 1.25 -2.5 %

Sodium saccharin 0.03 %

Sodium caprylate 0.4 %


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Solubilizing agents: Used to increase solubility of slightly soluble

drugs they acts by any one of the following:

solubilizers,

emulsifiers or

wetting agents. Examples:

Dimethylacetamide,

Ethyl alcohol

Glycerin

Lecithin

PEG 40 castor oil

PEG 300

Polysorbate 20, 40, 80


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Tonicity- adjusting agents: Used to reduce the pain of injection.

Buffers may acts as tonicity contributor as wellas stabilizers for the pH.

Isotonicity depends on permeability of a livingsemipermaeable membrane

Hypotonic : swelling of cells (enlargement)

Hypertonic: shrinking of cells (reduction)

Example: Glycerin

Lactose

Mannitol

Dextrose Sodium chloride

Sorbitol


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LABELING: Name of product

Quantity of the product % of drug or amount of drug in specified volume of

amount of drug and volume of liquid to be added

Name and quantity of all added substances

Mfg. license no. Batch no.

Manufacturer/Distributor

Mfg. & Expiration date

Retail price (incl. of all taxes)

Mfger. address

Veterinary product should be so labeled


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Must check each individual monogram for:

Type of container: Glass

Plastic

Rubber closure

Type of glass

Type I Type II

Type III

NP

Tests for glass containers Powdered Glass test

Water Attack test Package size

Special storage instructions


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Production facilities

Types : Emulsion

Suspension

Solutions

Preparation of

IV fluids

IV admixtures

TPN

Dialysis fluids

QC tests for parenteral


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Production facilities:

Clean- up area

Preparation area

Aseptic area

Quarantine area

Finishing and packaging area

Sterile area


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S

T

O

CK

R

O

O

M

COMPOUNDING

AREA

CLEAN UPAREA

ASEPTIC

AREA

QUARANTINE

AREA

STERILIZATION

STORAGE

AND

TRANSPORT

PACKING

AND

LABELLING

LAY OUT OF PARENTERAL MANUFACTURING AREA


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Clean- up area:

Non aseptic area

Free from dust ,fibres & micro-organisms

Constructed in such a way that should withstand

moisture, steam & detergent

Ceiling & walls are coated with material to preventaccumulation of dust & micro-organisms

Exhaust fans are fitted to remove heat & humidity

The area should be kept clean sodium that toavoid contamination to aseptic area

The containers & closures are washed & dried in

this area.


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Preparation area:

The ingredients are mixed & preparation is

prepared for filling

Not essential that the area is aseptic

Strict precaution is taken to prevent contaminationfrom outside

Cabinets & counters: SS

Ceiling & walls : sealed & painted


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Aseptic area:

Filtration & filling into final containers & sealing isdone

The entry of outside person is strictly prohibited

To maintain sterility, special trained persons are

only allowed to enter & work

Person who worked should wear sterile cloths

Should be subjected for physical examination to

ensure the fitness

Minimum movement should be there in this area

Ceiling & walls & floors : sealed & painted or

treated with aseptic solution and there should not

be any toxic effect of this treatment


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Cabinets & counters: SS

Mechanical equipments : SS AIR:

Free from fibres, dust & micro organisms

HEPA filters are used which removes particles

upto 0.3 micron

Fitted in laminar air flow system, in which air is

free from dust & micro organisms flows with

uniform velocity

Air supplied is under positive pressure which

prevents particulate contamination from

sweeping

UV lamps are fitted to maintain sterility


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Quarantine area: After filling, sealing & sterilization the

products or batch is kept in this area

The random samples are chosen and

given for analysis to QC dept.

The batch is send to packing after issuing

satisfactory reports of analysis from QC

If any problem is observed in aboveanalysis the decision is to be taken for

reprocessing or others..


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Finishing and packaging area:

After proper label, the product is given forpacking

Packing is done to protect the product fromexternal environment

The ideal Packing is that which protectsthe product during transportation, storage,shipping & handling.

The labeled container should be packed in

cardboard or plastic containers Ampoules should be packed in partitionedboxes.


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Preparations for IV Fluids:

LVPs which are administered by IVroute are commonly called as IV fluids.

Purposes :

Body fluids, Electrolyte replenisher

Volume supplied:

100 to 1000 ml


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Precautions / necessities in mfg.:

Free from foreign particles

Free from micro organisms

Isotonic with body fluids

As they are in LVP no bacteriostatic agentsare added

Free from pyrogens


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Examples:

Dextrose injection IP : available in 2 , 5 , 10 , 25 &50 % w/v solution.

Used for Fluids replenisher,

Electrolyte replenisher

Sodium chloride & Dextrose injection IP: (DNS) Contains

0.11 to 0.9 % Sodium chloride

2.5 to 5.0 % Dextrose

Used for

Fluids replenisher, Electrolyte replenisher

Nutrient replenisher


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Sodium chloride injection IP:

0.9 % conc.

Also known as normal saline solution

Used as

Isotonic vehicle

Fluids replenisher, Electrolyte replenisher

Sodium lactate injection IP:

Contains 1.75 to 1.95 % w/v of sodium lactate

Used as Fluids replenisher,

Electrolyte replenisher


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Mannitol injection IP:

Contains 5, 10 , 15, 20 % of mannitol Used as :

Diagnostic aid

Renal function determination

As a diuretic

Mannitol & Sodium chloride injection IP:

Contains 5, 10 , 15, 20 % of mannitol &0.45 % of Sodium chloride

Used as : As a diuretic


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Other solutions:

Ringer injection IP

Ringer lactate solution for injection IP

Common uses :

Used in surgery patients

In replacement therapy

Providing basic nutrition

For providing TPN As a vehicle for other drug subs.


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IV ADMIXTURES

Definition:

When two or more sterile products are addedto an IV fluid for their administration, the

resulting combination is known as IVadmixture.

In hospitals, prepared by nurses by combiningor mixing drugs to the transfusion fluids.

The drugs are incorporated in to bottles of LVtransfusion fluids.


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Care :

Microbial contamination

Incompatibility

Physical : change in color

Chemical : hydrolysis, oxidation, reduction etc..

Therapeutic: undesirable antagonistic or

synergistic effect


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Methods for safe & effective use of IVadmixture:

Proper training to nurses & pharmacist

Instruction regarding labeling

Information for stability & compatibility to

the hospital pharmacy dept.

Information for the formulation skills to the

pharmacist.


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TPN stands for Total Parenteral Nutrition.This is a complete form of nutrition,

containing protein, sugar, fat and addedvitamins and minerals as needed for eachindividual.

Total Parenteral Nutrition (TPN) may be

defined as provision of nutrition formetabolic

requirements and growth through the

parenteral route.

Total Parenteral NutritionTotal Parenteral Nutrition


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Total Parenteral Nutrition (TPN)

(Intravenous Nutrition)TPN refers to the provision

of all required nutrients,

exclusively by theIntravenous route.

Parenteral Nutrition (PN)can be used tosupplement ordinary or tube feeding.


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Components of TPN solutions:

(1) Protein as crystalline amino acids.(2) Fats as lipids.

(3) Carbohydrate as glucose.

(4) ElectrolytesSodium, potassium,

chloride, calcium and magnesium.

(5) Metals/Trace elementsZinc, copper,

manganese, chromium, selenium.

(6) Vitamins A, C, D, E, K, thiamine,riboflavin, niacin, pantothenic acid,

pyridoxine, biotin, choline and folic acid.


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TPN might be necessary if:

a patient is severely undernourished, and

needs to have surgery, radiotherapy or

chemotherapy;

a patient suffers from chronic diarrhea and

vomiting;

a baby's gut is too immature; a patient's (their "gastrointestinal tract") is

paralysed, for example after major surgery.

Why it is necessary?


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Normal Diet TPN

ProteinAmino Acids

CarbohydratesDextrose

Fat..Lipid Emulsion

VitaminsMultivitamin Infusion

MineralsElectrolytes & Trace Elements


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Nutritional Requirements

Amino acids

Glucose

Lipid Minerals

Vitamins

Water and electrolytes

Trace elements


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Total Parenteral Nutrition

Electrolytes

Electrolyt

e.

Daily Requirement Standard Concentration

Na 60-150 meq 35-50 meq/LK 40-240 meq 30-40 meq/L

Ca 3-30 meq 5 meq/L

Mg 10-45 meq 5-10 meq/L

Phos. 30-50 mM 12-15 mM/L


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TPN is normally used following surgery,

when feeding by mouth or using the gut

is not possible,

When a person's digestive system

cannot absorb nutrients due to chronic

disease, or, alternatively, if a person's

nutrient requirement cannot be met by

enteral feeding (tube feeding) and

supplementation.

When is it necessary?


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Short-term TPN may be used if a person's

digestive system has shut down (for instanceby Peritonitis), and they are at a low enough

weight to cause concerns about nutrition

during an extended hospital stay.

Long-term TPN is occasionally used to treat

people suffering the extended consequences

of an accident or surgery.

Most controversially, TPN has extended thelife of a small number of children born with

nonexistent or severely birth-deformed guts.


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GENERAL INDICATIONS Patient who cant eat

Patient who wont eat

Patient who shouldnt eat Patient who cant eat enough

If the gut works, use it.


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NOMENCLATURE TPN: Total Parenteral Nutrition

IVH: Intravenous Hyperalimentation

TNA: Total Nutrient Admixture

TPN: Total Parenteral Nutrition

3-In-1 Admixture

All-In-One Admixture

PPN: Peripheral Parenteral Admixture


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Indications for TPN

Short-term use Bowel injury /surgery

Bowel disease

Severe malnutrition

Nutritional preparation prior to surgery.

Malabsorption - bowel cancer

Long-term use

Prolonged Intestinal Failure

Crohns Disease

Bowel resection


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The preferred method of delivering TPN is

with a medical infusion pump.

A sterile bag of nutrient solution, between 500

mL and 4 L is provided.

The pump infuses a small amount (0.1 to 10

mL/hr) continuously in order to keep the veinopen.

Feeding schedules vary, but one common

regimen ramps up the nutrition over a few

hours, levels off the rate for a few hours, andthen ramps it down over a few more hours, in

order to simulate a normal set of meal times.


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The nutrient solution consists ofwater, glucose,

salts, amino acids, vitamins and (more

controversially) sometimes emulsified fats. Long term TPN patients sometimes suffer from

lack of trace nutrients orelectrolyte imbalances.

Because increased blood sugarcommonly

occurs with TPN, insulin may also be added tothe infusion.

Often though, an insignificant amount of insulin is

added, sometimes 10 units or less in 2 liters ofTPN.

In actuality, the patient will probably get less than

that.


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Complications of TPN

Sepsis

Air embolism

Clotted catheter line

Catheter displacement

Fluid overload

Hyperglycaemia

Rebound

Hypoglycaemia


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Dialysis is the process in which

substances are separated from one

another due to their difference indiffusibility (distribution) thr

membrane.

The fluids used in dialysis are knownas dialysis fluids.

DIALYSIS FLUIDS


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General uses : Renal failure

waste product is removed

Maintain electrolytes

Also called as haemodialysis orintraperitoneal

dialysis

Transplantation of kidney

Poisoning cases


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Haemodialysis:

To remove toxins from blood

In haemodialysis, the blood from artery is

passed thr artificial dialysis membrane,

bathed in dialysis fluid. The dialysis membrane is permeable to

urea, electrolytes & dextrose but not to

plasma proteins & lipids

So excess of urea is passed out from blood

thr dialysis fluid.


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After dialysis blood is returned back to thebody circulation thr vein.

A kidney unit may require more than 1200

litres of solution / week.

So haemodialysis fluid is prepared in conc.

Form then it is diluted with deionised water

or dist. water before use.


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Composition ofConcentrated Haemodialysis Fluid BPC

Dilute 1 liter of conc. solution with 39 liters of water to make 40litres.

Storage: store in warm place as it is liable to convert into crystalson storage.

COMPOSITION

Dextrose monohydrate -----------

Sodium acetate ---------------------

Lactic acid ---------------------------Sodium chloride -------------------

Potassium chloride ---------------

Freshly boiled & cooled water -q.s.

8.0 gm

19.04 gm

0.4 ml22.24 gm

0.4 gm

100 ml


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IntraperitonealDialysis: Peritoneal cavity is irrigated with dialysis

fluid.

Peritoneum acts as a semi permeable

membrane

Toxic subs. excreted by kidney are

removed.

Requirements: Sterile

Pyrogen free


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Composition of Fluid IntraperitonealDialysis IP 1985

Sterilize by autoclave immediately afterpreparation.

COMPOSITION

Sodium chloride -------------------

Sodium acetate ---------------------Calcium chloride -------------------

Magnesium chloride --------------

Sodium metabisulphite ----------

Dextrose (anhydrous) -----------

Purified water -----------q.s.-----

5.56 gm

4.76 gm

0.22 gm

0.152 gm

0.15 gm

17.30 gm

1000 ml


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STERILITY TESTING FOR

PARENTERAL PRODUCTS


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1. Sterility testing - definition

Sterility testing attempts to reveal the

presence or absence of viable micro-

organisms in a sample number of

containers taken from batch of product.Based on results obtained from testing the

sample a decision is made as to the

sterility of the batch.


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Sterility testing -

is made after the product exposition to the

one of the possible sterilization procedures

can only provide partial answers to the

state of sterility of the product batch under

test

is inadequate as an assurance of sterility

for a terminally sterilized product


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Major factors of importance in sterility

testing The environment in which the test is

conducted

The quality of the culture conditions provided

The test method

The sample size

The sampling procedure


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1.1.Environmental conditions

avoid accidental contamination of the

product during the test

the test is carried out under aseptic

conditions

regular microbiological monitoring should

be carried out


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1.2.Culture conditions

Appropriate conditions for the growth of

any surviving organism should be provided

by the culture media selection.


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1.2. Culture conditions

Factors affecting growth of bacteria

Phases of bacterial growth

Culture media for sterility testing

1 2 1 Factors affecting gro th of


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1.2.1. Factors affecting growth of

bacteria

Nutrition

Moisture

Air

Temperature

pH

Light

Osmotic pressure Growth inhibitors


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1.2.2. Phases of bacterial growth

Lag phase (A)

Log (logarithmic or

exponential) phase (B) Stationary phase (C)

Decline (death) phase

(D)


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1.2.3.Culture media for sterility testing

capable of initiating and maintaining the

vigorous growth of a small number of

organisms

sterile

Types of media:

Fluid thioglycollate medium

Soya-bean casein digest medium

other media


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1.2.3.1.Fluid thioglycollate medium

composition described in next slide.

specific role of some ingredients

primarily intended for the culture ofanaerobic bacteria

incubation of the media:

14 days at 30 -35C


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Fluid thioglycollate medium

1 2 3 2 Soya bean casein digest


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1.2.3.2.Soya-bean casein digest

medium

primarily intended for the culture of both

fungi and aerobic bacteria

specific role of some ingredients

incubation of the media:

14 days at 20 -25C


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Soya-bean casein digest medium


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1.2.3.3.Fertility control of the media

are they suitable for growth of each

micro-organism?

'Growth promotion test for aerobes,

anaerobes and fungi' ; inoculation of media tubes with a MO

incubation (T, t)

the media are suitable if a clearly visible growth ofthe micro-organisms occurs

1 2 3 4 Effectiveness of the media


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1.2.3.4.Effectiveness of the media

under test conditions

are culture conditions satisfactory in the

presence of the product being

examined?

comparing the rate of onset and the

density of growth of inoculated MO in

the presence and absence of the

material being examined

growth control;

1 3 The test method for sterility


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1.3.The test method for sterility

of the product

Membrane filtration

Direct inoculation of the culture medium

f


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1.3.1. Membrane filtration

Appropriate for : (advantage)

filterable aqueous preparations

alcoholic preparations

oily preparations preparations miscible with or soluble in

aqueous or oily (solvents with no

antimicrobial effect)

solutions to be examined must be introduced

and filtered under aseptic conditions

All steps of this procedure are performed

aseptically in a Class 100 Laminar Flow Hood

1 3 1 1 Selection of filters for


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1.3.1.1.Selection of filters for

membrane filtration

pore size of 0.45 Qm

effectiveness established in the retention

of micro-organisms

appropriate composition

the size of filter discs is about 50 mm in

diameter

1.3.1.2.The procedure of membrane


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p

filtration

sterilization of filtration system and membrane filtration of examined solution under aseptic

conditions (suitable volume, dissolution of solidparticles with suitable solvents, dilution if necessary)

one of two possible following procedures:

the membrane is removed, aseptically

transferred to container of appropriate

culture medium

passing the culture media through closed

system to the membrane, incubation in situ

in the filtration apparatus (sartorius,

millipore).


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1.3.2.Direct inoculation of the


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1.3.2.Direct inoculation of the

culture medium

suitable quantity of the preparation to be

examined is transferred directly into the

appropriate culture medium

volume of the product is not more than

10% of the volume of the medium

suitable method for aqueous solutions, oily

liquids, ointments an creams


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Scheme for sterility test by membrane filtration Scheme for sterility test by direct inoculation

Advantages of the filtration method


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Advantages of the filtration method

wide applications a large volume can be tested with one

filter

smaller volume of culture media isrequired

applicable to substances for which no

satisfactory inactivators are known

neutralization is possible on the filter

subculturing is often eliminated

shorter time of incubation compared

1.4. Observation and


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1.4. Observation and

interpretation of the results Examination at time intervals during the

incubation period and at its conclusion

When the sample passes the test and

when fails?

When the test may be considered as

invalid?

There is low incidence of accidentalcontamination or false positive results

1 5 S li


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1.5. Sampling

Selection of the samples

Sample size

Minimum number of items to be tested


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Instead of the conclusion - Guidelines


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Instead of the conclusion Guidelines

for using the test for sterility

Precautions against microbial

contamination

The level of assurance provided by a

satisfactory result of a test for sterility as

applied to the quality of the batch is a

function of:

The homogeneity of the batch The conditions of manufacture

Efficiency of the adopted sampling plan

Guidelines


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Guidelines

In the case of terminally sterilizedproducts: physical proofs, biologically basedand automatically documented, showing

correct treatment through the batch during

sterilization are of greater assurance than thesterility test

Products prepared under aseptic

conditions: sterility test is the only availableanalytical method

only analytical method available to the

authorities who have to examine a


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PYROGENS AND PYROGEN TESTINGPYROGENS AND PYROGEN TESTING


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I Love The Rabbit!

Pyrogens


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Pyrogens

Pyrogenic - means producing fever

Pyrogens - fever inducing substances

Having nature

Endogenous (inside body)

Exogenous (outside body)

Exogenous pyrogens

mainly lipopolysaccharides bacterial origin, but not necessary

Structure of endotoxins


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Structure of endotoxins

Produced mostly by gram-negative

bacteria

Endotoxin - complex of pyrogenic

lipopolysaccharide, a protein and inertlipid;

lipid part of the lipopolysaccharide is the

main pyrogenic agent; polysaccharide partincreases solubility

Generalized structure of endotoxins


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Ge e a ed st uctu e o e doto s

Generalized structure of Endotoxins


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Sources of pyrogen contamination


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Sources of pyrogen contamination

solvent - possibly the most important

source

the medicament

the apparatus

the method of storage between

preparation and sterilization

The endotoxin characteristics


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The endotoxin characteristics

thermostable

water-soluble

unaffected by the common bactericides

non-volatile

These are the reasons why pyrogens

are difficult to destroy once produced in

a product

Tests for pyrogenic activity


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Tests for pyrogenic activity

Test for pyrogens = Rabbit test

Bacterial endotoxins

T t f R bbit t t


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Test for pyrogens = Rabbit test

the development of the test for pyrogens

reach in 1920

a pyrogen test was introduced into the

USP XII (1942)

The test consists of measuring the rise in

body temperature in healthy rabbits by the

intravenous injection of a sterile solution ofthe substance under the test.

Why the Rabbit?


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Why the Rabbit?

Reproducible pyrogenic response

Other species not predictable

Rabbit vs. dog as model?

Rabbits: false positives

Dogs: false negatives

Similar threshold pyrogenic response to

humans

Rabbit Pyrogen Test


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Rabbit Pyrogen Test

Rabbits must be healthy and mature

New Zealand or Belgian Whites used

Either sex may be used

Length of use >48 hours within negative result

>2 weeks within a positive result

Must be individually housed between 20and 23C

Rabbit test -


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Rabbit test -

selection of animals (healthy, adult, not less

than 1,5 kg,)

housing of animals (environmental problems:

presence of strangers (unknown place),

noise, T, ) equipment and material used in test

(glassware, syringes, needles)

retaining boxes (comfortable for rabbits as

possible) thermometers (standardized position in

rectum, precision of 0.1C)


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Rabbit test


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Rabbit test

Preliminary test (Sham Test) intravenous injection of sterile pyrogen-free

saline solution

to exclude any animal showing an unusual

response to the trauma (shock) of injection

any animal showing a temperature variation

greater than 0.6rC is not used in the main

test

Rabbit test -


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main test:

group of 3 rabbits preparation and injection of the product:

warming the product

dissolving or dilution

duration of injection: not more than 4 min the injected volume: not less than 0.5 ml per 1

kg and not more than 10 ml per kg of body

mass

determination of the initial and maximumtemperature

all rabbits should have initial T: from 38.0 to

39.8rC

the differences in initial T should not differ from

Rabbit test


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Interpretation of the results: the test is carried out on the first group of 3

rabbits; if necessary on further groups of 3 rabbits

to a total of 4 groups, depending on the results

obtained

intervals of passing or failing of products are onthe basis of summed temperature response

The result of pyrogen test:


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The result of pyrogen test:

No.of Rabbits IndividualTempt. rise

(c)

Tempt.Rise in

group (c)

Test

3 rabbits 0.6 1.4 Passes

If above not passes

3+5 = 8 rabbits

0.6 3.7 Passes

If above test not passes perform the test again

If above test not passes, the sample is said to be pyrogenic

or go thr the sources of contamination of pyrogen.



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to detect or quantify endotoxins of gram-

negative bacterial origin

reagent: amoebocyte lysate from horseshoe

crab (Limulus polyphemus or Tachypleustridentatus).

The name of the test is also Limulus

amebocyte lysate (LAL) test

Limulus polyphemus = horseshoe crab


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Limulus polyphemus = horseshoe crab

Mechanism of LAL


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Mechanism of LAL

the test is based on the primitive blood-clotting mechanism of the horseshoe crab

enzymes located with the crab's amebocyte

blood cells

endotoxins

initiation of an enzymatic coagulation cascade

proteinaceous gel

Commercially derived LAL


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reagents bleeding adult crabs into an anticlotting

solution washing and centrifuging to collect the

amebocyte

lysing in 3% NaCl

lysate is washed and lyophilized for storage

activity varies on a seasonal basis andstandardization is necessary.

Test performance (short)


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Test performance (short)

avoid endotoxin contamination

Before the test:

interfering factors should not be present

equipment should be depyrogenated

the sensitivity of the lysate should be known Test:

equal V of LAL reagent and test solution (usually 0.1

ml of each) are mixed in a depyrogenated test-tube

incubation at 37C, 1 hour remove the tube - invert in one smooth motion (180) -

read (observe) the result

pass-fail test

LAL test


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LAL test

Three different techniques:

the gel-clot technique - gel formation

the turbidimetric technique - the

development of turbidity after cleavage ofan endogenous substrate

the chromogenic technique - the

development of color after cleavage of a

synthetic peptide-chromogen complex

LAL test


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LAL test

6 methods with different steps of accuracy of LAL

test results:

Method A: gel-clot method: limit test

Method B: gel-clot method: semi-quantitative test

Method C: turbidimetric kinetic method Method D: chromogenic kinetic method

Method E: chromogenic end-point method

Method F: turbidimetric end-point method

In the event of doubt or dispute, the final decisionis made upon Method A unless otherwise

indicated in the monograph.

Gel-cloth technique (Methods A, B)


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allows detection or

quantification ofendotoxins

clotting of the lysate in

the presence of

endotoxins. 1.Preparatory testing

Confirmation of the

labeled lysate

sensitivity Tests for interfering

factors

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Gel Clot

Invert Tube in Smooth

Motion

Gel-cloth technique (Methods A, B)-


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cont. 2

.Limit test (method A) procedure described on page. 24

a firm gel - positive result.

an intact gel is not formed - negative result.

the interpretation of the results

3. Semi-quantitative test (method B)

quantification of bacterial endotoxins in the test solutionby titration to an end-point.

procedure is similar as in the limit test

The results are expressed as concentration of endotoxin as less, equal or greater than P (labeledlysate sensitivity).

Turbidimetric technique (Methods C,

F)


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F) photometric test to measure the increase in

turbidity

end-point test (Method F): quantitativerelationship between the endotoxin concentration and

the turbidity (absorbance or transmission) of the

reaction mixture at the end of an incubation period.

kinetic test (Method C): a method to measureeither the time (onset time) needed for the reaction

mixture to reach a predetermined absorbance, or the

rate of turbidity development.

Chromogenic technique (Methods D,

E)


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E) measuring the chromophore released from a

suitable chromogenic peptide by the reactionof endotoxins with the lysate

end-point test (Method E): is based on the

quantitative relationship between the endotoxinconcentration and the quantity of chromophore

released at the end of an incubation period

kinetic test (Method D): a method to measure

either the time (onset time) needed for the reactionmixture to reach a predetermined absorbance, or the

rate of color development

Instead of the conclusion -

Guidelines for test for bacterial


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endotoxins

the absence of bacterial endotoxins in a

product implies the absence of pyrogenic

component

if you wish to replace rabbit test you shouldprove that you dont have interfering factors

if rabbit pyrogen test is replaced by endotoxin

test, the last one should be validated

methods from C to F require moreinstrumentation, but they are easier to

automate

test for bacterial endotoxins is preferred over

Advantages of LAL testF t 60 i t 180 i t


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g Fast - 60 minutes vs. 180 minutes

Greater Sensitivity

Less Variability Much Less False Positives

Much Less Expensive

Alternative to Animal Model

cheaper,

more accurate than other is performed in the pharmaceutical laboratory

specific for endotoxins of gram-negative origin

particularly useful for:

Radiopharmaceuticals and cytotoxic agents

Products with marked pharmacological or toxicologicalactivity in the rabbit (e.g. insulin)

Blood products which sometime give misleading results inthe rabbit

Water for injection where LAL test is potentially morestringent and readily applied


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Particulate Matter Monitoring

Definition:


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Definition:

Unwanted mobile insoluble matter other

than gas bubbles present in the given

product.

It may be dangerous when the particlesize is larger than R.B.C. & may block the

blood vessel.

This type of products are immediatelyrejected from the batch.


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The limit test for particulate matter is prescribedin I.P. 1996 (A- 125)

Applicable for:

100 ml or more volume containers of single dose LV

given by IV infusion

Not applicable for:

Multidose injections

Single dose SVP

Injectable solutions constituted from sterile solids

Permitted limits of particulate

tt


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matter

Particle size in micrometer Max.No.of particles

(equal to or larger than) per ml

10 50

25 5

50 Nil

Sources of particulate matter


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Sources of particulate matter

Contamination

Contaminant

Intrinsic contamination:

Originally present in products e.g. Barium ions may react or leach with Sulphur

ion which are already present in formulation may

produce barium sulphate crystals.


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Extrinsic contamination:

Material comes from outside or environment

e.g. coming off the material from body & cloths of

person Entry of particle from ceiling , walls & furniture

May be in the form of cotton, glass rubber,

plastics, tissues, insect fragments, bacterial

contamination, dust, papers etc

Methods of monitoring particulate matter

contamination


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contamination

Visual method

Coulter counter method

Filtration method

Light blockage method

Vi l th d


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Visual method:

Simple method Filled container are examined against strong

illuminated screen by holding neck & rotating it slowly

or inverted it to keep out the foreign matter.

Coulter counter method: It is used for detection of particles less than 0.1

micrometer in diameter.

Based on electrode resistance.

Sample is evaluated between two electrode & ifparticle found the resistance of electrode is increased.

Filtration method:


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It is used for counting the particles in hydraulic fluids.

Sample passed thr filter

Material is collected on filter

Evaluated under microscope.

Disadvantage:

Skilled & trained person is required

Light blockage method:

Used for hydraulic oils

Allows stream of fluid under test to pass between abright white light source & photoiodide sensor.

Identification of Particulate

Matter


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Matter Microscopy

X- ray powder diffraction

Mass microscopy

Microchemical tests

Electron microscopy etc

Significance of Particulate Matter monitoring


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Its presence may causes:

Septicemia

Fever & blockage of blood vessels

Quality of product may affect


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As per USP

LVP : NMT 50 particles/ ml (size 10 or morethan 10 micrometer) & 5 particles/ ml (size

more than 25 micrometer)

SVP: 10,000 particles/ container of size 10

micrometer or greater & NMT 1000 particles/

container greater than 25 micrometer.

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13054 Parenterals Complete