Parenteral Sro A

7/27/2019 Parenteral Sro A

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PARENTERAL DRUG

DELIVERY


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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: outsideenteron: 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 only be givenby parenteral coz they are inactivated in GIT if given

orally.5) New drugs require to maintain potency & specificity

so that they are given by parenteral.


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Advantages:

Quick onset of action

Suitable for the drugs which are not

administered by oral route

Useful for unconscious or vomiting patients.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 of injection

Only trained person is required

If given by wrong route, difficult to control

adverse effect

Difficult to save patient if overdose

Sensitivity or allergic reaction at the site of

injectionRequires strict control of sterility & nonpyrogenicity than other formulation.


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Necessities of Parenteral preparations:

Sterility(must)

Pyrogen(must)

Free from particulate matter(must)

Clarity(must)

Stability(must)

Isotonicity(should)

Solvents or vehicles used must meet special purity and other standards.

Restrictions on buffers, stabilizers, antimicrobial preservative. 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

Subcutaneous

tissue


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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. Intrapleural8. 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

InsulinScopolamine

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 to

restore blood volume & toprevent tissuedehydration.

Combination of parenteral dosage forms foradministration as a unit product is known as an IVadmixture.

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 artery2 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-spinalDirectly given into the spinal cord

1 to 4 ml

24 to 28 gaugeMust be isotonic

Given:

LA

Analgesics

Neuroleptics


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Intraarticular:

Given directly into the joints

2 to 20 ml5 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 channel blockers


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Intradermal:

Also called as diagnostic testing0.05 ml

inch, 25 to 26 gauge needle

Should be isotonic Given:

Diagnostic agents


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Official Types of Injections:

1. Solutions of Medicinal

Example: Codeine Phosphate InjectionInsulin Injection

2. Dry solids or liquid concentrate does not

contain diluents etc.Example: Sterile Ampicillin Sodium

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

injectionExample: Methicillin Sodium for injection


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4. Suspensions

"Sterile....Suspension"

Example: Sterile Dexamethasone AcetateSuspension

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 for Suspension

6. Injectable Emulsions:

Example: Propofol injection


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Formulation of Parenteral:1. Therapeutic agents

2. Vehiclesi. Water

ii. Water miscible vehicles

iii. Non- aqueous vehicles

3. Added substances (Additives)

i. Antimicrobialsii. 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 for Quality 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 vehicleso 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 orconcentration

Used to prevent the multiplication of micro-organisms

Ex..

Benzyl alcohol ------ 0.510 %

Benzethonium chloride -- 0.01 %

Methyl paraben ---- 0.010.18 %

Propyl paraben --- 0.0050.035 %

Phenol --- 0.0650.5 %



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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 -- 0.020.1 %

Sodium bisulphite-- 0.10.15 %

Sodium metabisulphite-- 0.10.15 %

Thiourea - 0.005 %

B) Blocking agents:

Ascorbic acid esters- 0.010.015%

BHT- 0.0050.02 %

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 the metallicions present in the formulation so that the

ions will not interfere during mfg. of

formulation.

They form a complex which gets dissolved inthe solvents.

Examples:

Disodium edetate0.00368 - .05 % Disodium calcium edetate - 0.04 %

Tetrasodium edetate0.01 %


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Stabilizers:

As parenterals are available in solution form

they are most prone to unstabilize

Used to stabilize the formulation

Maintain stable

Examples:Creatinine0.5- 0.8 %

Glycerin1.52.25 %

Niacinamide1.25 -2.5 %

Sodium saccharin0.03 %

Sodium caprylate0.4 %


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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

PEG40 castor oil

PEG300

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 well as

stabilizers for the pH. Isotonicity depends on permeability of a living

semipermaeable 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

Sterile area


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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

C

K

R

O

O

M

COMPOUNDING

AREA

CLEAN UP

AREA

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 so that to avoid

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 forfilling

Not essential that the area is aseptic

Strict precaution is taken to prevent contamination fromoutside

Cabinets & counters: SS

Ceiling & walls : sealed & painted


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Aseptic area: Filtration & filling into final containers & sealing is

done 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 above analysisthe decision is to be taken for reprocessing or

others..


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Finishing and packaging area:

After proper label, the product is given for

packingPacking is done to protect the product from

external environment

The ideal Packing is that which protects theproduct during transportation, storage, shipping& handling.

The labeled container should be packed incardboard or plastic containers

Ampoules should be packed in partitionedboxes.


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Preparations for IV Fluids:

LVPs which are administered by IV routeare 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 agents areadded

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 mannitolUsed 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 IPRinger lactate solution for injection IP

Common uses :

Used in surgery patientsIn replacement therapy

Providing basic nutrition

For providing TPNAs a vehicle for other drug subs.


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IV ADMIXTURES

Definition:

When two or more sterile products are added to

an IV fluid for their administration, theresulting combination is known as IVadmixture.

In hospitals, prepared by nurses by combining

or mixing drugs to the transfusion fluids.The drugs are incorporated in to bottles of LV

transfusion 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 IV

admixture:

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. Thisis a complete form of nutrition, containing

protein, sugar, fat and added vitamins andminerals as needed for each individual.

Total Parenteral Nutrition (TPN) may be

defined as provision of nutrition for metabolic

requirements and growth through the parenteralroute.

Total Parenteral Nutrition

Total Parenteral Nutrition (TPN)


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Total Parenteral Nutrition (TPN)

(Intravenous Nutrition)

TPN refers to the provision

of all required nutrients,

exclusively by the

Intravenous route.

Parenteral Nutrition (PN)can be used to

supplement 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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Electrolytes

Electrolyt

e.

Daily Requirement Standard Concentration

Na 60-150 meq 35-50 meq/L

K 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?
http://en.wikipedia.org/wiki/Guthttp://en.wikipedia.org/wiki/Chronic_%28medicine%29http://en.wikipedia.org/wiki/Enteral_feedinghttp://en.wikipedia.org/wiki/Enteral_feedinghttp://en.wikipedia.org/wiki/Enteral_feedinghttp://en.wikipedia.org/wiki/Enteral_feedinghttp://en.wikipedia.org/wiki/Chronic_%28medicine%29http://en.wikipedia.org/wiki/Gut


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

digestive system has shut down (for instance by

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 the life ofa 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 Nutrition3-In-1 Admixture

All-In-One Admixture

PPN: Peripheral Parenteral Admixture

I di ti f TPN


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Indications for TPNShort-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 mLand 4 L is provided.

The pump infuses a small amount (0.1 to 10

mL/hr) continuously in order to keep the vein

open.

Feeding schedules vary, but one common regimen

ramps up the nutrition over a few hours, levels off

the rate for a few hours, and then ramps it downover a few more hours, in order to simulate a

normal set of meal times.

The nutrient solution consists of water glucose salts
http://en.wikipedia.org/wiki/Infusion_pumphttp://en.wikipedia.org/wiki/Sterilization_%28microbiology%29http://en.wikipedia.org/wiki/Sterilization_%28microbiology%29http://en.wikipedia.org/wiki/Infusion_pumphttp://en.wikipedia.org/wiki/Waterhttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Salthttp://en.wikipedia.org/wiki/Salthttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Water


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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

increasedblood sugarcommonly occurs with TPN,

insulin may also be added to the infusion. Often though, an insignificant amount of insulin is

added, sometimes 10 units or less in 2 liters of TPN.

In actuality, the patient will probably get less thanthat.

Occasionally, other drugs are added as well,

sometimes unnecessarily.

Complications of TPN
http://en.wikipedia.org/wiki/Waterhttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Salthttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Vitaminhttp://en.wikipedia.org/wiki/Fathttp://en.wikipedia.org/wiki/Electrolytehttp://en.wikipedia.org/wiki/Blood_sugarhttp://en.wikipedia.org/wiki/Insulinhttp://en.wikipedia.org/wiki/Insulinhttp://en.wikipedia.org/wiki/Blood_sugarhttp://en.wikipedia.org/wiki/Electrolytehttp://en.wikipedia.org/wiki/Fathttp://en.wikipedia.org/wiki/Vitaminhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Salthttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Water


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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 in

diffusibility (distribution) thr

membrane.

The fluids used in dialysis are known as

dialysis fluids.

DIALYSIS FLUIDS


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General uses :Renal failure waste product is removed

Maintain electrolytes

Also called as haemodialysis orintraperitonealdialysis

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 the

body 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 of Concentrated Haemodialysis Fluid BPC

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

Storage: store in warm place as it is liable to convert into crystals onstorage.

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 ml

22.24 gm

0.4 gm

100 ml


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IntraperitonealDialysis:

Peritoneal cavity is irrigated with dialysis fluid.

Peritoneumacts as a semi permeable membrane

Toxic subs. excreted by kidney are removed.

Requirements:

Sterile

Pyrogen free


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Composition of Fluid I ntraper itonealDialysis 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 containerstaken 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 stateof 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 asepticconditions

regular microbiological monitoring should

be carried out

1 2 C l di i


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1.2.Culture conditions

Appropriate conditions for the growth of

any surviving organism should be providedby the culture media selection.

1 2 C lt diti


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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 growth 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 C lt di f t ilit t ti


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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

1 2 3 1 Fl id thi l ll t di


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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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1 2 3 2 S b i di t di


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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

1 2 3 3 Fertility control of the media


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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 of themicro-organisms occurs

1.2.3.4.Effectiveness of the media


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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 of


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y

the product

Membrane filtration

Direct inoculation of the culture medium

1 3 1 Membrane filtration


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1.3.1. Membrane filtration

Appropriate for : (advantage)filterable aqueous preparations

alcoholic preparations

oily preparationspreparations 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 membrane


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filtration

pore size of 0.45 m

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

filt ti


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filtration sterilization of filtration system and membrane

filtration of examined solution under aseptic

conditions(suitable volume, dissolution of solid particleswith 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 systemto 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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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, oilyliquids, 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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g

wide applications

a large volume can be tested with one filter

smaller volume of culture media is required

applicable to substances for which nosatisfactory inactivators are known

neutralization is possible on the filter

subculturing is often eliminated shorter time of incubation compared with

direct inoculation

1.4. Observation and


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interpretation of the results

Examination at time intervals during the

incubation period and at its conclusion

When the sample passes the test and whenfails?

When the test may be considered as invalid?

There is low incidence of accidentalcontamination or false positive results

1 5 Sampling


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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 for


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using the test for sterility

Precautions against microbial contamination

The level of assurance provided by a

satisfactory result of a test for sterility asapplied to the quality of the batch is a

function of:

The homogeneity of the batchThe conditions of manufacture

Efficiency of the adopted sampling plan

Guidelines


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Gu de es

In the case of terminally sterilized products:physical proofs, biologically based and

automatically documented, showing correct

treatment through the batch during sterilization are

of greater assurance than the sterility test Products prepared under aseptic conditions:

sterility test is the only available analytical method

only analytical method available to theauthorities who have to examine a specimen

of a product for sterility.


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PYROGENS AND PYROGEN TESTING


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I Love The Rabbit!

Pyrogens


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y g

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


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Generalized structure of endotoxins


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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

Test for pyrogens = Rabbit test


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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 USPXII (1942)

The test consists of measuring the rise in

body temperature in healthy rabbits by theintravenous injection of a sterile solution of

the 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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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 0.6C is not used in the main test

Rabbit test -i


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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 maximum temperature

all rabbits should have initial T: from 38.0 to 39.8C the differences in initial T should not differ from

one another by more than 1C

Rabbit test


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Interpretation of the results:

the test is carried out on the first group of 3 rabbits; ifnecessary 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 on the

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 horseshoecrab (Limulus polyphemus or Tachypleus

tridentatus).

The name of the test is alsoLimulus amebocyte

lysate (LAL) test

Limulus polyphemus = horseshoe crab


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p yp

Mechanism of LAL


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the test is based on the primitive blood-

clotting mechanism of the horseshoe crab

enzymes located with the crab's amebocyte

blood cellsendotoxins

initiation of an enzymatic coagulation cascade

proteinaceous gel


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Test performance (short)


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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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Three different techniques: the gel-clot technique - gel formation

the turbidimetric technique - the development

of turbidity after cleavage of an endogenoussubstrate

the chromogenic technique - the development

of color after cleavage of a synthetic peptide-

chromogen complex

LAL test


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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 decision ismade upon Method A unless otherwise indicated in

the monograph.

Gel-cloth technique (Methods A, B)


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allows detection or

quantification of

endotoxins

clotting of the lysate in the

presence of endotoxins.

1.Preparatory testing Confirmation of the

labeled lysate sensitivity

Tests for interfering

factors

Gel Clot

Invert Tube in Smooth

Motion

Gel-cloth technique (Methods A, B)-

t


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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 solution bytitration to an end-point.

procedure is similar as in the limit test

The results are expressed as concentration of endotoxin asless, equal or greater than (labeled lysate sensitivity).

Turbidimetric technique (Methods C,F)


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) photometric test to measure the increase in

turbidity

end-point test (Method F): quantitative relationshipbetween the endotoxin concentration and the turbidity

(absorbance or transmission) of the reaction mixture at theend of an incubation period.

kinetic test (Method C): a method to measure either thetime (onset time) needed for the reaction mixture to reach a

predetermined absorbance, or the rate of turbidity

development.


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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 should

prove 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

the test for pyrogens

Advantages of LAL test Fast - 60 minutes vs. 180 minutes


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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 toxicological activityin the rabbit (e.g. insulin)

Blood products which sometime give misleading results in therabbit

Water for injection where LAL test is potentially more stringent

and readily applied


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Particulate Matter Monitoring

Definition:


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Unwanted mobile insoluble matter other

than gas bubbles present in the given

product.

It may be dangerous when the particle size

is larger than R.B.C. & may block the blood

vessel.

This type of products are immediately

rejected from the batch.


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The limit test for particulate matter is prescribed in

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 matter


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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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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

Visual method:


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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 & if particlefound 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 a bright

white light source & photoiodide sensor.

Identification of Particulate Matter


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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

As per USP


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As per USP

LVP : NMT 50 particles/ ml (size 10 or more than10 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.

Parenteral Sro A

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Transcript of Parenteral Sro A