in process quality control tests (ipqc) for parenteral or sterile dosage forms

IPQC for Parenterals or Sterile Dosage Forms | Mr. Sagar Kishor Savale

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In Process Quality Control Tests (IPQC) For Parenteral

or Sterile Dosage Forms


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In Process Quality Control Tests (IPQC) For Parenteral

or Sterile Dosage Forms

Mr. Sagar Kishor Savale

M. Pharm (Pharmaceutics)


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Parenteral Route

It is a route of administration other than the oral route, this route of administration

bypasses the alimentary canal.

It includes, I.V., I. M., Subcutaneous route for parenteral administration.

Types of Parenteral

1. Based on types of packaging

a) Single dose units: ampoules, infusions and prefilled disposable syringes.

b) Multiple dose units: multiple dose vials.

2. Based on the production and control

a) Small volume parenterals: volume < 100 ml,

b) Large volume parenterals: volume ≥ 100 ml,

In Process Quality Control Tests (IPQC) for Sterile Dosage Form

Quality Assurance: The planned and systematic activities implemented in a quality system so

that quality requirements for a product or service will be fulfilled.

Quality Control: The procedure or set of procedures intended to ensure that a manufactured

product or performed service adheres to a defined set of quality criteria or meets the

requirements of the client or customer.

IPQC

IPQC means controlling the procedures involved in manufacturing of the dosage forms

starting from raw materials purchase to dispatch of the quality product in ideal packaging.

It monitors all the features of the product that may affect its quality and prevents errors

during processing.

These are the tests performed between QA and QC and provides for the authorization of

approved raw materials for manufacturing based on actual laboratory testing generally

called as IPQC such as physical, chemical, microbiologic and biologic tests.

IPQC is concerned with providing accurate, specific & definite descriptions of the

procedures to be employed, from, the receipt of raw materials to the release of the finished

dosage forms.


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1. Leakage Test (visual inspection, bubble test, dye tests and vacuum ionization test)

Leakage test is employed to test the package integrity. Package integrity reflects its ability to

keep the product in and to keep potential contamination out”. It is because leakage occurs when

a discontinuity exists in the wall of a package that can allow the passage of gas under pressure

or concentration differential existing across the wall. Leakage test can be done by dye bath test.

Dye Bath Test

The test container is immersed in a dye bath. Vacuum and pressure is applied for some time.

The container is removed from the dye bath and washed. The container is then inspected for

the presence of dye either visually or by means of UV spectroscopy. The dye used may be of

blue, green, yellowish-green color. The dye test can be optimized by use of a surfactant and or

a low viscosity fluid in the dye solution to increase the capillary migration through the pores.

The dye test is widely accepted in industry and is approved in drug use. The test is inexpensive

and is requires no special equipment required for visual dye detection. However, the test is

qualitative, destructive and slow. The test is used for ampoules and vials.

2. Clarity Test

Clarity testing is carried out to check the particulate matter in the sample. In this test transparent

particles or white particles observed against the black background and the black or dark

particles observed against the white background.

3. pH

Checking the bulk solution, before filling for drug content, pH, color, clarity and completeness

of solution.

The pH of a formulation must be considered from following standpoint:

The effect on the body when the solution is administered

The effect on stability of the product

The effect on container-closure system

pH measurement

pH is measured by using a pH meter .

pH meter is initially calibrated with respective buffer capsule then the pH of the

preparation is measured.


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4. Particulate matter in injections

The preparations intended for parenteral use should be free from particulate matter and should

be clear when inspected visually. Two methods are described by USP according to the filled

volume of the product to be tested. For large volume parenterals (LVP's), a filtration followed

by microscopical examination procedure is used. For small volume parenterals (SVP's) a light

obscuration based sensor containing electronic liquid borne particle counter system is used.

The USP standards are met if the LVP's under test contain NMT 50 particles per ml of 10μ m,

and NMT 5 particles per ml of 25μm in an effective linear dimensional fashion. The USP

standards are met if the SVP's under test contain NMT 10,000 particles per container of 10 μm,

and NMT 1000 particles per container of 25μm in an effective spherical diameter.

Table 1: Limits for particle number as per IP, BP, EP and JP

Volume of solution Particle

size ≥ 10

μm

Particle

size ≥25

μm

Small volume injections

(< 100 ml)

3000 per

container

300 per

container

Large volume injections

(> 100 ml)

12 per ml 2 per ml

5. Sterility Test

Sterility can be defined as the freedom from the presence of viable microorganisms.

It is done for detecting the presence of viable forms of bacteria, fungi and yeast in

parenteral products.

The test for Sterility must be carried out under strict aseptic conditions in order to avoid

accidental contamination of the product during test.

All glassware's required for the test must be Sterile.

Sterility testing attempts to reveal the presence or absence of viable microorganisms 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.

Major factors of importance in sterility testing:

The environment in which the test is conducted

The quality of the culture conditions provided


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The test method

The sample size

The sampling procedure

Environmental conditions:

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.

Culture conditions:

Appropriate conditions for the growth of any surviving organism should be provided

by the culture media selection.

Factors affecting growth of bacteria:

Nutrition

Moisture

Air

Temperature

pH

Light

Osmotic pressure

Growth inhibitors

Culture media used for sterility testing:

1. Fluid thioglycolate medium

2. Soybean casein digest medium

1. Fluid thioglycolate medium (FTM):

FTM provides both aerobic and anaerobic environments within the same medium.

FTM is an excellent medium for the detection of bacterial contamination.

Thioglycolate has the advantage of neutralizing the bacteriostatic properties of

mercurial preservatives.

Composition:

L-cysteine, trypticase peptone, dextrose, yeast extract, sodium chloride, sodium

thioglycolate, resazurin, agar, purified water, final pH 7.1


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Specific role of some ingredients primarily intended for the culture of anaerobic

bacteria.

Incubation of the media: 14 days at 30 -35°C

2. Soybean casein digest medium:

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 -25°C

Composition

Trypticase soya broth, trypticase peptone, phytone peptone, sodium chloride, dipotassium

phosphate, dextrose, purified water, final pH 7.3

Sterility test methods

[1] Direct inoculation method

[2] Membrane filtration method

[1] Membrane filtration methods

Selection of filters for membrane filtration:

Pore size of 0.45µ effectiveness established in the retention of microorganism’s appropriate

composition the size of filter discs is about 50 mm in diameter

The procedure of membrane filtration

Sterilization of filtration system and membrane filtration of examined solution under

aseptic conditions.

Filtration of the sample through a membrane filter having the nominal size of 0.45µ

and a diameter of 47mm.

After filtration the membrane is removed aseptically from the metallic holder and

divided into two halves.

The first half is transferred into 100 ml of culture media meant for fungi and

incubated at 20˚ to 25 ˚c for not less than seven days.

The other half is transferred into 100ml of fluid thioglycolate medium and incubated

at 30 to 35 ˚c for not less than 7 days.


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Advantages of membrane filtration over direct inoculation method Greater sensitivity

The entire contents can be tested providing an advantage in the sterility testing of

LVP and increasing the ability to detect contamination.

The antimicrobial agent and antimicrobial solutes in the product sample can be

eliminated by rinsing prior transferring the filter into test tubes of media

Thereby minimizing the incidence of false-negative test results.

Organisms present in an oleaginous product can be separated from the product during

filtration and cultured in a more desirable aqueous medium.

Disadvantages

This method cannot differentiate the extent of contamination between units if present

because all product contents are combined and filtered through a single filter and

cultured in single test tube.

There exists a higher probability of inadvertent contamination in manual operations.

Samples size to be taken

[2] Direct inoculation method

Required quantities of liquid is removed from the test containers with a sterile pipette /

sterile syringe.

Aseptically transfer the specified volume of the material from each container to vessel

of culture medium


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Mix the liquid with medium but not aerate excessively.

Incubate the inoculated media for not less than 14 days, unless otherwise specified in

the monograph at 300c - 350c in the case of fluid thioglycolate medium and 200c - 250

c for soybean casein digest medium.

When materials examined renders the medium turbid so presence / absence of microbial

growth cannot be determined readily by visual examination transfer suitable portions

of medium to fresh vessels of the same medium between 3 rd. and 7 th day after test is

started.

Continue incubation of the transfer vessel for not less than 7 additional days after

transfer and total of NLT 14 days.

Interpretation of results

At the end of the incubation period the following observations are possible:

No evidence of growth; hence the preparation being examined passes the test for

sterility.

If there is evidence of growth, retesting is performed using the same number of samples,

volumes to be tested and the media as in the original test. If no evidence of microbial

growth is then found, the preparation being examined passes the test for sterility.

If there is again evidence of the microbial growth then isolate and identify the

organisms. If they are not readily distinguishable from those growing in the containers

of the first test then the preparation being examined fails the test for sterility.

If they are distinguishable from the organisms of the first test then again do the test

using twice the number of samples. The preparation being examined passes the test for

sterility in case there is no evidence of microbial growth. In case there is evidence of

growth of any microorganisms in second re –test, the preparation being examined fails

the tests for sterility.

Test for packaging containers

Powdered glass Test:

Use crushed glass containers in 250-ml conical flask, add 50 ml high purity water, and

cap the flask with borosilicate glass beaker

Place the containers in the autoclave and close it securely hold temperature at

1210c±20c for 30 min., counting from the time this temperature is reached.


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Cool the flask, decant the water from the flask into a clean vessel, and wash the

residual powdered glass with high purity water, add 5 drops methyl red solution,

titrate immediately with 0.02 N sulphuric acid.

Record the volume of 0.02N Sulphuric acid used to neutralize the extract from 10 g of

the prepared specimen of glass.

Water attack at 121°C

Rinse 3 or more containers with high purity water. Fill each container to 90% of its

capacity with high purity water.

Cap all the flasks with borosilicate glass beaker, place in the autoclave at 121 C for 60

min.

Test for plastics

Leakage test: 10 containers are filled with the parenteral fluid and inverted for 24 hrs. And

checked for any leakage.

Transparency: Dilute the preparations and compare the cloudiness with the control that is

water.

Water vapour permeability: Containers stored at 20-25o c at 60±5% Rh for 14 days and

check for water vapour permeability.

Rubber closure tests

Sterilization


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Fragmentation

Self sealability

Clarity and color

Labels and labelling

The label states the name of the preparation (in case of a liquid preparation)

The percentage content of drug in a specified volume (in case of dry preparation)

The route of administration

Storage condition

Expiration date

Name of the manufacturer

The lot number

Containers for injection that are intended for use as dialysis, or irrigation solution are

labelled to indicate that the contents are not intended for use by IV infusion.

Injection intended for veterinary use are labelled to that effect the containers are so

labelled that a sufficient area of the container remains uncovered for its full length to

permit inspection of the contents.

6. Pyrogen test

Pyrogen: “Pyro” (Greek = Fire) + “gen” (Greek = beginning).

Fever producing, metabolic by-products of microbial growth and death.

Bacterial pyrogens are called “Endotoxins”. Gram negative bacteria produce more

potent endotoxins than gram + bacteria and fungi.

Endotoxins are heat stable lipopolysaccharides (LPS) present in bacterial cell walls, not

present in cell-free bacterial filtrates.

Stable to at least 175ºC; steam sterilization ineffective.

Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm) so endotoxins can

easily pass through 0.22μm filters.

Sources: Water (main), raw materials, equipment, process environment, people, and

protein expression systems if using gram negative bacteria.

Other Source: Equipment, Containers (Glass, plastic, metal), Solvent and Solute.


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Biological properties of endotoxin

Pyrogen elevate the circulatory levels of inflammatory cytokines which may be

followed by fever, blood coagulation, hypotension

Low doses of Pyrogen: asymptomatic inflammation reaction Moderate doses: fever &

changes in plasma composition

High doses: cardiovascular dysfunction, vasodilation, vasoconstriction, endothelium

dysfunction, multiple organ failure & finally death.

Elimination of pyrogens

1. Dry heat sterilization: For glass wares, metal equipments, powders, waxes, oils, heat stable

drugs.

650 o C temp - 1 min



2. Ultra filtration

3. Reverse osmosis: RO membrane is composed of cellulose acetate phthalate/ polyamide

4. Distillation

5. Adsorption method

6.1. Rabbit Test

This test basically involves the injection Sample solution which is to be tested into a Rabbits

which are used as test animals through ear vein.

The Temperature sensing probe (Clinical Thermometer, Thermosestor or similar probe) into

a rectum cavity of Rabbit at the depth of 7.5 cm, the test solution must be warmed at 37º

prior to injection.

Then Rectal temperature is recorded at 1, 2, 3 hr subsequent to injection.

This test is performed in separate area designed solely for this purpose under environmental

conditions similar to animal house should be free from disturbances that likely to excite

them.

Initially this test is performed on 3 Rabbits but if required results are not obtained this test

is repeated on 5 additional Rabbits with same sample solution administer to initial 3 rabbits.

Prior to 1hr of injecting sample solutions the control temperatures of rabbits are determined.

Use only those rabbits whose control temperature is no vary by more than 1 ºc.


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Table 2: Interpretation of Result (Rabbit Test)

6.2. Bacterial endotoxin test

LAL (Limulus Amebocyte Lysate) test is used to characterize the bacterial endotoxin that

may be present. The USP reference standard contains 10,000 USP endotoxins per vial. The

LAL reagent is used for gel-clot formation. The test is performed using stated amounts of

volumes of products, standard, positive control, negative control of endotoxin. The tubes are

incubated at 37±1ºC FOR 60 ±2 minutes. When the tubes are inverted at 180ºC angle,

formation of firm gel confirms positive reaction. While formation of a viscous gel that doesn't

maintain its integrity or absence of a firm gel confirms negative reaction. The test is invalid if

the standard endotoxin or positive product control doesn't show end point within ± 1. Two fold

dilution from label claim sensitivity of LAL reagent or if the negative control shows gel-clot

end point.

The following methods can be used to monitor the endotoxin concentration:

Method A - Gel- clot limit test method

Method B -Semi quantitative gel clot method

Method C - Kinetic turbidimetric method

Method D - Kinetic chromogenic method

Method E - End point chromogenic method

7. Content Uniformity and Weight

Determine the content of the active ingredient of each of 10 containers taken at random. The

preparation under examination complies with the test if the individual values thus obtained are

all between 85 and 115 percent of the average value. The preparation under the examination

fails to comply with the test if more than one individual value is outside the limits 85 to 115


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percent of the average value or if any one individual value is outside the limits 75 to 125 percent

of the average value. If one individual value is outside the limits 85 to 115 percent but within

the limits 75 to 125 percent of the average value, repeat the determination using another 20

containers taken at random. The preparation under examination complies with the test if in the

total sample of 30 containers not more than one individual value is outside the limits 85 to 115

percent and none is outside the limits 75 to 125 percent of the average value.

Table 3: Limits for Uniformity of Weight

Pharmaceutical

Formulation

Average

Mass

Percentage

Deviation (%)

Powders for

parenteral use

More than 40

mg

10

Powders for eye

drops

Less than 300

mg

10

Powders for eye

lotions

300 mg or

more

7.5

8. Extractable Volume

a) Single Dose Containers

Method I: Where the nominal volume does not exceed 5ml. Use 6 containers, 5 for the tests

and 1 for rinsing the syringe used. Using a syringe with appropriate capacity, rinse the syringe

and withdraw as much as possible the contents of one of the containers reserved for the test

and transfer, without emptying the needle, to a dry graduated cylinder of such capacity that the

total combined volume to be measured occupies not less than 40% of the nominal volume of

the cylinder. Repeat the procedure until the contents of the 5 containers have been transferred

and measure the volume. The average content of the 5 containers is not less than the nominal

volume and not more than 115% of the nominal volume. Alternatively the volume of contents

in milliliter can be calculated as mass in grams divided by the density.

Method II: Where the nominal volume is more than 5ml. Transfer the contents of not less than

3 containers separately to dry graduated cylinders such that the volume to be measured

occupies not less than 40% of the nominal volume of the cylinder and measure the volume

transferred. The contents of each container are not less than the nominal volume and not more

than 110% of the nominal volume.


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9. Volume Filled

Volume in container

An injection container is filled with a volume in slight excess of the labelled size Physical and

chemical tests.

Determination of filled volume:

10 mL or more: 1 container

3-10 mL: 3 containers

Less than 3 mL: 5 containers

Other Quality Control Test

Test Apparatus pH pH Meter

Viscosity Ostwald viscometers

Osmolality Osmometer (count of the number of particles in a fluid sample)

Conductivity Conductometer (conductivity of vehicle used in sterile

preparation) (Pure Water 0.55mS/cm)

Temperature For Heat

Sterilization

Thermometer, Digital Thermometer (To maintain the constant

temperature during heat sterilization of product)


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Environmental Control Test

Traffic control: A carefully designed arrangement to control traffic. Personnel should be

permitted into aseptic area. Only after following rigidly prescribed procedures.

Surface disinfection: Must be inherently neat, orderly, reliable and alert. Should be in good

health.

Air control (HEPA filters): It is composed of glass fibers and filters. It is 99.97% efficient

removes particles of 0.3 um size and larger. (Velocity is 100+/- 20 ft/min).


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Aseptic Techniques:

Aseptic area: the area which are used to kill the pathogenic microorganisms.

HEPA filter integrity test

Air flow pattern

Air velocity study

Heat sensitization study

Heat distribution study


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Instrumental Methods

This is also called as the particle count method particle counting may be based on any one

of the following principles; change in

Electrical resistance

Light absorption

Light scattering


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Physical and Chemical test

Identity tests

These tests are qualitative chemical methods used to conform the actual presence of compound

for example color formation, precipitation.

Quality tests

These tests are the physical methods used to measure accurately the characteristic properties

of drug. For example: Absorbance, refractive index.

Purity tests

Purity tests are designed to estimate the level of all known and significant impurities and

contaminants in the drug substance under evaluation. For example: Tests for clarity of

solutions, Acidity, Alkalinity.

Potency tests

Potency tests are assays that estimate the quantity of an active ingredient in the drug.


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Aqueous vehicle For Sterile Product

Quality Tests Water for Injection

Chloride Test

Calcium Test

Heavy Metal Test

Sulphate Test

Ammonia test

Other test for Water for Injection

Conductivity Test: Limit (not more than 3 mS).

pH: Limit (5 to 7).

Total Organic Carbon (TOC): Limit not more than 500 PPB (part per billion).

Water for injection

• Not sterilized and pyrogen free

• It is intended to be used within 24 hours after

collection

• Total solid contents not more than 1 mg/100 ml

Sterile water for injection

• It must be pyrogen free

• endotoxin level is not more than 0.25 USP

• single dose containers not larger than 1 liter

Bacteriostatic water for injection

• One or more suitable antimicrobial agents

• It is packaged in prefilled syringes or in

vials

• Not more than 30 ml of the water

• Bacteriostatic agents such as benzyl alcohol

may cause gasping syndrome (multiorgan

failure)).


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Processing of parenteral preparation

Lay out of Parenteral Manufacturing Area

Cleaning of containers and

Equipment

Collection of materials

Preparation of parenteral products

Filtration

Filling the preparation in final

container

Sealing the container

Sterilization

Evaluation of the parenteral

preparation

Labeling & packaging


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Grades for Evaluation of Sterile products

Microbial count according to Grade

Microbial count of Viable Micro-organism

Microbial count of Non-Viable Micro-organism


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Lyophilization or freeze drying

Lyophilization or freeze drying is a process in which water is removed from a product after it

is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapour

without passing through a liquid phase.

The process consists of three separate, unique, and interdependent processes;

Freezing,

Primary drying (sublimation), and

Secondary drying (desorption).

The Lyophilization process generally includes the following steps

Dissolving the drug and excipients in a suitable solvent, generally water for injection

(WFI).

Sterilizing the bulk solution by passing it through a 0.22 micron bacteria-retentive

filter.

Filling into individual sterile containers and partially stoppering the containers under

aseptic conditions.

Transporting the partially stoppered containers to the lyophilizer and loading into the

chamber under aseptic conditions.

Freezing the solution by placing the partially stoppered containers on cooled shelves

in a freeze-drying chamber or pre-freezing in another chamber.

Applying a vacuum to the chamber and heating the shelves in order to evaporate the

water from the frozen state.

Complete stoppering of the vials usually by hydraulic or screw rod stoppering

mechanisms installed in the lyophilizers.

There are many new parenteral products, including anti-infectives, biotechnology

derived products, and in-vitro diagnostics which are manufactured as lyophilized

products.

Additionally, inspections have disclosed potency, sterility and stability problems

associated with the manufacture and control of lyophilized products.


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Biological indicators (BIs)

Biological indicators (BIs) are the most accepted means of monitoring the sterilization

process because they directly determine whether the most resistant microorganisms

(e.g., Geobacillus or Bacillus species) are present rather than merely determine whether

the physical and chemical conditions necessary for sterilization are met.

Biological indicators (BIs) are used for determination of Bacillus species.

BIs such as, ethylene oxide, ethylene chlorohydrin, ethylene hydroxide and

halogenated ethylenehydrine.

BIs was determined by Z value and D Value.

Z value is define as Temperature is required to higher death rate.

D value is define as reduction of microbial population by 90 % of the factor 10.

F0 value is define as the time required at temperature to produced lethality is similar to

produce 121ºc at stated time.

D value is 10 factor greater than Z value.

e.g. D value is 121 ºc as the Z value is 10 ºc.


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Sterile Products

Injections

Injections are sterile solutions, emulsions or suspensions prepared by dissolving emulsifying

or suspending the active ingredients and other additives in water for injection or other suitable

non-aqueous vehicle or in mixture of two, if they are miscible.

Powder for injection or Infusion

Powder for injections are sterile solid substances (including freeze dried material) which are

distributed in their final containers which, when shaken with the prescribed volume of the

appropriate sterile liquid, rapidly form clear and practically particle-free solutions or uniform

suspension.

Intravenous Infusion

These are sterile aqueous solutions or emulsions with water as continuous phase. When a drug

is infused intravenously at a constant rate, a plateau concentration will be reached progressively

in the most frequently most of the cases follows first order kinetics. On starting the infusion,

there is no drug in the body and therefore, no elimination. The amount of drug in the body then

rises, but as the drug concentration increases, so does the rate of elimination. Thus, the rate of

elimination will keep rising until it matches the rate of infusion. The amount of drug in the

body is then constant and is said to have reached a steady state or plateau.

Implants

Implants are sterile solid preparations of size and shape for implantation into body tissues so

as to release active ingredient over an extended period of time. An implant is a medical device

manufactured to replace a missing biological structure, support a damaged biological structure,

or enhance an existing biological structure. Medical implants are man-made devices, in contrast

to a transplant, which is a transplanted biomedical tissue. The surface of implants that contact

the body might be made of a biomedical material such as titanium, silicone or apatite depending

on what is the most functional. In some cases implants contain electronics e.g. artificial

pacemaker and cochlear implants. Some implants are bioactive, such as subcutaneous drug

delivery devices in the form of implantable pills or drug-eluting stents.


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Concentrated Solutions for Injections

Concentrated solutions for injections are sterile solutions that are intended for administration

by injection or by IV infusion only after dilution with suitable dilution with a suitable liquid.

After dilutions these preparations should comply with the requirements of tests for injection or

intravenous infusions as appropriate.

***************************************************************************

Contact

Mr. Sagar Kishor Savale,

M. Pharm (Pharmaceutics),

Mobile No.: +91 9960885333,

Email: [email protected]

in process quality control tests (ipqc) for parenteral or sterile dosage forms

Health & Medicine