CONTENTS

• Water Testing

• Moisture content determination

• TOC Analysis

• Bacterial Endotoxin Test

• Environment Monitoring
 Water Testing
Water plays a very crucial role in pharmaceutical
industries and is divided into 3 types based on its
applications :

3. Raw water (RW)

5. Demineralized water (DMW)

7. Water For Injection (WFI)

Raw Water
is potable water filtered through gravel
contains ions and suspended particles
is further processed into DM water and WFI

Analysis
Samples of raw water are collected and analyzed for the
following parameters –

• Chemical
• Microbiological
 Chemical Analysis

1. Odour - Examine the taste of raw water sample physically. It should

be odourless

3. pH - Measure by suitable and calibrated pH meter. pH range is 6.5-

8.5.

5. Conductivity - Measure suitable and calibrated conductivity meter.

Limit – NMT 750µs/cm

4. Hardness - To 100 ml water add 2 ml of ammonia buffer pH 10, 50

mg of mordant black II and 0.01M disodium edetate until a pure blue
colour is produced. Measure the volume of disodium edetate used and
calculate the hardness by the following formula: -

Hardness (mg/liter) = EDTA used (ml) 1000 / Sample Vol.

Microbial Analysis

1. Total Plate Count

c) Pour plate method - Dispense 1 ml of each sample into two sterile

petridishes and add 15-20 ml of melted Plate Count Agar at 40- 45C.
Invert the petridishes and incubated at 30-35C for 48-72 hours.
Count the number of colonies and express in term of CFU/ml .
Limit of viable microbial count is NMT 500 CFU/ml

h) Membrane Filtration Method -Load the membrane(.45 m) onto

the filter holder.Sterilize this apparatus by autoclaving. Pour 1ml of
sample directly into funnel Apply vacuum to filter sample and transfer
immediately to plate count Agar.Incubate plates in inverted position
at 30-35C,48-72hrs . Count no. of colonies and express as cfu/ml .
Pathogens

The sample is mainly tested for the following four pathogens :

a) Salmonella species
b) Escherichia coli
c) Pseudomonas aeruginosa
d) Staphylococcus aureus

First filter 100 ml sample through sterile funnel and inoculate membrane
in Soyabean Casein digest Medium (SCDM) and incubate at 30 -35
C,24-48 hrs. After incubation inoculate sterile 10-ml foll. enrichment
broth using 1ml incubated SCDM.

i.) Selenite F. Broth for Salmonella species

ii) Mac Conkeys Broth for E. coli
iii) Cetrimide Broth for Pseudomonas aeruginosa
Test for Salmonella species:
If growth is present in Selenite F. Broth, presumptive test is done by
inoculating and incubating the following selective media at 30-35C for
24 hrs. After incubation detect colour of colonies.
Test for Escherichia coli :

If acid and gas production occur in MacConkey’s Broth tube, then

presumptive test using the foll. selective media is done :
Test for Pseudomonas aeruginosa :

If the inoculated Cetrimide Broth tube shows growth with the

greenish/ bluish pigmentation, inoculate the following selective media
plates and incubate at 30-35C for 24-28 hours for presumptive
identification of the pathogen.
Test for Staphylococcus aureus-

If black settled growth is present at the bottom of the Giolitti Cantoni

Broth under anaerobic condition. Inoculate the following selective
media and incubate at 30-35C for 24-28 hours for presumptive
identification of pathogen .
Demineralized Water
also called Purified water
prepared by means of ion exchange
total viable count is NMT 100 cfu/ml
Not suitable for preparations intented for parenteral
administration

Analysis
Samples of DM water are collected and analyzed for the
following parameters –

• Chemical
• Microbiological
Chemical Analysis

1. Description - When physically examined (Organoleptic test) DMW

should be clear, colourless, odourless, and tasteless liquid .

3. Residues on evaporation- Evaporate 100 ml of sample to dryness

on a water bath dry to a constant weight at 105C. The residues

weight is not more than 1mg (0.001%).

5. Total Organic Carbon- Analyze the sample for total organic carbon

in a calibrated TOC analyzer Limit: NMT 500 ppb

10. Conductivity- Measure by suitable conductivity meter.

Limit: NMT 1.3µS/cm at 25C.
Water for Injection (WFI)

apyrogenic distilled water

intended for use in the preparation of medicines for parenteral
administration
obtained by distilling demineralised water from a neutral glass
, quartz or suitable metal still fitted with a device for
preventing moisture
packaged in sterile vials / ampoules and is terminally sterilized
Total viable microbial count limit is NMT 10 cfu/100 ml
 MOISTURE CONTENT DETERMINATION
Karl-Fischer Titration :

a method for quantifying water content in a variety of products.

Principle– it is based on rxn. between iodine and sulfur dioxide in nonaqueous

medium . KF reagent consists of foll. components -

* methanol (containing excess SO2) – used as a solvent
* pyridine – buffering agent
* iodine – oxidizing agent
 Theory
•
Reaction :
•

ROH + SO2+ R’N

• [R’NH]SO3 + H2O + I2 + 2R’N 2[R’NH]I + [R’NH]SO4R
[alcohol]
•
[base] [alkylsulfite salt] [iodine] [hydroiodicAcid Salt] [alkylsulfate salt]

•
Alcohol reacts with SO2 and base to form intermediate alkyl sulphite which
•
then oxidizes into alkyl sulphate salt . That signals the end-point of the titration
•
titrator’s indicator electrode. Water and iodine are consumed in a 1:1 ratio in the
•
above reaction. Once all of the water present is consumed, the presence of excess
•
iodine is detected voltametrically by the titrator’s indicator electrode. That signals the
•
end-point of the titration .
•

•
Titration Monitoring – The use of specially formulated water standards enables the
•
efficient monitoring of titrator performance .
•
 TOC Analysis
Total organic carbon (TOC) is the amount of carbon bound in an organic
comp. and is often used as a nonspecific indicator of water quality or
cleanliness of pharmaceutical manufacturing equipment .

A typical analysis for TOC measures both the total carbon

present as well as the inorganic carbon (IC). Subtracting
the inorganic carbon from the total carbon yields TOC.
Another common variant of TOC analysis involves removing
the IC portion first and then measuring the leftover carbon.
This method involves purging an acidified sample with
carbon-free air or nitrogen prior to measurement, and so
is more accurately called NPOC
 Theory
2 types of methods are used for calculation of TOC -

• TC – IC :It measures the amount of inorganic carbon (IC) evolved from an

acidified aliquot of a sample and also the amount of total carbon (TC) present in
the sample. TOC is calculated by subtraction of the IC value from the TC the
sample
• IC – NPOC : employs acidification of the sample to evolve CO2 and measuring it
as IC ,then oxidizing and measuring the remaining NPOC

Acidification The removal and venting of IC and POC gases from the liquid sample
by acidification and sparging occurs in the following manner ---
•
Oxidation
•
The second stage is the oxidation of the carbon in the remaining sample in the
form of carbon dioxide (CO2) and other gases. Modern TOC analyzers perform this
oxidation step by one several processes .
•

•
Non-dispersive infrared (NDIR)
•
The non-dispersive infrared analysis (NDIR) method offers the only practical
interference-free method for detecting CO2 in TOC analysis. The principal advantage of
using NDIR is that it directly and specifically measures the CO2 generated by oxidation
of the organic carbon in the oxidation reactor, rather than relying on a measurement
of a secondary effect .
•

•
Applications
•
important in detecting contaminants in drinking water, cooling water, water used
in semiconductor manufacturing, and water for pharma use.
•