1.Microbial Testing
Purpose: Ensures that water is free from harmful microorganisms and meets microbiological quality standards.
Key Chapters: USP <61>, <62>, <1231>.
a)Total Aerobic Microbial Count (TAMC)
Objective: Quantifies the number of viable aerobic microorganisms in water.
Methods:
Membrane Filtration: Pass 100 mL of water through a 0.45 µm filter, then incubate the filter on agar.
Pour Plate Method: Mix water sample with molten agar and incubate.
Spread Plate Method: Spread the sample onto the agar surface.
Acceptance Limits:
Purified Water (PW): ≤ 100 CFU/mL.
Water for Injection (WFI): ≤ 10 CFU/100 mL.
b)Pathogen Testing (Specified Microorganisms)
Objective: Detect harmful pathogens in water.
Target Microorganisms:
1) Escherichia coli
2) Pseudomonas aeruginosa
3) Salmonella spp.
4) Staphylococcus aureus
5) Candida albicans
Methodology:
Enrichment in selective media.
Identification via biochemical or molecular tests.
Acceptance Limits: Absence in specified volumes.
2.Endotoxin Testing
Purpose: Detects bacterial endotoxins (pyrogens) in water.
Key Chapter: USP <85>.
Method:
Limulus Amebocyte Lysate (LAL) Test: Uses blood cells from the horseshoe crab to detect endotoxins.
Three approaches:
1)Gel Clot Method: Formation of a gel indicates endotoxins.
2)Turbidimetric Method: Measures changes in turbidity.
3)Chromogenic Method: Color change indicates endotoxin presence.
Acceptance Limits:
WFI: ≤ 0.25 EU/mL.
PW: Not routinely tested but should comply when used in formulations.
3.Conductivity Testing:
Purpose: Ensures ionic purity by measuring the water’s ability to conduct electricity.
Key Chapter: USP <645>.
Methodology:
Measure conductivity at 25°C using a conductivity meter.
For WFI and PW, the limit depends on the temperature. At 25°C: ≤ 1.3 µS/cm.
Stage 3 Test: Measures conductivity after sequential additions of acid and base if Stage 1 and 2 fail.
4.pH Testing
Purpose: Verifies compliance with pH specifications.
Key Chapter: USP <791>.
Methodology:
Use a calibrated pH meter.
Purified Water and WFI should have pH in the range 5.0 to 7.0.
5.Total Organic Carbon (TOC) Testing
Purpose: Measures the amount of organic impurities in water.
Key Chapter: USP <643>.
Methodology:
Use a TOC analyzer that oxidizes organic carbon into CO2 and measures it.
Oxidation methods: UV light, chemical oxidants, or heat.
Acceptance Limits: ≤ 500 ppb (µg/L).
6.Nitrate Testing (Specific to WFI)
Purpose: Detects nitrate impurities.
Key Chapter: USP <1231>.
Methodology:
Spectrophotometric analysis or ion chromatography.
Acceptance Limit: Nitrates should be below detectable limits for WFI.
Testing Frequency and Sampling
Frequency:
Daily for microbial testing in critical points.
Weekly or monthly for TOC and conductivity.
Annually for endotoxins (if system is well-validated).
Sampling Points:
Storage tanks.
Distribution loops.
User points (e.g., faucets).
Sampling must be representative and aseptic.
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