Photometric Quantitative Techniques
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Photometric Quantitative Techniques
Turbidimetric Technique
USP 85 specifies a photometric assay to detect endotoxins in pharmaceutical samples. There are two types of photometric assays – endpoint-turbidimetric assay and kinetic-turbidimetric assay.
The endpoint-turbidimetric assay is based on the quantitative relationship between the concentration of endotoxins and the turbidity (absorbance or transmission) of the reaction mixture after the incubation period.
While, kinetic-turbidimetric assay quantifies either the duration (onset time) needed to attain a specific level of absorbance or transmission within the reaction mixture, or it measures the speed at which turbidity formation occurs.
The test is conducted at the recommended incubation temperature provided by the lysate manufacturer, which is typically around 37 ± 1 °C.
Chromogenic Technique
This technique measures the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with lysate. Based on the assay principle used, the technique can be classified as an endpoint-chromogenic assay or a kinetic-chromogenic assay.
The endpoint-chromogenic assay is based on the principle of detecting endotoxin concentrations and the release of a chromophore following a defined incubation period.
On the other hand, the kinetic-chromogenic assay evaluates either the time required to achieve a set absorbance level or the rate at which color change occurs. This assay is conducted at the incubation temperature suggested by the lysate manufacturer, usually maintained at approximately 37 ± 1 °C.
Preparatory testing
Preparatory testing is performed to ensure the accuracy and reliability of the turbidimetric and chromogenic techniques. This test helps to confirm that the criteria for the standard curve is valid and also determines if the sample solution interferes with the test. Additionally, the test method needs validation if any change in conditions could potentially affect the accuracy of the test outcomes.
Acceptance criteria –
The test should be carried out for each lot of lysate reagent.
- The standard endotoxin solution is used to prepare at least three endotoxin concentrations within the range specified by the lysate manufacturer to establish the standard curve.
- The assay is performed by following the manufacturer’s instructions for the lysate, including volume ratios, incubation time, temperature, pH, and other relevant parameters. Each standard endotoxin concentration is tested in at least three replicates.
- In kinetic methods where the desired range exceeds two logs, it is necessary to include additional standards to cover each log increase within the range of the standard curve.
Test for Interfering Factor
Solutions (A–D) are prepared as indicated in table 4. Subsequently, the test is conducted in duplicate, following the instructions specific to the lysate used.
Table 4. Solutions preparation for the Inhibition/Enhancement Test for Photometric Techniques
Solution | Endotoxin Concentration | Solution to Which Endotoxin Is Added | Number of Replicates |
Aa | None | Sample Solution | Not less than 2 |
Bb | Middle concentration of the standard curve | Sample Solution | Not less than 2 |
Cc | Three concentrations (lowest concentration is designated as λ ) | Water for BET | Each not less than 2 |
Dd | None | Water for BET | Not less than 2 |
Acceptance criteria
- The absolute value of the correlation coefficient of the standard curve generated using Solution C should be greater than or equal to 0.980.
- For Solution D, the result should not exceed the blank value limit specified in the description of the employed lysate reagent or should be below the endotoxin detection limit of the lysate reagent used.
To ensure that the assay is not affected by interfering factors, the measured concentration of the endotoxin added to the Sample Solution should range within 50%–200% of the known added endotoxin concentration. This is determined after subtracting any endotoxin detected in the solution without added endotoxin.
At MIS, we offer USP 85 testing services using both The gel-clot technique and Photometric Quantitative Techniques.
Our services are tailored to meet the stringent requirements of the pharmaceutical and medical industries. For further information about our USP 85 testing services, please consult with our experts here.
FAQs
USP 85 provides methods for the detection and quantification of endotoxin contamination in pharmaceutical products, medical devices, and similar materials. In USP 85, product samples are screened for endotoxins by using Limulus Amebocyte Lysate (LAL) test. LAL test can be performed using three techniques: Gel-clot, Turbidimetric, and Chromogenic Standard Endotoxin Stock Solution.
USP 85 test can be carried out for biologics, medical devices, injectable medications, ophthalmic products and intravenous solutions etc.
USP 85 test 3-4 weeks to complete.
Contact us for more information
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