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An Overview Of The Turbidimetric Endotoxin Method

An Overview Of The Turbidimetric Endotoxin Method

The Turbidimetric Endotoxin Method

As we continue our focus on endotoxins testing for the month of July, this week our focus is on the Turbidimetric Method of Endotoxins testing. The turbidimetric method is a widely used technique for the detection and quantitative analysis of endotoxin concentrations. Endotoxins are lipopolysaccharides (LPS) found in the outer membrane of gram-negative bacteria and can cause significant immune responses and other adverse effects in humans.

The turbidimetric method is based on the principle that endotoxins cause the aggregation of a compound called Limulus amebocyte lysate (LAL), which is derived from the horseshoe crab’s blood cells. NJ Labs previously published an article about extraction of this compound from horseshoe crab blood.  You can read that article here. When endotoxins are present in a sample, they interact with the LAL and induce a gel formation or clotting reaction, leading to an increase in turbidity (cloudiness) in the solution. Here’s an overview of the turbidimetric method:

  1. Sample Preparation: The turbidimetric method, much like the gel clot method, requires samples to be put into solution, and then diluted if necessary. If the sample is already a liquid it can just be diluted.

    The Maximum Valid Dilution is then calculated by the following formula:

    (Sample Specification) x (Sample Concentration)
                              (Lysate Sensitivity)

    so a sample with a specification of NMT 3 EU/g
    being resuspended with 1g in 4ml of LAL Reagent Water, with a lysate sensitivity of 0.03 EU/ml would be

    (3) x (0.25 g/ml) = 25
               0.03
    Meaning the sample could be diluted up to 1:25 while not exceeding the client specification. Hence the name “Maximum Valid Dilution”. We have more flexibility with the turbidimetric method with regards to our Lysate sensitivity because Turbidimetric REQUIRES a standard curve to be run, and then the endotoxin calculation is performed off the standard curve , giving quantitative results. Lysate sensitivity is simply the lowest concentration on the standard curve, which can be low as 0.001 EU/ml
  2. LAL Reagent Preparation: The Limulus amoebocyte lysate (LAL) reagent is prepared by reconstituting a lyophilized form of the lysate with a reconstitution buffer provided by the manufacturer. The LAL reagent contains enzymes and clotting factors necessary for the reaction.
  3. Test Setup: First, it will be necessary to reconstitute and dilute the CSE or Control Standard Endotoxin, which is an accepted Reference Standard of known Endotoxin value, usually reconstituted to 1000 EU/ml and then diluted down to a working solution for sample testing. Unlike gel clot however, we don’t dilute down to 2λ. Because λ, which is the lysate sensitivity, is not used in Turbidimetric, since the lysate sensitivity is not a fixed number. It is simply the lowest concentration on the standard curve. And, unlike gel-clot, a standard curve is REQUIRED, as it is the basis from which we calculate endotoxin concentration in the sample. Multiple test tubes or wells are then prepared, each containing a different concentration of the test sample. To determine the endotoxin concentration in the sample, a standard curve is constructed using a series of known endotoxin concentrations. The turbidity values obtained from the standard curve are used to quantify the endotoxin concentration in the test sample. The standard curve needs to be at least 3 points for routine testing, or 5 points for method validation, and this done at a MINIMUM in duplicate (for routine testing) or triplicate (for validation). The standard curve also needs to have the correlation coefficient be greater than 0.980. The control tubes consist of positive and negative controls. The positive control contains a known amount of endotoxin, while the negative control does not contain any endotoxin. The positive control is not just a tube with known concentration of endotoxin that would be a point on the standard curve. Rather, it is a Positive PRODUCT control, which is a spiked solution of the sample to yield an endotoxin concentration at the midpoint of the standard curve, and that must be within 50-200% to be considered passing.  
  4. Reaction: A specific volume of LAL reagent is added to each tube or well, including the control tubes. The tubes are then mixed to ensure thorough mixing of the LAL reagent and the sample. For example, a Positive Product control spiked to yield a 1.0 EU/ml solution would need to yield a result of between 0.5EU/ml and 2.0 EU/ml to be considered passing. Also, this test does not stop at a defined period of time like the gel-clot which is finished in 60 minutes. The test concludes when the lowest point on the standard curve is yields a result. Since it has the lowest concentration, it should be the last point to finish.
  5. Turbidity Measurement: The turbidity of each tube is measured using a spectrophotometer at a specific wavelength (commonly 340 nm). The turbidity of the solution is caused by the formation of a gelatinous clot in the presence of endotoxins. The Turbidimetric method utilizes absorbance values vs a function of TIME to calculate endotoxin concentration. Which means the sample does not get MORE Turbid, it simply gets Turbid faster. All samples will eventually reach the same turbidity (i.e. the point at which the sample will clot), but higher sample concentrations will get there faster. For example: a standard curve of:

    0.16 EU/ml
    0.08 EU/ml
    0.04 EU/ml

    will all reach the same absorbance values of 20+, but will take different times to reach them:

    0.16 EU/ml = 10 minutes
    0.08 EU/ml = 20 minutes
    0.04 EU/ml = 40 minutes
  6. Calculation: The endotoxin concentration of the test sample is calculated based on the turbidity measurements and the standard curve. The results are typically expressed in endotoxin units per milliliter (EU/mL) or endotoxin units per gram (EU/g) depending on the units of the sample.

In summary, the turbidimetric method allows for accurate quantification of endotoxin concentrations in test samples, making it suitable for determining the potency or compliance of pharmaceutical products, medical devices, or other products where endotoxin contamination is a concern.

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