Recovery @ Detection level

Dear All,
With the available instrumentation [Double beam UV spectrophotometer] we are able to develop methdology only upto the level of detection of detergent residues.
Limit of detergent is 10ug/swab of 4.sq.inch
Swab desorption volume =10ml
Target concentration of detergent =10ug/10ml = 1ppm

UV reading @ Target level (ie) Standard preparation of 1ppm, has value of only 0.006 Absorbance.
Any reading below 0.02 Abs is not considered to be quantifiable value in case of UV-Visible spectroscopy.

[1]If the case is as explained above, at what level recovery [spiking of detegent] shall be performed to support the validation data?
[2]If recovery is not possible at this detection level of analytical technique , [u]whether it is wise to relax the limit of detergent according to LD 50value?
Expecting provide your valuable replies/comments

Dear Mr T Arun,

There are two approaches which you can use for performing recovery studies when the LOD (detection limit) value is used as CV acceptance criteria.

  • [b]Spike at LOQ level[/b], for example, assuming that expected recovery is 70% then spike at LOQ+(100-70)%*LOQ level. If you are not confident about recovery, then you may spike at 150% (50% is considered acceptable as the least possible recovery) or 200% of LOQ value. Atleast by this mean you will make sure that the amount you recover is quantifiable and well above LOQ. The recovery factor thus obtained will have higher level of confidence (& justifiable). Verify % recovery (at LOD level) by spiking at LOD+(100-Recovery)%*LOD level. Analyze samples and check if you can detect the residue.
  • [b]Spike at 150% or 200% of LOD value [/b](by doing this you will make sure the amount detected is above LOD), analyse the samples and determine % recovery. The amount spiked at 100% of LOD, most probably will not be detected (unless the %recovery is 100%) due to loss of residue during sampling and extraction phases. Although you will be able to detect and determine the amount recovered but the analysis results below LOQ level are generally not considered acceptable due to high level of uncertainty and lower degree of confidence. This can be used as worst-case approach to determine %recovery (considering the recovery factor will be lower than the one obtained using Approach#1). Which method you are using for setting acceptance limit for detergent residues? I hope using the above mentioned approaches you will be able to solve your issue (without any compromise and any need to relax any of the criterion). Thanks
  • Dear Ovais,
    Intially, We have worked approach #2.(ie) Spiking @ 200% to LOD level or 200% to the acceptance criteria.Recovery was nearly 46%
    Next, we have tried approach #1.Spiking @ 200% to LOQ level
    Recovery was nearly 60%.
    Recovery is verified @ LOD level.Results are satisfactory.
    But recovery factor employed is very high[1.67]

    1.The CV limits should be practically achievable and verifiable at every end [Both at Manufacturing & at Analytical part].
    2.It is important to define the sensitivity of the analytical methods in order to set reasonable limits

    On what basis, we can scientifically justify approach#1?

    Note: We are following Toxicity based approach, using LD 50values for setting limits to detergent residues. After calculation , we have got MAR/MACO value as 9.57, which has been then fixed to 10ug/swab.

    Expecting your valuable comments/suggestions.

    Dear Mr T Arun,

    I am bit confused here, why are you using a recovery factor of 1.67? The recovery factor should be either 0.6 (for 60% recovery) or 0.46 (for 46% recovery), based on what you have stated above. For employing a recovery factor of 1.67, the recovery should be more than 160%. Another thing, you spiked at 200% of LOD and the recovery was 46% but when you spiked at LOD+(100-60)%*LOD level (i.e. using % recovery which you obtained after spiking at 200% of LOQ), you managed to detect the residue (i.e. verify the recovery). That means recovery at 200% of LOD was 46% and at 140% of LOD was ~60%.

    The guideline actually states, “The analytical methods used to detect residuals or contaminants should be specific for the substance to be assayed and provide a sensitivity that reflects the level of cleanliness determined to be acceptable by the company.” If I use this guideline for your situation then the sensitivity requirement is not fulfilled. In simple words, assuming that the CV acceptance limit is 0.957μg/mL and the detection limit of analytical method is 1μg/mL, the stated requirement is not met because the analytical method is not sensitive enough to prove that the required level of cleanliness is achieved (as the LOD and MACO are same). In this case you would not be able to justify your “claim” (of residue removal) just by saying that you are not able to detect the residue (“not detected” will not mean that the residue level is below 0.957 or 1μg/mL). For example, if the residue on a surface is present at an amount of 12μg (higher than MACO of 9.57 or 10μg) and the swab recovery is 70%, then I should be able to detect around 8μg. However, in your case the result would be “not detected” (as the amount required to be detected is lower than LOD). Meaning, I can’t justify that the equipment surface is devoid of residue above MACO level. Auditors are simply going to reject the idea of setting LOD limit at MACO level.

    After reading your initial post, I supposed that you are setting limit for detergent residues based on LOD of analytical method (and not based on MACO calculation) thatswhy I suggested those approaches. I would like to suggest you two things (1) Change the analytical method (shift from UV to HPLC or other, nowadays analytical methods are available which can detect residues at ng or ppb levels) or make it more sensitive (LOD should be far below MACO limit). This is very important if you want to justify that the CV limit is achievable and verifiable (1ppm is not a level which you can’t achieve). I would like to highlight one more requirement (as per regulatory guideline - -ICH/FDA), which states, “The efficiency of cleaning procedures for the removal of detergent residues should be evaluated. Acceptable limits should be defined for levels of detergent after cleaning. Ideally, there should be no residues detected.” This is what regulators expect when it comes to detergent residues i.e “there should be no residues detected.” (2) Make your sampling method more precise and accurate: Detergent residues are generally water-soluble and should give high recoveries.

    Thanks and cheers