The lab results show 0.00043 mg/m2of “Q” on the surface of the critical component or witness sample. The customer, FDA, EPA, OSHA, clinician, forensic scientist, or military requires that the level of “Q” not exceed 0.00044; the detection limit is 0.0004. Should you breath a sigh of relief? Maybe not. Understanding and respecting the lower limits of the method is essential for regulatory compliance as well as to evaluate, validate, and monitor critical cleaning and contamination control protocols.
Results near the detection limit are less certain than are those closer to the mid-range. At low levels, noise masks the presence or absence of the analyte (the chemical being tested). The detection limit is the lowest concentration that produces a response detectable above the noise limit, typically a signal to noise ratio (S/N) of 3. The quantitation limit may be defined as the lowest analyte concentration that can be determined with acceptable precision and accuracy, often a S/N of 10.1,2 Consider subtleties of definitions in the context of the specific standard, guidance document, or report.
NOISE AND COMPLEXITY
Detectors and Mixtures
Many factors contribute to the noise and therefore to the detection limit and quantitation limit. One is the sensitivity and specificity of the detector. Two labs measure “Q” by high pressure liquid chromatography (HPLC); one uses a less sensitive detector. Also, detectors may lose sensitivity withage.
If the sample contains only “Q,” detection and quantification are relatively simple. If “Q” is one of several organic chemicals, speciation (identifying “Q”) and quantification are more complex because detectors may not completely speciate. The impact is magnified at lowlevels.
Separation and Extraction
Separating “Q” from other chemicals is often accomplished by chromatography,based on differential elution (release) from a sorbent.3 One source of noise in chromatography is the presence of co-eluting compounds. If “R” elutes close to “Q,” it might effectively “swamp out” lowlevels of “Q”so the detector does not see it.
One approach to eliminating “R” is to add an extraction based on differential solubility in two immiscible liquids. However, introducing more steps introduces more uncertainty near the lowerlimits.
REFERENCE STANDARDS, INTERNAL STANDARDS, CONTROLS
Standards and control samples are essential near the detection limit to adjust for changes in separation and/or variations in response of detectors. An internal standard, a compound that is not expected in the sample but that behaves similarly to the analyte of interest, may be added at the beginning of extraction to correct for losses. Where a regulatory agency indicates the option between using low level reference standards or the signal to noiseratio, standards are usually the preferable choice.4
BACK AWAY FROM THE DETECTION LIMIT
Pushing any analytical method to the limits (low or high) is asking for trouble. Even subtle differences among analysts and laboratories are magnified near the detection limit. If the required maximum level of “Q” is near the method detection limit, wave red flags. Change the method to a more sensitive one. Determine if the requirement is truly a requirement. Customers, regulatory agencies, and QC groups specifying maximum limits of minute levels of analytes ought to actively explore the practicality and necessity of theirrequirements.
- FDA, Center for Drug Evaluation and Research (CDER), Reviewer Guidance, Validation of Chromatographic Methods, November, 1994.
- J. M. Green, “A Practical Guide to Analytical Method Validation, Analytical Chemistry, 1996 (68): 305A. http://pubs.acs.org/hotartcl/ac/96/may/may.html.
- Kanegsberg and Kanegsberg, “Chromatography Systems, Parts I –III, A2C2 Magazine (now Controlled Environments Magazine), January – March, 2005.
- Detection Limits, West Coast Analytical Services (Bodycote) http://www.wcaslab.com/TECH/DETLIM.HTM.
Barbara Kanegsberg and Ed Kanegsberg are independent consultants in critical and precision cleaning, surface preparation, and contamination control. They are the editors of The Handbook for Critical Cleaning, CRC Press. Contact them at BFK Solutions LLC., 310-459-3614; firstname.lastname@example.org; www.bfksolutions.com.