[Readers Insight] Is "Not Detected" Truly "Not Detected"? (Not An Article about LOD)

This article is written by Welch's contract writer Chromatography Mound. The content of the article presents a point of view from the author solely.

When we encounter the term "Not Detected" (ND) in analytical chemistry, does it truly mean the substance is absent? This is a compelling question that warrants closer scrutiny.

The immediate reaction for most professionals is to associate "Not Detected" with the Limit of Detection (LOD). If the concentration of a sample is below the LOD, it IS reported as "Not Detected." Even if an instrument is capable of achieving a much lower LOD than what is required by a specific regulation, any value falling below the regulatory LOD must still be officially reported as "not detected."

But no, this article is not about Limit of Detection. The scope of this discussion lies differently.

The presence of an additive or residue in a sample usually has an underlying cause, which provides the rationale of why we test for a substance. In the analytical process, we generally determine the identity (qualitive) of a target compound by utilizing reference standards to perform the test based on retention time (RT), spectral data, and characteristic ions; then, we perform the test to test the amount (quantitative) based on the response intensity.

However, if one or more of these parameters deviate from the standard, how do we make a qualitive judgement?

Let's take an example of the "Nafils": PDE5 inhibitors such as Sildenafil, Tadalafil, and Vardenafil. If we analyze a sample for these three specific compounds and find they are below the LOD, we can only conclude that these specific targets were "Not Detected." But can we conclude that "no other PDE5 inhibitors are present"? The answer is a definitive NO.

Unscrupulous manufacturers are also aware of this. Now that regulations target specific molecular structures, by performing simple modifications to the molecular functional groups (for example, substituting a methyl group with an ethyl group), they can create structural analogs that effectively bypass a large quantity of conventional detection methods.

To fight against such manufacturers, one of the most common regulatory responses is to continuously expand the scope of target compounds in official standards. For example, in China, BJS 201805 (Determination of Nafil Compounds in Food) specified as many as 90 target "Nafil compounds", and BJS 202405 (Determination of Compounds such as Sildenafil and Tadalafil in Food) increased the number to 95.

While necessary, this approach is resource-intensive and fails to fundamentally eliminate the issue, as it essentially remains a "cat-and-mouse" game against chemical modifications.

To address this challenge fundamentally, the author conducted a different strategy. Use Diflorasone as a model. By leveraging the scanning capabilities of high-resolution Time-of-Flight Information Dependent Acquisition Mass Spectrometry (TOF-IDA/MS), the author implemented targeted screening of all Diflorasone derivatives without the need for reference standards for every individual analog ^[1].

The principle of the methodology includes first identifying the characteristic fragment ions shared by Diflorasone and its ester derivatives. Using these characteristic fragments as a baseline, the system employs a Precursor Ion Scan to reverse-scan for parent ions. This allows for the identification and confirmation of potential new parent ion.

This methodology establishes a systematic structural recognition pathway for Diflorasone and its related esters, with the overall technical workflow shown below.

Suppose an illicit manufacturer modifies a Diflorasone derivative—such as changing the number of ester groups or the type of fatty acid chain—the compound will have a different retention time and an unknown mass-to-charge ratio (m/z) for its precursor ion. In a routine targeted analysis, this compound would be "invisible."

However, these compounds still produce characteristic fragment ions, such as m/z 121 and m/z 335, during fragmentation. By conducting a "blanket search" for these specific fragments and tracing them back to their parent ions, the unknown derivatives can be localized and identified.

This strategy enables the precise identification of all structural derivatives. It significantly raises the barrier for illicit chemical modification while simplifying the experimental complexity for analysts, ultimately strengthening market supervision and regulatory efficacy.