I was fired. Was I wrong?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Walked in today and was walked out. Toxicology lab has been losing samples for a while now I suspect it was largely fiscal. Whatever.

What I want to pick your brains about is the reason they gave, was that I "failed" my 'training' in relation to processing properly LC/MS/MS chromatography data.

A month ago I was given a number of previously reported samples to analyze independently with Masshunter (Agilent LCMS software), to be later 'graded' against the reported values. I used the same methods they did and generated samples with values that were based on the calibration curve generated that day of the run. Basic stuff. However, I 'failed' to follow a particular practice they have been using, and I feel it is yielding in about 1 in 10 samples going out with false negative results, daily.

What I would like to do is for you to tell me, who is correct in the following scenario:

LCMS Curve is generated using increasing concentrations of anayltes and their deuterated internal standards. Levels 1-9

The L1 is considered their Lower Limit of Detection
The L2 is considered the cut-off.
Any sample's value, with proper chromatography, above the cut-off is considered a positive result. Any below the cut-off is considered negative.

Basic stuff so far.

The calibration curve plots the values of analyte's response, in relation to the internal standard response. In theory the internal standard is added in equal concentration in all samples.

So, in essence, we are generating values based on the RATIO of the response to the internal standard's response, both measured by area-under-the-curve. Which, I believe, is the whole point of using internal standards in LCMS. The idea being that by using the ratio and not just the analyte's response alone, you can help weed out the effects of ion suppression. So that if something causes a lower response to the target analyte, it will likely also happen to the internal standard. So even if a sample randomly gets half the total response as it normally "should" due to ion suppression, it will still yield a correct concentration value due to the ratio being used to make the concentration value and not just the analytes response alone.

So far am I right? I think this is still page 1 of analytical chemistry textbooks, right?

Ok, so here is where they change it up. They claim that if the sample's response is LOWER than the L2's response, that even if the calculated concentration value is above the cut-off, it is to be called negative and not positive.

So for example:

Morphine Calibration Curve's L2 Analyte Response = 10,000 with a concentration of 100. (So cut-off is 100.)

Morphine of Test Sample's Response = 8,000 with a calculated concentration of 155.

I say the 155 is correct and the sample is positive because there was ion suppression. The use of an internal standard protected this sample from being called negative because its value was made from a ratio of both.

They say, the sample, despite being above the cut-off, is negative because the analyte's response was lower than the L2's.

I feel they are totally ignoring the existence of ion suppression in LCMS by this practice and are calling samples with properly calculated values, well above the established cut-off, negative. Thus resulting in them reporting to physicians a "false-negative" result.

This is a problem because we do urine toxicology and most of our samples are for drug compliance. As in, "is Jane Doe taking her meds or selling them?" I feel that because of their random "rule", they are reporting out that people who ARE taking their meds are not. And this will result in people getting fired from their doctors and from receiving their meds because of supposed 'non-compliance' when in reality it was they were just unlucky enough to have had their sample have ion suppression.

Who is right here? I got fired for calling those test values positive. What would you do? What is your call? Literally tens of thousands of people have been effected by this 'rule'. Is is scientifically sound?

Thanks guys!
More information is needed.

Is there a standard operating procedure (SOP) for this analysis? Did you read this SOP? Did you sign any protocol confirming that you read this SOP? Is it only your responsibility that you "failed training" or is it the responsibility of your supervisor?

What are the signal-to-noise ratios (SNR) for typical responses of the standard and the internal standard (IS) at level L2? Are SNRs = 10? (5? 50?). What about the SNRs at level L1? Prescision of the responses below L2 might be too low to use these responses for the calculation of the response ratio and further for the calculation of the concentration.

Are there any limits for the variation of the responses of the standard and IS in the SOP? Are the responses (not their ratio) allowed to change by 10% (20%? 50%? 100%?) from their typical (average) values? Of course, IS serves to compensate for the variation, but there must be limits for the allowed variation of the responses.

Is the difference between the concentrations 100 and 155 significant from a medical (not chemical) viewpoint? Is the medically (not analytically) relevant cut-off concentration 100 or is this medically relevant concentration much higher (1000)?
confusedchemist wrote:
So for example:

Morphine Calibration Curve's L2 Analyte Response = 10,000 with a concentration of 100. (So cut-off is 100.)

Morphine of Test Sample's Response = 8,000 with a calculated concentration of 155.

I say the 155 is correct and the sample is positive because there was ion suppression. The use of an internal standard protected this sample from being called negative because its value was made from a ratio of both.

They say, the sample, despite being above the cut-off, is negative because the analyte's response was lower than the L2's.

I feel they are totally ignoring the existence of ion suppression in LCMS by this practice and are calling samples with properly calculated values, well above the established cut-off, negative. Thus resulting in them reporting to physicians a "false-negative" result.


I would say that in this example, '155' is the right value to be reported, and you are right. Note that I'm not into toxicological/medical analysis, this is just my scientific point of view as an analytical chemist working with LCMS. I do not know what is generally accepted or common practice in your sector.

It is possible that, somewhere in the QC section of the SOP, it is written that samples need a certain amount of internal standard recovery. In your example, the recovery is quite low (20-ish %?), and can be either due to matrix suppression or losses in the sample preparation. Larger errors occur the further the internal standard is from 100% (=same signal as in calibration curve), and at some point it can be below what you would call a peak. In such a case, you can not report anything on that sample, including a negative result.

Hope you find you new job soon.
I agree that the decision should be made on the basis of the ratio of the responses (or directly on the basis of the concentration calculated by using this ratio), NOT on the basis of the analyte response alone. Response ratio or calculated concentration should be used as the cut-off value. However, there should be limits for the variation of the responses, and the lowest analyte response falling within these limits should have the SNR not less than some pre-defined value (e.g. SNR not less than 10) or should be precise enough.

confusedchemist wrote:
Morphine Calibration Curve's L2 Analyte Response = 10,000 with a concentration of 100. (So cut-off is 100.)
Morphine of Test Sample's Response = 8,000 with a calculated concentration of 155.

Let's assume the IS response at conc. of 100 is 20,000. Then, assuming a straight calibration line passing through zero, at conc. of 155, you should see the analyte response of 15,500 and the IS response of 20,000. Actually, you see the analyte response of 8,000 (conc. is 155) and presumably you see the IS response of about 8,000 x 20,000/15,500 = 10,323. So, the recovery of IS is only 100 x 10,323/20,000 = 52%. Is this value acceptable? If the lab did not re-analyse this sample and the lab used these particular data for reporting the "negative" result for this sample, there can be two answers: 1) recovery 52% is acceptable; 2) the lab does not check the recovery.

Also I have to refine my earlier question about the SNRs. Of course, the SNR of IS at level L2 is not a problem since the SNR of IS must be high enough and be nearly the same at every calibration level because the concentration of IS in all the calibration solutions is constant.
I have extensive experience of working in a toxicology lab and you are certainly not in the wrong. Using the response ratio is vital especially when using the internal standard; after all, as you said, that is the reason for having it; especially when ion suppression is common with many drugs of abuse. What is equally as concerning that if there are two conflicting values eg.. (so not a clear positive or negative) is that the sample isn't sent for repeated analysis (presuming more sample is left).

I would not just walk away from the job, contact HR/ work unions or even an employment lawyer. From the information you have gave, you haven't failed any training. You are questioning the accuracy of the results, and as an analytical chemist rightly so! The lab sounds like it has been sending inaccurate results out for a while and they are aware of it but do not want it highlighted.

Good Luck!
my vote is with rndirk and the rest; (1) you're right that the calculated value using the internal standard is the best estimate that you have, and if it's above the cut-off then the result should not be reported as negative.
(2) There should, by the way, be something in the SOP specifying some sort of minimum S/N ratio, or some sort of minimum internal standard recovery, so that in the case of outrageously high ion suppression you don't report anything at all (the ratio becomes meaningless as the situation degenerates towards zero-divided-by-zero). Rndirk's right about that.
(3) Caveat, I also don't work in drug analysis
(4) What you do about it is up to you, and local law. If this were a matter of failing a test in competence, in some jurisdictions you'd expect follow-on training and some sort of competence procedure (that could ultimately lead to dismissal) rather than summary dismissal on the spot, but I know many countries have far less satisfactory employment law. Whether you feel you want to be a whistle-blower for what looks on the face of it to be a disreputable procedure is also up to you - and you might be able to get advice on how to do it, the likely consequences, and how to protect yourself from trouble.
(5) But whatever the situation, you're probably well out of it. Good luck with your future. You don't want to be working for people who cheat. Sooner or later the "stuff" will hit the fan in the lab you've just left, and all sorts of trouble will happen: It's better not to have a big history working for a company like this when an auditor prods something and the whole house of cards comes tumbling down.
Thank you for all of your detailed responses. I appreciate it.

To answer a few questions:

1. Yes there is an SOP and yes this rule was in it.

2. Yes, there was a minimum Signal/Noise ratio required (10), as well as a minimum IS response required per sample (1,000), as well as an analyte specific identification-qualifier-ion-ratio window the sample had to fall into. Per the SOP.

3. The samples that were called negative, met all of the above required criteria for a positive call. So yes for S/N above 10, yes for 1,000+ IS, yes for Qualifier ratio within windows. The only reason the samples were called negative was because the sample's response was lower than the calibration curve's, of which was significantly cleaner having been made from a synthetic urine substitute and not a human sample. (I don't take issue with the calibration made that way, I just think it explains why we would see higher counts because there was significantly less junk in the sample than found in testing samples.)

4. We had access to the patient's prescribed medications and could see that in most cases, they were prescribed the drug in question. This caused a crisis of conscience with one of our "certifying scientist" almost daily and she would fight with the person responsible for making/enforcing this rule regularly, often resulting in tears. "They're taking it! It's right here! And I'm supposed to tell the doctor 'No they're not?!' We're hurting people!" 2 other employees tried pushing back against this practice as well, but got nowhere so they just did what they were told. They openly talked about this being wrong as well.


5. In some cases (as this issue comes up about 5-10% of all of the daily samples), the calculated concentration of the sample will be 2x+ the cut-off. So as for the medical significance, I can't say as I'm not a physician but I would imagine it would vary drug to drug. We did report quantitative values on most of the drugs so the physician could make that call.
This situation rings so many alarm bells. If your certifying analyst isn't being listened to, and is in tears, something has gone seriously wrong with internal quality. You are well out of this. Sooner or later it's going to come out, and it will be a big, big mess.
Double posting. Deleted. See the post below.
Just for my curiosity, what is the SNR for the response of 10,000 of the L2 calibration standard? I got the idea that the SNR is not less than 10, but I am interested in the actual value of this SNR (how much it is higher than 10).
What is the recovery of IS corresponding to the allowed minimum IS response of 1,000? What is the usual IS response in the calibration?

Not trying to justify your employer and the SOP for this analysis, I'd like to discuss the question of the medical significance of the results a little more. Is the difference between the concentrations of 90 (reported as "negative"), 110 (reported as "110"), and 200 (reported as "200") significant for a physician? Is it possible that the true "negative" results (conc. below 100), the lower "positive" results (conc. above 100 but not higher than, say, 300), and the false "negative" results (actual conc. from 100 to 300, but erroneously reported as "negative") are all considered as "negative" or as "insignificant traces" by a physician? The cut-off conc. in the analytical method is 100. The cut-off conc. for a physician that leads to some medical decisions might be much higher (e.g. 1000). In this case, erroneous reporting of some results, which are somewhat higher than 100 (2x-3x, but not 10x), as "negative" might be not such a heavy crime as it is considered from the point of view of an analyst.
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