Starting material for a calibration curve

Discussions about GC and other "gas phase" separation techniques.

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I'm new to GC/MS, and working on a university project to analyze short chain fatty acid (SCFA) levels in mouse serum.

I am looking at acetic, propanoic, butyric, isobutyric, valeric, isovaleric, and using heptanoic acid as a surrogate. Naphthalene is being used for the internal standard.

I am evaluating two separate derivation methods as part of the project, BSTFA and PFBBr. So far I've diluted the SCFA's in diethyl ether and derivatized them. I'm getting good separation, and now it's time to build a calibration curve.

The PI suggested spiking the SCFA's into serum to build the calibration curve (he's a microbiologist, not a chemist—but then, I'm just an undergrad, so...), but since I'm extracting out into diethyl ether, and my evaluation is of the organic layer, that doesn't make sense to me, since there might be variable loss in the process, and then, what are you really calibrating?

My thought is that it should be derivatized SCFAs in diethyl ether, but I believe Peter Apps mentioned in one of the posts that analytes in solvent weren't a good starting point for calibration. Anyone care to elucidate?

My second question is, I suspect I want to make my dilutions for calibration from the derivatized sample. The other alternative is to make the dilutions of the SCFAs in solvent, and then to add the derivatizing reagent to each dilution. Which to do?

Thirdly, do I want to treat each SCFA separately for building the calibration curve(s), or do I want to have a single set of dilutions that contains all of the SCFAs in one batch?

And finally, since Naphthalene is being added at the end of the extraction process, do I need to add it to the calibration dilutions?

Cheers,
Chris Gursche
Hi Chris,

I can offer my insight. Your PI is correct. You should be making calibration standards in serum and derivitizing them from that calibration stock in serum. There are many reasons for this; however, the main reason is that you want your samples and standards to be analyzed under near identical conditions. You do not know the extraction efficiency the diethyl ether has for your analytes, and you do not know if there are things in the serum which will compete for derivitization or will negatively or positively effect this process. When building a calibration curve for PK studies, the standards must be treated under near identical conditions as the samples so it is a clear and direct comparison.

If I were doing your experiment, I would build 8-10 individual standards with a series of blanks. The concentration will depend on your experiment. To create each standard, I would spike the serum with a known amount of SCFA (using a solvent both the plasma and SCFAs are miscible in), add your internal standard, mix very thoroughly, and then proceed with your derivation processes. I would repeat this for each standard I make, and I would highly discourage you from doing this once and diluting from a stock.

It is important to add the internal standard before the derivation process and not after because, again, you do not know the extraction efficiency of your process. This will help normalize things if there is deviation. Additionally, making each standard individually rather than diluting from a stock will allow you to see if you made any bad standards. If you dilute from a stock, it is very difficult to determine if your standards are near their true value unless you made a pipetting error.

I would also suggest using an internal standard that more closely matches the rest of your SCFAs so it undergoes the derivation process as well.

I hope this helps.

Aaron
Hi Aaron

Thanks for your comments.

I have two questions —

1. Given that the process uses heptanoic acid as a surrogate (heptanoic acid doesn't appear in biological samples, and it undergoes the derivation process, since it is a medium chain fatty acid, so the point is that the recovery rate of heptanoic acid is an indicator of the process loss), wouldn't it be better to treat the naphthalene as an internal standard, and add it at the end of the process, so that it can function as a measure of instrument efficiency? That is to say, is the GCMS measuring things the same way today as it was last week? And if not, what is the variance?

2. So the point of the calibration curve is, it is the process that is being calibrated and not the machine. 10 µM of propanoic acid in the original shows up as x, 100 µM shows up as y, etc. Is that the correct understanding?

The only concern I have is that the SCFAs other than heptanoic are all expected to be in the sample to one degree or another. How does one get a true calibration then? If I spike the serum with acetic acid, how do I know how much of the acetic acid is mine, and how much was there to begin with? Right now, I'm mimicking the process with a saline buffer in place of the serum, but that, as you pointed out, lacks potential interfering factors.

I guess I could measure a saline sample against a serum sample for heptanoic acid, and figure out what the potential interference level is.

Cheers,
Chris Gursche
I agree with you about the naphthalene. The heptanoic acid is you assessment of the quality of your extraction. Naphthalene is just to help you assess the quality of your injection technique.

The "machine" is part of the process. Pet peeve of mine - machines do work, instruments make measurements. I had a professor once who just went off every time someone would call an analytical instrument a "machine".

What you are calibrating is the entire measurement - from beginning to end. If you can demonstrate the repeatability of your extraction as being good, it becomes a "known-to-be-good" part of your method. An automatic if you will.

Look up information on the "Method of Standard Addition". Basically, it works for detectors that respond linearly with changes in concentration. This is true for the vast majority of GC detectors.

Ro = response for unknown concentration of analyte
Co = concentration of unknown analyte in sample
R1 = response for analyte in the spiked sample
C1 = known amount of analyte added

Ro = m*Co (m is the instrument response factor, slope of the line relating response to concentration)
R1 = m*(Co + C1) (total response due to the concentration of the analyte)

R1 - Ro = m*Co + m*C1 - m*Co
(R1 - Ro)/C1 = m
Co = Ro/m
Co = Ro*C1/(R1 - Ro)

This is how you can estimate the concentration of the analyte in your sample. You want to be sure that you added enough analyte so that R1>Ro but still in the linear range of your detector. If R1 is too close to Ro, you're essentially dividing by zero. Not good. It's also best if you can add your analyte to the matrix without changing the matrix a great deal. Don't add your standard in 1 mL of methanol to 2 mL of sample. Also not good.
Here's some example data. I wanted to determine what the minimum detectable quantity of toluene-in-room-air I could determine using thermal desorption sampling (GCMS). What you see here is the data from triplicate analysis of my Tenax tubes (after baking them out) and 3 additions of a known toluene standard. This is essentially a tube-blank measurement (ppb in air).

Toluene in Room Air

C R

0 71075
0 164823
0 46803
54 7565524
163 26035434
272 40884784

0.51
0.45
0.47
1.20
1.04
1.10
0.34
0.29
0.31

Ave (ppb) = 0.64
StdDev = 0.37
StdMean = 0.12

Example:

0.51 = 71075*54/(7565524 - 71075)

etc. Much less than 1 ppb in is not above the blank within the 95% level of confidence.
So if I understand this correctly, R1 and C1 are known, as is m (from my calibration curve), so that's how I figure out R0:

(R1-x)/C1 = m
m x C1 = R1 - x
R1 - ( m x C1) = x
x = R0

and then my only unknown is C0, which I can now figure out as well. Or

(R1 - mC1)/m = C0, and then I can figure out R0

Cheers,
Chris Gursche
Just use the equations I have provided. I've laid it all out.

Ro = analyte response in the unspiked sample (you measure this)
Co = unknown concentration of the analyte
R1 = response for the analyte in the spiked solution (it's actually Radded + Ro, you measure this)
C1 = the concentration of the analyte that you know you've added

Co = Ro*C1/(R1-Ro)

That's it. I'm sorry if I confused you with the derivation.

Remember, your analyte fortification must be large enough so that R1 is easily bigger than Ro BUT still in the linear range of the detector. In my example, the first fortification was ~10X greater than the blank. If you plot my data, you'll see that it's all easily in the linear dynamic range of the detector. The first 3 points in my set are blank TD tubes (0 ppb toluene added). The first spike was such that the added concentration was 54 ppb in the air, etc.

Co = Ro*C1/(R1-Ro) = 71075*54/(7565524-71075) = 0.51 ppb

in the blank. I used the 3 fortifications and the 3 blank analyses to get 9 estimates of the "typical" blank concentration for this method.

If you want to read more about this calibration method, consult any junior-level text book on analytical chemistry.
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