Chromatography Forum

Hello everyone, this is my first day in the forum. I am not very comfortable with my English but I will do my best. I am doing my Masters Degree under the title"Chemical Analysis - Quality Control", and I have some queries.

I have no prior experience in GCMS. I am analysing small organic acids in urine for diagnosis of inborn errors of metabolism (methylmalonic, propionic, glutaric, acidemias etc), using an Agilent 5975C GC-MSD with column: DB-5 30m x 0.25mm i.d.=0.25mm.

1. I prepare a standard solution of each acid in methanol:
_Are there any known issues in the esterification of the acids with methanol? meaning, will this result in an esterification product? Is methanol the proper solvent?

2. A second thing.
_Can anyone help me with the analytical conditions required for running MS in full scan mode?

3. And a third question: My colleague is analyzing drugs (codeine, morphine and 6MAM) with the same column in urine.
_ Is it likely to have any problems by using the same column?

Any help would be much appreciated.

Many thanks:)

1 - Expect esterification. It will not be instantaneous, but if you let your solutions stand, they will change.

2 - If your colleague who is running drugs can not show you how to run full scan, I would suggest that you see if there is someone you can talk with who can talk you through it and make sure that you do not miss one of the little details. It is not hard.

3 - You should be able to use the same column for multiple methods. DB-5 is not my favorite column for acids. But, on a shared instrument I would see if I get the analytical range I need before getting into the issue of changing to another column. For acids measured as methyl esters, by the way, DB-5 is just fine. The thing to watch out for is inlet maintenance. Be sure you start with a clean inlet liner, a new o-ring and change the septum frequently. (Discard the liner and o-ring when you remove them from the inlet and use new ones.) Check for leaks with a leak detector. Most problems with a GC/MS system are with the inlet system.

Don Hilton,
I would like to thank you for your reply. I really appreciate it.
I have some questions about the things you said

1. I prepare stock solutions of each acid in MeOH 10.000, 1000, 100 and 10 ug/ml and I am keeping them at the refrigerator (-20 oC).
When you say: ''It will not be instantaneous, but if you let your solutions stand, they will change''
-Do you mean that solutions can change during storage or during sample preparation (evaporation, TMS derivatization etc)? For how long can I keep my stock solutions at the refrigerator?

2. I will try:P

3. When you say: ''Be sure you start with a clean inlet liner, a new o-ring and change the septum frequently.''
- Should I use new liner and o-ring every time, after my colleague's analysis? It is really costly and I don't think I can do it more often than one time in a month (or maybe after 100 injections).

Thanks a lot

The acids will esterify over time in methanol. The rate of change will depend on the presence (or lack of) acid or anything else that can act as a catalyst. Esterification will occur during storage.

To find out how long your stock solutions are stable in the refrigerator, keep your stock solution in the refrigerator and periodically make up a fresh solution and run it as an unknown. At some point the answer for your freshly made solution will not agree with results from the stored standard. If you make replicate injections for calibration and for reading the unknown, you can measure the accuracy of your method and set tolerances for your needs for a standard.

If you are making TMS derivatives of the acids, I assume you are evaporating standards to dryness before derivatizing. Could you use another solvent, such as methylene chloride?

-- on new liners.

If stuff collects in the inlet liner it tends to form active sites. Active sites are spots with chemical reactivity - that react with reactive molecules. I demonstrated this to myself one day on an instrument that I used only for teaching classes. I knew that I could use an inlet liner in the instrument for two or three weeks - because the class made few injections and the solutions were all very clean. Every class stated with an exercise in setting the detector voltage of the mass spectrometer. The students would inject 200 pg hexacholrobenzene onto the column to estimate the instrument sensitivity and make the correct adjustment to see 2 pg on column. There are a couple of reasons for the selection of 200 pg HCB in this exercise. 1) Any instrument on the planet can find 200 pg HCB, in the dark, and with its eyes closed. 2) HCB is not particularly affected by active sites, so you can get a fix on the sensitivity of an instrument, even if the inlet liner is not in the best possible shape.

So, I had a vanilla bean that had been soaking in about 20 mL vodka for about a year and had run a profile of the mixture shortly after I'd put the vanilla bean in the vodka. This instrument had easily seen the 2 pg HCB earlier in the week and I knew that the instrument was in great condition. Class was over for the week and so I made one injection of the vanilla solution, injected one microliter, and took my data file to my desk and left the instruments to sit the weekend before the next class. (I did not check them for condition because they had been in great shape and the never degraded from one week to the next between classes.)

Monday came and to make a long story short - the instrument on which I had made this one injection could not see the 200 pg HCB - even with the detector cranked up to the max. New liner - and we were back up and running.

The point to the story: Liners die and may die quickly. If you are doing metabolimics work, you are working with biological extracts. They tend to have high boiling stuff in them. This stuff collects in the liner and on the seal-plate on the bottom of the inlet. I am running TMS derivitized urine extracts these days and don't expect more than about 20 to 30 injections out of a liner. I am still doing development of the technique and will look to see if I can make a liner last longer - or if I have to make it shorter.

As you develop and run your method, find a solution with analytes at a low level and periodically run it to be sure that you can see your peaks with the same signal to noise for the peaks that you had when you stared the work. If this degrades and you do not notice it, you will publish one of those papers that shows a method developed with this fantastic limit of detection and study results that show no statistically significant difference -- only to find out later that your instrument had "gone blind" and you did not notice. Your paper will be cited frequently - as the work with which everyone else disagrees.

Yes, replacement of liners gets expensive – but poor results are more expensive, so don’t try to get more out of the liner than is there! (Science is an expensive proposition.)

You have been more than welcoming to me and I want to thank you. You clearly took some time to write all those things down and I feel obliged in a way.

I will take into consideration all that you've given me and make up some final decisions (+talk with my professor) before I get started with my project.

I will be around and maybe I'll have some more questions:)

many thanks again.

Hi everybody

I want to prepare urine samples, spiked with organic acids in order to have a calibration curve from normal to abnormal concentrations.

For example, I want to spike urine with methylmalonic acid.
In Human Metabolome Data Base (http://www.hmdb.ca/metabolites/HMDB00202) I found the following normal concentration for the acid:

Biofluid Urine
Value 2.0 +/- 0.7 umol/mmol creatinine
Age Infant:0-30 days old
Sex Both

1. What does it mean: umol/mmol creatinine?
2. How much acid should I add to urine samples?

The typical reference range of creatinine (>18yrs) for women is considered about 45-90 umol/l and for men 60-110 umol/l.
In unknown samples I have to give the result in ug acid/mg creatinine.

Any help would be much appreciated.

Many thanks:)

Because the quantity of water in urine depends on a number of factors, like how much water a person drinks, the concentrations of metabolites in urine are not always good indicators of what is going on in the metabolism of the person. Creatinine is a metabolite from the body that is presumed to be formed at a relatively constant rate in a person. So, creatanine is used as a reference to give a more meaningful value for metabolimic study.

I would suggest 1) use urine from several subjects 2) obtain creatinine values on each collection of urine and 2) spike from nothing added to a level above that you would expect in a high concentration sample.

Don Hilton
many many thanks for your reply
we'll be in touch

Hello everyone
I have a recovery issue.
I'm checking recovery and LOD.
I analysed twice the following samples:
a)standard solution with a mix of 9 org. acids 50ppb in MeOH
b)blank urine
c)spiked urine with 9 acids 50ppb (ng/ml).
I am doing LLE in urine with 3ml ethyl acetate twice.
I calculated the mass of the acids in order to be the same in mix standard and in spiked urine (1,36ng/ul = 0,136ng = 136pg in MSD with a split 1:10)
I spiked 3ml urine with 150ul from 1ug/ml mix standard solution in MeOH.
I evaporated 150ul from 1ug/ml mix standard solution in MeOH. Then derivatization with 110ul (BSTFA+TMCS)
I added 30ul pentadecanoic acid, PDA (IS) from 1000ug/ml (10ug/ml) in all samples.
The problem is that, after manual integration, the area in urine is much bigger than in standard.

example:
succinic
area blank = 1128081
area spiked = 1540188
area spiked-blank= 412107
area std = 28741

PDA
area blank = 4826169
area spiked = 4221880
area std = 4920984

I used new liner and septum. Solutions are 1 month old.

Any help would be much appreciated.

Many thanks:)

If I understand what you have done, for the standard you evaporated the methaol solution - I presue to dryness. And for the urine extracts, you evaporated the ethyl acetate extracts to dryness.

Did you add the internal standard before evaporation?

Have you looked for the fatty acid methyl esters in the chromatogram?

Have you located peaks for the mono and di TMS derivatives of succinic acid? (Incomplete derivitation will give you a mixture - and apparent loss of the succinic acid if you are looking for only the di-TMS derivative.)

If you are trying to evaporate the methanol from the standard, I would suggest that you add ethyl acetate to the evaporation to avoid trapping methanol in the residue. Any excess methanol will react with the TMS.

So, I can give yo a few ideas - without a bit more detail about your procedure and your chromatogram, these may be all irrelevant.

Hi Don Hilton. Many thanks for your reply.

1.Did you add the internal standard before evaporation?
Yes, I added IS before evaporation in standard sample and before LLE presudure in urine samples.

2. Have you looked for the fatty acid methyl esters in the chromatogram?
Yes, i found my acid-2TMS in both standard and urine chromatographs.

3. Have you located peaks for the mono and di TMS derivatives of succinic acid?
I only searched for di TMS derivatives of succinic according to the retention time.
(RT has changed a bit in urine samples in relation to standard, but my proffesor doen't want to use retention indices by the moment, I don't know why not)
Bibliography suggest 100ul mix of BSTFA+TMCS (1:10). I use 100ul BSTFA and 10ul TMCS, because I have the reagents separately. The only thing that others change is temperature and time of derivatization (60-80oC for 30-60min). I made the reaction in 60oC for 30min. So I suppose that 110ul are enough for derivatization.

4. If you are trying to evaporate the methanol from the standard, I would suggest that you add ethyl acetate to the evaporation to avoid trapping methanol in the residue. Any excess methanol will react with the TMS.
At the end of evaporation in standard sample I don't see any MeOH in the vial (no bubbles in the glass).

I would like to thank you again for your reply. I really appreciate your help

Hi everybody

I am checking every week for the stability of my stock solutions which are stored in the frige at -24oC.
According to data, the stock solution (mix of 10 acids) in methanol is stable for 15 days.
In every analysis I had the same retention time for every 2TMS-acid.
But when I checked the mix stock on 22th day, retention times have changed.
The only thing that has changed is that I 've replaced the liner and septum with new ones and that 2 collegues of mine have used GC/MS. They injected biological samples (serum, urine) in the same column.
We use the column Optima-5-ms, 340ºC, 30 m x 250 μm x 0.25 μm, Scitech Scientific, Duren-Germany (8 months old)
These are the results for RT (sim mode, ?=lost peaks):


standard sample 15th day 22th day
acid RT RT min
glycolic 10,324 10,092
3-OH-propionic 12,438 12,202
malonic 14,393 14,127
methylmalonic 14,791 ?
ethylmalonic 17,34 ?
succinic 18,602 18,298
methylsuccinic 19,117 ?
glutaric 22,048 21,764
adipic 25,138 24,961
pimelic 26,824 26,682
IS (PDA) 30,164 30,041


The picture below refers to glycolic acid: peaks went at front after 4 replicate injections. I have the same image for all acids. I've never had this before.

I've checked for airleaks after the replacement of liner and everything looks fine.
Is it a chromatograph problem (dirty column)?
Should I make a column conditioning at high temperature for some hours?

Any help would be much appreciated.
Many thanks:)
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Very puzzling - a progressive decline in both retention and peak area, with no deterioration of peak shape.

Smaller areas could be due to deteriorating standards, or declining sensitivity of the MS.

Shorter retentions can be due only to one or more of; an increase in gas flow, a decrease in the amount of stationary phase in the column, a shorter column, an increase in temperature. None of these would affect peak area. Are you sure that your colleagues did not trim a piece from the end of the column ? Are you sure that before you did the maintenance there were no leaks ? What temperature is the column at when the GC is is standby ?

Peter

Peter Apps,
Thank you for your replay.

1. Are you sure that your colleagues did not trim a piece from the end of the column.
Yes, I was present the last time we cut a small piece from the column and I made the appropriate corrections in my method (SIM).

2. Are you sure that before you did the maintenance there were no leaks?
I injected MeOH and I run my current method in order to clean liner and column. I checked for airleaks (manual tune) with my method loaded. relelative abundance of both m/z 18 and 28 very low (around 1-1,3%)

3. What temperature is the column at when the GC is is standby?
Inlet 110oC and column 50oC


I baked out MS (ion source) a month ago and from 13/4/2010 till 6/5/2010 I was the only one who has worked in GC/MS.
I've made all my experiments (calibration curve, intra and inter day, stability tests) with the same liner cause I dilute urine very much, that's why liner is quite clean. Everything was OK.
The last test of std stock was on 12th of May
Sorry, I don't have new data

Hello everybody!

About the problem I had, I would like to inform you that it was because of the dirty column. I cut a piece and everything was fine.
I hope this will be helpfull for someone who will probably have a familiar problem.

I have finally finished my project and I am getting my Msc.
I would like to thank this forum for the great help and experience that provides, especially to the new scentists, like me.
Many thanks especially to Don Hilton for his help. He was great with me.

Keep up the good work.
I wish you all good luck and good results to your projects.
We will be in touch.
Su