Avoiding contamination with plastic pipette tips

[Fat Jonny]

This is going to sound like a stupid question, but I'm more used to inorganic analysis so want opinions of organic people!

What is the best way of preparing calibration standards in hexane for a GCMS assay if I want to avoid contaminats from plastic pipette tips?

I'm monitoring mass 149 with one particular assay and getting repeat contaminant peaks every 30 seconds or so along the chromatogram which I'm guessing is phthalates from the plastic pipette tips used to prepare standards, since a hexane blank dispensed into a glass autosampler vial with a glass tranfer pipette is blank.

Why isn't there such a thing as disposable glass pipette tips? How do I transfer 50uL of a stock solution without plastic??

[Paulhurleyuk]

I'm no expert, so this is off the tyop of my head, but could you make up your solutions using standard glass disposable bulb pipettes and a balance ?

Paul.

[chromatographer1]

Syringes will work.

There are pipetting devices which use glass tips instead of polypropylene.

There are also glass capillary tubes that will hold 50 µL volume.

Certainly syringes are the cheapest and easiest solution.

best wishes,

Rod

[MikeD]

If you don't use a syringe the calibration of your pipette device might be a bit out for hexane. Calibration usually assumes water.

[AICMM]

Fat Johnny,

Wiretrol makes a convenient little dispenser for small volumes over and over again, teflon and glass. Otherwise, Hamilton or SGE make the appropriate syringes very typically used in the organic world. Wiretrol available from Fischer or VWR while SGE or Hamilton from almost all GC houses (Supelco, Alltech, Restek, etc....)

Best regards.

[Consumer Products Guy]

Manual pipetting using glass pipets and volumetric flasks, or Hamilton diluter. Personally, Fat Jon, I wouldn't use a pipetter for anything this critical, doubt you'd get the precision you'd really like.

[Fat Jonny]

Thanks for the suggestions... the problem is I'm used to prepping inorganic analysis batches and using the plastic disposable pipette tips is just so damn easy and convenient.

I'm no expert, so this is off the tyop of my head, but could you make up your solutions using standard glass disposable bulb pipettes and a balance ?

Paul.

For an analytical batch (i.e. GMP validation) that may contain two calibration curves with 7 points in each, plus 5 replicates of QC's at 5 levels... that's one hell of a long sample prep! That's why I was thinking there must be something easier/quicker.

If you don't use a syringe the calibration of your pipette device might be a bit out for hexane. Calibration usually assumes water.

This might also sound stupid... but I figured volume was volume by the time you reach ng/mL levels and are dealing with the same sample matrix. e.g. if I weigh out my reference solid and dissolve in hexane to make a 10,000 ug/mL stock and then process my samples by dissolving in hexane, isn't diluting with hexane going to be fine with a pipette since it's all hexane?

Plus I checked the accuracy of the plastic pipettes with an analytical balance correcting for the density of hexane at 0.6657. All volumes from 100uL to 5mL were within 2% of nominal. As long as you wet the pipette before first use, these pipettes are accurate. It's just the contamination issue!

I like the little disposable glass micropipettes. $16 for 100 isn't too bad either. May have to get some of those!

[Peter Apps]

Try a different brand of pipette tip. Several years ago I torture tested tips from both Eppendorf and Gilson by soxhlet extracting several of them and then running the extract. They were clean.

Peter

[Fat Jonny]

ooh now that sounds like a fun experiment....

Maybe I'll try that!

[Bruce Hamilton]

I never found air displacement pipettes to be consistently accurate with any volatile organic. 2% usually isn't good enough for most quantitative work, but it's fine for qualitative work.

Air displacement pipettes would always surprise me with a gradual creep as the "air" volume/temperature/composition changed. Positive displacement pipettes are a little better than air displacement, but I've always preferred using syringes for volatile organics.

Also, most name-brands of pipette tips I've tested don't have significant extractable plasticisers, however prolonged use of organic solvents often washes traces of the pipettor piston or seal lubricant into tips and samples.

In my experience, plasticiser leaching from tips is very low compared to leaching from other plasticware, such as centrifuge tubes. Warm ethanol in an ultrasonci bath is a useful solvent to evaluate leaching, because it also grabs any antioxidants as well as most plasticisers.

Bruce Hamilton

[HW Mueller]

Rod, Bruce,

what syringes do you use? I have never seen one that I would trust. I can imagine that one could get good repetition with a syringe that has teflon (or whatever) seals on the piston and a piston guide set to a desired volume. But what do you do if you need a different volume at ~ each use?

On air displacement syringes: I used to prepare controls to be used by clinical laboratory technicians. Once in a while I put pure ehyl ether in some control vials. They never got any ether all the way to the analyzers, so couldn´t foul up anything, though some tried hard. (Actually, they also played some jokes on me, it was all about evened out).

[Fat Jonny]

OK some conclusions:

mass 149 is also a mass associated with column bleed... The repeated 149 peaks are most likely due to that. Since conditioning did nothing I bought a new column and this seems to be under control now!

there are phthalates in our distilled deionised water (therefore there are phthalates in our saline stock solutions) producing background peaks... now using Sigma HPLC grade water to prepare saline!

After leaching overnight in hexane, there are minimal extractables in pipette tips

I'd suggest a 2% relative error limit is fine for pipette use. This is our standard SOP acceptance criteria for inorganic pipette checks and has never been a problem so far.... If I can show I get less than 2% error with water and am within the same limits for hexane, what's the big deal? If GLP/GMP acceptance criteria is +/- 15% (20% at LLOQ)....

[Bruce Hamilton]

I use SGE Gas Tight syringes for quantitatively dispensing samples that are predominantly volatile organic solvents. I find them really easy to use, once you understand the parallax error, and need for multiple flushes. Also, I don't have to calibrate them as often as pipettors.

I have the range of 25, 50, 100, 250, 500, and 1000 uL. They are PTFE-faced plungers with removeable needles - so I can strip them down and ultrasonicate for cleaning when dealing with cytotoxins. I don't use the luer lock versions. nor the PTFE sealed versions ( too much trouble to clean, and the seals bind after a while with solvents ).

For smaller volumes ( < 10 uL ) I tend to use the 5 and 10 uL guided plunger-in-barrel series, which don't have a PTFE face, but I find the long design allows me to quickly set and dispense the volume. For really small volumes <1 uL, I often use a 0.5 uL or 1 uL plunger-in-needle SGE syringe, but usually would change the dilution to use a larger volume.

In general, I use the same syringe for preparation of standards and samples, so the actual volume conformance specification ( 1% ), is not a major issue. It's important to have several sizes, so graduations are relevant, and samples are near the nominal capacity.

Normally, I quickly flush the syringe with each sample about 10 times ( only takes a few seconds), then slowly draw up the sample past the graduation, and then push down to the mark. I then flush the syringe with sample solvent about 10 times before next sample, unless I know it's a time series experinent, when I ignore the solvent flush.

I quickly wipe the needle and tip with a tissue, and withdraw the plunger a mm or two, however the usual recommendation is to withdraw the plunger and then wipe the needle.

It's really important to quickly, but carefully, wipe the outside of the needle that has contacted the sample, especially if you are making large single dilutions of very small ( < 10 ul ) aliquots. Otherwise you will find calibration curves don't pass through zero.

With regard to the 2% relative error limit of the air displacement pipettor, the specification for the 100 and 1000 uL models I use ( Eppendorf ) is an inaccuracy of +-0.8 ( 100uL) or +-0.6% (1000uL ), and an imprecision of <=0.2%.

As noted earlier, I've never been able to consistently get an acceptable value for use of an air displacement ( tried three different brands ), or even a positive displacement ( Gilson ) pipettor with hexane, or any other volatile, low viscosity, solvent.

If 2% works for you, fine.

Bruce Hamilton

[Fat Jonny]

OK some conclusions:
there are phthalates in our distilled deionised water (therefore there are phthalates in our saline stock solutions) producing background peaks... now using Sigma HPLC grade water to prepare saline....

Nope that's no good... there's phthalates in the Sigma HPLC Grade water!!

[Bruce Hamilton]

Nope that's no good... there's phthalates in the Sigma HPLC Grade water!!

Perhaps you would like to share the product code?. A cursory check revealed that Sigma sells several brands of HPLC water, at least one of which offers grades espcially for LC-MS.

I'd be a little surprised if their gradient HPLC or LC-MS grades have significant phthalates, as they prsumably are widely used for trace organics.

Once you have ensured that you could not have contaminated the samples, you should report your results and concerns to Sigma, and ask for an explanation.

Bruce Hamilton