Surviving TFA

By: SP Genevac

What’s So Special About TFA?

TFA (Trifluoroacetic acid) has several properties that make it very important that you take the right precautions when evaporating it, in order to get optimum performance and prevent damage.

Firstly, it’s an acid. This means that if not handled with care, it will corrode components of a system over time. Next, it is quite volatile (with a boiling point / pressure characteristic pretty similar to methanol). In combination with other solvents it can present a quite “bump-prone” mixture. Thirdly, (and perhaps most infamously), it exhibits “creep”. In simple terms, this means that liquid TFA can climb the sides of a vessel to the height that any TFA vapor reaches. What is more, it can carry with it dissolved compounds, which may then end up actually leaving the vessel. Fourthly, and particularly worryingly for users who run into this problem, it can seep through polypropylene (including some brands of 96 well microtiter plates). This is not evidence of a failure to seal the bottom of the well during molding of the plate – it actually goes through the plastic itself, due to it having an extremely low surface tension. TFA in sufficient concentration will also attack silicone (both silicone rubber seals and the special oil in a standard Genevac Cole pump). Finally, it can soften a PTFE coating on an item that is exposed to liquid TFA for any great length of time.

So how can we deal with all this?

It’s quite simple when you know how. All you need to do (for an HT system) is:

  1. choose a sample vessel it will not leak through
  2. prevent creep occurring before evaporation begins
  3. evaporate it without bumping, without forming any condensation anywhere and without any liquid being thrown or sprayed onto the PFTE coated chamber
  4. ensure that the TFA vapor is always re-captured in the first condenser pot and not sent through the pump in any great quantity
  5. ensure that when defrosting the condenser the TFA does not creep back into the evaporator

Choose The Right Hardware

Polyprop plates

It has been conclusively demonstrated at a number of user sites that TFA travels through the 96 well polypropylene plates. The simple test to prove this goes as follows:

Load the plates into a suitable Genevac swing (i.e. with a metal base beneath the plate*). Sandwich a piece of filter paper between the base of the plate and the metal swing. Run the machine for a short while (long enough to get up to full rotational speed for 30 minutes) with just TFA in each well of the MTP. If when you open the system up there are 96 telltale marks on the filter paper, this indicates TFA has passed through the bases of the wells. There is no other mechanism by which this pattern could be made on the filter paper.

Experience so far seems to suggest that conical wells that taper to a point are most vulnerable to this effect. To date an official list of good / bad plates has not been compiled though Genevac would welcome the feedback of any users who have experience with particular brands.

Sometimes this effect comes to light in a different way – the user is running the plates in Genevac “open swings” (not advised*) with the lamps shining directly on the base of the plates. When the plates leak, a set of 8 lines can be seen all around the chamber for each level on the rotor (i.e. 3 sets of 8 lines in an HT12). What is actually happening is that liquid TFA is leaking through the wells, flying off at a tangent, hitting the chamber, and (over time) softening the PTFE coating. Though this softening is to some effect temporary, the continuing impingement by drops of liquid TFA at 60 mph (while the coating is soft) starts to damage the chamber coating.

The Right Swings

The effect known as creep results in TFA climbing up the walls of a vessel. This will happen in time if samples are just left in a microtiter plate. Therefore, one of the best things you can do to prevent problems is not to leave samples sitting in plates any longer than you have to. For example, when loading samples including a lot of TFA into an evaporator, make sure to start the run as soon as possible afterwards.

If creep still occurs (i.e. TFA in small quantities climbs out of the wells) there are special swings available from Genevac. These are designed to catch small amounts of TFA as it leaves the plate (or other vessel) and retains it until it turns to vapor, rather than let it be thrown against the chamber wall as liquid droplets. Both the Fast-Stack deep well (used for carrying two plates per rotor position) and the SideBridge swing are available with this modification. Look for “anti-creep” variants in the Accessories Brochure.

Choose the right lamp glass

One effect of splattering TFA (and some compound) onto the walls of the chamber is that you will also find it gets splattered onto the small rectangular windows that separate the infra red lamps from the chamber. There is a potential failure sequence that goes like this:

  1. TFA and compound gets splattered on the glass.
  2. Solvent evaporates leaving a dried deposit.
  3. Deposit gets hot, particularly in center of the lamp beam where intensity is greatest.
  4. Compound carburizes and turns black.
  5. Black patches absorb even more lamp heat.
  6. Heat leaves glass by conduction from hot spots to edges. This means there is a temperature gradient.
  7. This means that there is differential expansion.
  8. The glass breaks, causing (at best) downtime or (at worst) an implosion of glass into the chamber.

It is possible to get a different glass arrangement, especially for use in systems suffering from this problem. The system has two different layers of glass. The inner most (facing the chamber) has an extremely low coefficient of thermal expansion. This means that the list above looks more like this:

  1. Heat leaves glass by conduction from hot spots to edges. This means there is a temperature gradient.
  2. So what? (**)

This dual glass system is retrofittable to existing evaporators, or can be specified at manufacture time. There is a third option, however:

  1. TFA and compound gets splattered on the glass
  2. Solvent evaporates leaving a dried deposit
  3. Conscientious operator wipes glass clean before next run.
  4. Problem solved.

Note that the glass can be cleaned with a lint free cloth and some acetone. It is VERY IMPORTANT that you do not press on the glass while doing this. It sits on a sprung seal and pushing against it can push it off the seal and allow dirt to get into the seal.
(**) It is of course good practice to keep the glass clean whatever type you have.

Program Your System Optimally

Prevent Bumping

If you have a mixture of solvents including TFA, you will certainly want to select Dri-Pure™ as an anti-bumping precaution. If TFA is the most volatile of your solvents, and if you have “Variable Dri-Pure™” enabled on your system, you can shorten the vacuum ramp.
(Contact your sales rep for a document explaining how to do this to best effect). If however the TFA is mixed with another, more volatile, solvent (like methylene chloride) then the standard vacuum ramp may be appropriate.

Give the Condenser a Chance

As with all volatile solvents, it is important to choose the correct pressure to optimize the way the condenser deals with the vapor.

For example, if you run TFA with a target pressure of 0.5 mbar, the following happens. At 0.5mbar, the boiling point is –60 deg C. This means it is too cold to condense in the condenser first stage and must go through the pump and be caught in the second stage of the condenser. As vapor is being produced in the evaporator far faster than the pump can pump it out, the pump becomes a serious bottleneck, and a backlog of vapor builds up. This means the target pressure is not achieved, and will probably not go much below 4 or 5 mbar for some time. Since the target is 0.5 mbar, the pump continues pumping in an attempt to lower the pressure, and thus TFA is sent through the pump in great quantity. The pump will suffer over time.

If you instead choose a higher pressure, (12 mbar is a good figure) the boiling point will be –20 deg C, and the condenser will be able to condense the vapor at the same rate at which it is being produced. This means that the target pressure can be achieved, and hence the pump can shut its inlet valve and let the condenser deal with the vapor. This will extend pump life.This means overall that the amount of vapor caught in the first condenser pot is increased, and the amount that travels through the pump to the second pot is decreased.

Of course, there will be some cases where the TFA binds tightly to the compound and so it is impossible to dry the samples completely without dropping the pressure lower than 12mbar. Here’s where some experimentation comes in. Lets say all the TFA that can be removed at 12 mbar is captured in the condenser, and the condenser temperature has dropped back to –45 degrees, but the samples still have some un-evaporated TFA in. It is possible to then drop the pressure to perhaps 4mbar without significant TFA from the first condenser pot migrating through the pump, which may remove the last bits of TFA from the samples. But if you drop the pressure to 2 mbar and below, you will start to see significant amounts of TFA travelling through the pump to the second pot (and beyond).

If dropping this lower is the only way that you can get your samples fully dry, then of course, it has to be done.

Initial Transients

So far the situation we’re describing relates to “steady state” drying once the pressure in the chamber has settled down. But there is more to consider. At the start of a run, there is often a fairly rapid onset of boiling producing a lot of vapor when the working level of vacuum is first approached. At these times, the condenser can be temporarily “overwhelmed”, which means that it cannot condense the vapor at the same rate it is being produced (and so some of that vapor goes through the pump). This situation can be prevented by the use of a gentle vacuum ramp, making the onset of boiling less severe.

Vacuum ramping is not just for preventing bumping!

You will know if the vacuum ramp is sufficiently gentle if the system manages to pretty much achieve its target pressure all the way through the ramp. If not, then the condenser is not condensing everything as it arrives, and so it is going through the pump.

Pump Life

For small quantities of TFA, run at the right pressure, the pump will not suffer unduly. However, if strong solutions (20% and more) are run regularly, there will inevitably be some effect on the pump. The silicone oil in the Genevac Cole pump will degrade in time with exposure to a lot of TFA, and in these circumstances Genevac recommend that the pump be “upgraded” to the “CPC” specification. This simply means that a different (more expensive) oil is used and the pump rotational speed is changed to reflect the different viscosity of the CPC oil.

This upgraded can be carried out in the field to an existing pump.

Caring for your condenser

Perhaps the most obvious bit of advice about the condenser is “remember to empty it”. Some users do forget and though the system still (eventually) dries the samples by pulling the solvent out through the pump, the drying times are very long. Therefore it is only likely to be on longer than necessary overnight runs that an un-emptied condenser goes unnoticed.

Samples will only get dry in these circumstances if the run is programmed for a period way longer than it really needs to be, and that in itself might be said to be bad practice. Even if you have all night to do a run, you should still aim to program the system to run only long enough to get the samples dry, because needless prolonged running ages a system prematurely. A system that does an 8 hour run every night then switches off for 8 hours will live 50% longer than one that runs right through to morning for no reason.

Full condensers often occur not so much because they were not emptied, but because they were not emptied fully. This might be because the condenser was not fully defrosted before draining, or because some sort of residue or sludge has built up which is obstructing the draining process. Though not that common, this is still something you should take steps to prevent.
On a system running mostly TFA (with not much of any other solvent), it is wise to “flush” the condenser periodically to prevent such build-ups. To do the best job of this, use a flushing kit. This is a factory option for an HT-4/DD-4 but can be retro-fitted to external condensers (i.e. HT-8/12 and Megas). It consists principally of a “probe” which reaches down into the condenser pot and is used to spray methanol onto the walls of the pot as part of a semi-automated cleaning process.

A slightly less effective, more time consuming, but still worthwhile method (on a system without a flushing kit) is to evaporate a full “dummy run” of pure methanol. On an HT-8 or 12, this should amount to a litre or more (300 mls on an HT-4) and the run should be carried out at a pressure of at least 12 mbar to ensure the methanol is all condensed in the first condenser pot, where most rinsing is required.

IMPORTANT – How to defrost

This section applies equally to Series 1 and Series 2 systems.

The process of defrosting a condenser involves heating the contents of it until all the ice is melted. On a standard Genevac condenser this means that heat is applied for a

predetermined length of time. With TFA, however, there is a risk that you may end up producing hot liquid TFA in the condenser before the defrost cycle is over. This potentially leads to accelerated creep, meaning that the TFA that you have already trapped in the condenser can make its way back up the tubing into the evaporator chamber, causing quite a mess.

There are two ways to prevent this. On a new system that is specified in advance to be for TFA use, a special “defrost” temperature sensor is fitted by Genevac which ensures that defrost heating stops when the liquid in the condenser rises above a certain temperature. This means the TFA can never get hot enough to cause a problem.

But this is only available as a factory option, not as a retrofit.

For systems without this extra sensor, the best way to defrost with TFA is to open the drain valves before commencing the defrost. This ensures that as soon as liquid TFA is formed, it drains out of the condenser, and there is never a large body of liquid TFA being heated.

This technique should only be used when TFA is present in the condenser, not with other solvents. If you are using other aqueous solutions you will find that ice in the condenser thaws a lot quicker if surrounded by warm water, than if you have a dry lump of ice touching the warmed condenser walls in a few points. Hence you should keep the drain valves shut during defrost when not using TFA.

On an HT-4 and DD-4, it is a bit more awkward to defrost with the drain open. On an HT- 4, open the lid, open the drain valves, then close the lid far enough to see the screen (and hence navigate the defrost menu) but not far enough that the lid re-closes the drain valve.

On a DD-4, lower the lid so that you can get at the defrost button, start the defrost cycle, then leave it fully open again, ensuring the drain valves are not closed by the lid.

Automatic Shutdown

The Genevac series 2 systems have a feature called “Autoshutdown”, which ensures that after a run has finished, the system goes into a “sleep” mode. This is the same state that the system boots up in, and the same state the system goes into when you hit STOP twice from the main menu.

The “sleep” state means that
• The condenser is no longer refrigerating
• The pump switches off (after 30 mins of purging to ensure no vapours remain which could condense in the pump).

People often ask “why not go straight into defrost automatically, as well?” The answer is that your samples are still in the evaporator chamber, and the last thing you want is for solvent vapour to re-evaporate from the condenser and condense on your samples.

Autoshutdown is a “system option” (not a “program option”), which means that if it is enabled, it is enabled for all programs. It cannot be selected on a program by program basis. It can be found by choosing “Options” on the main menu screen and then choosing “options” again. Hitting the enter key toggles its status.

It doesn’t matter (wear wise) that the evaporation is over at midnight, if the system continues to run till 8am when you return, that’s 8 hrs more wear. Autoshutdown therefore makes your system live longer.

Another advantage of Autoshutdown is that if the condenser has been off all night it will have got most of the way back to room temperature, and you might find that it is almost ready to drain without any defrost being required. Of course, you should verify that this is the case before relying on it, because not fully draining a condenser before restarting will (over a few runs) potentially lead to a cumulative problem. To verify if overnight shutdown is thawing your condenser, simply drain it in the morning, then do a defrost cycle and see if anything else comes out. If not, the overnight shutdown was enough to thaw it.

Under normal circumstances Genevac would always recommend that this option be enabled, particularly if the system is run overnight.
The one exception to this rule is with TFA.
You might enable Autoshutdown, then find that you open the door of the evaporator and are met with TFA fumes (or worse, liquid TFA in the chamber) the morning after the run, because of creep from the condenser.

In this case, you may find it preferable to disable Autoshutdown, so that the condenser continues to hold the TFA safely refrigerated in the condenser until you return to the system the next morning.

Look after the Seals

Don’t dispose of TFA in the Arctic.

But seriously, nitrile seals can swell on prolonged contact with TFA. In extreme cases, a swollen door seal will prevent the door from closing and then the evaporator is unusable until the door seal is replaced. If there are any spillages of solvent onto the door seals, (or lid seals on an HT-4/DD-4, or the window seal on an HT-12 door) clean them up with methanol immediately. Ensure all staff are aware of this, and you should not have a problem.

You might even want to consider obtaining a spare door/lid seal just in case, if you frequently suffer spillages.

Finally, some tricks of the Trade

Often evaporation does not go the same when there are compounds present as when you are “practising” using just solvents. For one thing, it is often slower.

Another example is as follows. Whereas you might expect a volatile solvent like TFA to be evaporated before water (in a TFA / water mixture), it can be the case that significant amounts of TFA remain bound to the compound at the end of the evaporation of aqueous TFA.

In some cases the problem can be resolved by using other additional solvents. Toluene is popular for assisting in the removal of TFA, and any experience gleaned in this area from using a rotary evaporator will be relevant when using a Genevac.

Another lesser known fact is that the whereas pure TFA suffers creep, addition of water to the mixture reduces the creep (up to about 40% water by which time it has pretty much stopped completely). So if it is possible to run with an aqueous mixture, this will be better from the point of view of creep.

Surviving TFA – A summary

As we have already seen, correct programming will reduce both the condensation and the amount of TFA that travels through the pump (or travels back into the chamber later). Careful choice of microtitre plates and (if necessary) sample swings will ensure that the chamber is never sprayed with liquid TFA. Defrosting the condenser with the drain valve open and also flushing it on a regular basis will greatly reduce problems. Wiping the lamp window glass clean if anything accumulates on it will prevent build up of hard baked on deposits that could cause the glass to break. And by cleaning up all spillages you can prevent swelling seals that could cause the system to lose vacuum.

But if there’s one overriding thing to remember, it is this.
If you have a problem, contact Genevac.
• If you need assistance setting up the system, email
• If you think there is a fault, call the service department.

Don’t suffer in silence.

Evaluation of an Improved Sample Preparation Method for Quantative Analysis of Very Low Levels of Airborne Polycyclic Aromatic Hydrocarbons for Worker Protection and Health Screening

By: Nicolas Falquet, Gilles d’Esperonnat & Rob Darrington


Polycyclic Aromatic Hydrocarbons (PAHs) are large class of compounds comprising two or more fused aromatic rings. PAHs are naturally occurring in fossil fuels and their derived products and can be formed during incomplete combustion of carbon based fuels. As such they are a by-product of many industrial processes. PAHs vary greatly in size, nature and hazard to human health, some are not classified as toxic, where as others are known carcinogens. The IARC specified 16 as being of particular interest, others have subsequently added this list. In all, over 100 PAHs have been described.

Given the risks and potential risks to human health presented by PAHs, many high risk organisations, such as Foundries, Bitumen Works & Smoke Houses routinely monitor workers and their environment for PAH levels. Typically PAHs are trapped using filters (particulate forms) or resins such as XAD2 (gaseous forms) through which work place environmental air is drawn. Filters may be situated in a small device attached to the workers overalls, or from larger units measuring the air in a wider area. Potential problems exist when recovering the PAHs from the filters and preparing the samples for analysis, principally, losses due to PAH volatility are reported for bi- and tri-cyclic PAHs (ISO11338-2:2003). Therefore, ITGA undertook a study to improve sample recovery and therefore PAH determination when working with low and very low levels of analytes.

Sample Preparation Methodology

Methods for workplace sampling are well described in the literature (NFX43-294 and Method Metropol 011) and result in samples trapped on glass or quartz fibre filters. The filters are preserved and delivered to the analytical laboratory. The whole filter placed into a barcoded vial, 10ml dichloromethane (DCM) is added and the tube placed in an ultrasonic bath at room temperature for 15 minutes to extract the analytes. This operation is repeated once with 10ml of DCM to optimise extraction. Following extraction the sample is concentrated to 1ml using a nitrogen blowing system and then analysed via HPLC coupled to a Fluorescence detector. XAD2 resin tubes may be used as an alternative to fibre filters.

Evaluation of new Sample Preparation Methodology

A standard solution containing the US-EPA 16 PAHs (as defined by IARC, 1987) was spiked onto quartz fibre filters or XAD2 resin tubes and allowed to air dry. The filters / tubes were then extracted twice using 7ml DCM and sonnication in the ultrasonic bath for 15 minutes at room temperature. The combined sample (14ml) had a 100ul aliquot removed. This was made up to 1ml with acetonitrile was taken and injected into HPLC-Fluorescence to provide a 100% reference. The remaining DCM had 100l 2-pentanol added as a solvent keep and was evaporated via centrifugal vacuum evaporation in the Genevac EZ-2 Envi (Figure 1). Temperature and pressure during evaporation were controlled such that the DCM evaporates but the 2-pentanol does not, as previously described by Marsico (2006) and Massat et al. (2007).

Figure 1 (right) – Genevac EZ-2 Envi

The samples were then made up to 1ml using acetonitrile and injected into HPLCFluorescence for analysis. Recoveries for all analytes, even the most volatile were in excess of 90% and the fit of the analytical curve to the reference sample was very good, and shown in figure 2 below.

The samples were then made up to 1ml using acetonitrile and injected into HPLCFluorescence for analysis. Recoveries for all analytes, even the most volatile were in excess of 90% and the fit of the analytical curve to the reference sample was very good, and shown in figure 2 below.

Figure 2 – HPLC-Fluorescence Chromatogram Overlay of Reference Sample to Post Concentration Sample

Red – the reference point. Blue – other chromatograms refer to the PAH compounds Naphthalene, Acenaphthene, Fluorene, Phenanthrene

Validation of the Process

Having delivered similar results to the existing method, and being beneficial in the sense of “automation” of the concentration process, statistical validation of the process and equipment was required. Using the above methodology, a solution containing 14 PAH samples was spiked onto quartz fibre filters and also on to XAD2 resin tubes. Filters were spiked at 100ng and 10ng. These were allowed to dry and extracted, concentrated and analysed. The process was repeated on six distinct occasions using new samples and solutions on each occasion. The results are presented in Figure 3.

Figure 3 – Data from Validation Studies 

Mass Recovered (ng) and Recovery % are averages from each of the 6 repetitions performed. SD is the standard deviation across repetitions.

The results generally show excellent recovery and good standard deviation figures. Due to a contamination from XAD2 resin, for two compounds (naphthalene and acenaphtene) limits of quantification have been validated at 50ng instead of 10ng.


The new method of sample preparation was found to be superior to the existing methods. Recoveries are seemingly a little lower for the 10ng studies because this  approaches the limit of detection of the analytical method. Following successful validation and external audit by COFRAC (Comité français d’accréditation) the new method and systems have been adopted into routine daily use.

About the Authors

Nicolas Falquet is Testing Manager at ITGA, a leading independent analytical testing laboratory, based at Le polygone, 46 rue de la Télèmatique, 42000 St-Etienne, France.  ITGA is part of the Carso Group.

Gilles d’Esperonnat is responsible for sales and service of Genevac evaporators in France and based in the Lyon area Rob Darrington is Product Manager at the Genevac head office, Farthing Road, Ipswich, IP1 5AP, UK.


IARC. 1987. IARC Monographs on the evaluation of carcinogenic risks to humans, supplement 7, Overall evaluation of carcinogenicity: an updating of IARC monographs 1-42. Lyon: International Agency for Research on Cancer

Marsico, Anna Maria, 2006. Improving Analysis of Pesticides – a new method development protocol to increase recovery of volatile compounds. First published in Lab Asia, August 2006 & available via

Massat, F, Planel, B & Venezia, A, 2007, Evaluation of Evaporative Sample Preparation Techniques. First published in International Environmental Technology, March/April 2008, pp 36, and also available via

NF X 43-294. June 1995. Sampling and analysis of polycyclic aromatic hydrocarbons INRS. 2007. Method Metropol 011. Polycyclic Aromatic Hydrocarbons. NF ISO 11338-2. March 2004. Determination of gas and particle-phase polycyclic aromatic hydrocarbons – Part 2 : sample preparation, clean-up and determination.

Evaluation of Evaporative Sample Preparation Techniques for Alcohol Markers and Drugs of Abuse in Hair Samples

By: Dr Eleanor I Miller & Dr Simon P Elliott ROAR Forensics, Malvern, UK


Hair analysis can be a useful tool in many forensic and clinical applications to establish drug use, trends of use and in the assessment of chronic alcohol consumption. For example, it can be utilised as part of a medico-legal investigation into drug-related deaths (as a complement to testing other post-mortem biological samples), drug-facilitated crimes, as part of programme compliance for those participating in drug or alcohol dependency treatment or as part of workplace or health insurance screening.

Drugs and drug metabolites can become encapsulated within body hair and analysis for these drug residues provides an accurate assessment of an individual’s retrospective drug intake over a period of time (typically months prior to sample collection) delivering more information than an ‘on the spot’ test, e.g. blood or urine, which only offer a snapshot of drug use. A further limitation of a blood or urine sample is that it has to be collected in close proximity to when the drug is taken, or suspected to have been taken, whereas hair for analysis may be collected many weeks later.

Drugs and drug metabolites circulating in the bloodstream pass into the hair follicle and these can become locked into hair strands when they are formed beneath the skin. As the hair

strands grow out, the segment containing any drug metabolites grows with it. It can take several months to grow out in order to allow for an appropriate hair sample collection which is targeting the correct time period under investigation. After this time, testing can potentially determine which drugs were taken and also indicate approximately when they were taken providing evidence of regular, acute or isolated drug use.

Equipment Validation

When introducing any new piece of instrumentation or equipment into a highly controlled environment such as a forensic analysis laboratory, the new unit must be evaluated to ensure that it does not create any artefact in the samples or cross-contaminate the tubes, potentially invalidating the analysis. The Genevac® EZ-2 is a centrifugal vacuum evaporator which can accept many samples, and therefore can be useful in a busy laboratory. With regard to evaporative sample concentration technology the most important issues are prevention of cross contamination and sample recovery, especially for very volatile analytes as some are only present in picogram (pg) quantities.

As part of their evaluation of new equipment, ROAR Forensics evaluated the Genevac EZ-2 before introduction to their processes. A summary of the data is presented in this report, which investigates the potential for cross contamination using a hair alcohol marker and a drugs of abuse (DoA) solution, and, evaluates recovery of amphetamine, which is renowned for its volatility.

Cross Contamination Study

The aim was to determine if any cross-contamination occurred during the evaporation process in the Genevac EZ-2 system for a hair alcohol marker and commonly targeted drugs of abuse in a forensic toxicology hair testing laboratory, at relevant concentrations.

The methodology was based on a previous cross-contamination study involving 96 well micro-titre plates which found that the sample travel was always observed to be horizontal1.

“Blank” tubes containing no analytes were positioned in the sample holder along the row containing the spiked sample, in some adjacent positions and also in a few positions at the furthest points from the spiked sample position. 13mm diameter x 100mm height tubes were used in a Genevac 10-5002 sample holder. The arrangement is shown in Figure 2.

Trials were carried out as follows:

1. 1500pg of a hair alcohol marker – ethylglucuronide (EtG) in 2ml of solid phase extraction (SPE) eluent, a mixture of methanol and formic acid. Two identical sample holders were evaporated in the EZ-2 using method 2, “Low BP”, with the sample holder temperature set to 40°C.

2. 1000ng of a DoA standard solution in 7ml of SPE eluent, a mixture of acetone, dichloromethane, ethyl acetate, and ammonium hydroxide. Two identical sample holders were evaporated in the EZ-2 using method 5, “Low BP Mix” with the sample holder temperature set to 40°C.

The DoA solution contained; ecgonine methyl ester, cocaine, benzoylecgonine, norcocaine, cocaethylene, morphine, 6-monoacetylmorphine, codeine, dihydrocodeine, methadone, EDDP, amphetamine, methamphetamine, MDA, MDMA, MDEA and MBDB.

The levels of analytes were selected because they are the concentrations that produce the highest hair calibrator in each method; 50pg/mg equivalent for EtG and 50ng/mg equivalent for DoA. After evaporation the tubes were reconstituted with 100l of mobile phase and analysed via LC-MS-MS.

Cross Contamination Study Results

No EtG or DOA analytes were detected from analysis of any of the tube contents which were evaporated in positions P1, P4, P12, P17, P19, P21, P23, P24, P28, P36 and P40. Position P20 (“positive” control) showed expected analytes having been spiked with 1500pg EtG or

1000ng DOA.

Analyte Recovery Study

Amphetamine was used for this study due to its renowned volatility. The concentration selected for assessment is the equivalent of 0.2 ng/mg (the current proposed Society of Hair Testing (SoHT) cut-off for an indication of active amphetamine use)2. 4ng of amphetamine in methanol was pipetted into 13 x 100 mm test tubes. Three tubes were evaporated in the EZ-2 using method 5 “Low BP Mix” with the sample holder temperature set at 40°C. Three tubes were allowed sufficient time to evaporate in a fumehood at room temperature. After evaporation the tubes were reconstituted with 100l of mobile phase and analysed via LCMS- MS.

The peak areas for amphetamine were compared for the two different sets of evaporation conditions.

The percentage relative recovery was calculated using the equation below:

% RRecovery = average peak area for amphetamine evaporated using Genevac EZ-2 x 100 average peak area for amphetamine evaporated at room temperature

The recovery for the tubes evaporated in the Genevac EZ-2 relative to the tubes evaporated at room temperature was calculated to be 115 %.


No cross-contamination was observed during the evaporation processes selected for EtG or DOA for the concentrations tested.

Based on the limited data, it would appear that the evaporation system is suitable for evaporating the SPE eluent containing amphetamine, with no loss observed. It would also appear that the Genevac evaporation programme used for DOA produces excellent recovery for amphetamine, which is renowned for its volatility. From an interpretative perspective, it would appear that samples containing amphetamine at the SoHT recommended cut-off of 0.2 ng/mg would be determined at this level.


1. Dri-Pure Sample Integrity Protection System. An Evaluation by GlaxoWellcome. Dr Martin Deal (1999), available via

2. Society of Hair Testing. Recommendations for hair testing in forensic cases. Forensic.Sci.Int. 145:83-84 (2004).


The authors would like to acknowledge Eleanor Menzies of King’s College London for her contribution to this evaluation study.

About the Authors

Dr Eleanor I Miller is a Specialist Forensic Toxicologist & Dr Simon P Elliott is Managing Director, at ROAR Forensics, Malvern Hills Science Park, Geraldine Road, Malvern, WR14 3SZ, UK. (

Advances in Sample Preparation for Metabolite Profiling

By: Flavio Cinato – Research Scientist, Nerviano Medical Sciences, Milan, Italy Rob Darrington* – Product Manager, Genevac Ltd, Ipswich, UK Steve Knight – Marketing Manager, Genevac Ltd, Ipswich, UK

Project Outline: 

The aim of the project was to set up a procedure, based on a semi-preparative LC-MS-MS system to allow the determination of the biological activity and the definitive structure of metabolites taken from in vitro assays or in vivostudies. Purified metabolites could then be tested in several ways, including:

·       Tested on target enzymes for activity

·       Tested on non-target enzymes for activity

·       Definitively identified with NMR

·       Entered into toxicological studies

·       Assessed for possible drug-drug interaction

·       Tested for reactivity

·       Used as standards for pharmacokinetic determinations 

There is a significant advantage in being able to extract the sample from the original assay, purify and identify it so that it can then be used for further study. Typically, metabolites that require further study are synthesised followingdetermination, which extends the time taken for metabolite evaluation considerably. To enable further studies to take place, the goal of the project was to establish a system to provide 50 to 100ul of a 1mM solution of purified identified metabolite in DMSO solution. This solution can then be screened for activity against a number of different targets. The procedure was established using a series of Tyrosine Kinases targets and a compound with well-established metabolism leading to two main metabolites, one of which is active, and another which is inactiveagainst specific targets.


Samples were taken from the assay and an aliquot presented to the LC-MS-MS system, first running an analytical column to determine the optimal conditions for preparative separation. Next, the bulk of the sample is separated using the preparative column, and the fractions collected. LC solvents were water and methanol, containing 0.1% formic acid as a modifier. After separation the samples were dried and then diluted to known concentration and reanalysed by LC-MS-MS and NMR. 

It is at the evaporation stage of the process that problems were initially encountered. 

First, one of the drying methods trialled blew nitrogen onto the samples to hasten evaporation, however this resultedin much of the sample drying and sticking to the sides of the tube which made dissolution in minimal (50 to 100ul) DMSO very difficult. This lead to use of a centrifugal concentration system which evaporates the dried sample into a small area at the base of the tube, which is far better for redissolving in minimal solvent. 

The second drying issue that was encountered was in screening samples post drying. At first, blank samples (containing no compound) were run through the whole process and via both drying methods showed up false positives in the screening trials, this was attributed to residual modifier from the LC solvents. 

Thirdly, with both of the methods tried, some compound degradation was observed, attributable to poor temperature control of the samples in the evaporator. Comparisons between standards and pilot samples showed lower activity of the samples that has been dried. Clearly further method development needed to be done as sample degradation is unacceptable due to the sensitive and exacting nature of the toxicologists work. 

These issues led Nerviano to search for a more efficient evaporation system, one that concentrated the sample to the bottom of the tube leaving little or none on the tube walls, removed all the modifier from the LC solvents thereby eliminating a cause of false positives in the screens, and did not degrade the compounds with excessive heat.   Trials with the Genevac EZ-2 for the evaporation of the purified parent and metabolite fractions proved very successful with the activity results very close to those obtained with the respective analytical standards. False positives were eliminated and a low extent of compound degradation was observed. Minor variations seen were acceptable considering the intrinsic variability of the activity as determined by high throughput screening. Above and beyond the greatly improved screening results, an additional benefit was that the evaporation time was sensibly lower compared with the other centrifugal system tried, or the blow down method.  


The novel semi-preparative auto-purification system including on-line LC-MS-MS analysis was successfullyinstalled and setup. The new system allows the isolation and identification of pharmacologically active compounds and their metabolites. The Genevac evaporator provided the optimal balance between speed and minimal compound degradation during the evaporation step, and eliminated interferences with mobile phase buffers from the LC solvents. The validity of this procedure was confirmed by subsequent biological activity tests and by proton NMR. Validation of sample preparation techniques is as important as analytical methods to because they either may be the source of erroneous results.