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Ion suppression in mass spectrometry has been described recently in detail and should always be considered during analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) in a drug metabolism and pharmacokinetics (DMPK) environment. At best, ion suppression leads to decreased sensitivity but at worst could lead to incorrectly determined pharmacokinetic (PK) parameters. Our investigations centred on polyethylene glycol (PEG 400), an excipient often used in pre-clinical dosing vehicles. PEG was also found to be present in large quantities in the blood collection tubes used for pre-clinical PK studies. Ion suppression was observed for many analytes, either due to the use of PEG in the dosing vehicle or in blood collection tubes. The elimination of large ion suppression effects was attained by simple chromatographic gradient changes and the use of alternative blood collection tubes. The effect of the above was to increase the detected plasma concentration levels, which resulted in a change in key PK parameters. Copyright © 2006 John Wiley & Sons, Ltd.
There has been an explosion in the use of liquid chromatography/tandem mass spectrometry as a universal analytical endpoint for drug metabolism and pharmacokinetics (DMPK) applications in recent years, e.g. pharmacokinetic (PK) analysis, intrinsic clearance, CYP inhibition/induction, PAMPA and Caco 2 permeability, metabolite identification, plasma stability studies, etc. Although the technique is extremely sensitive and robust there is potential for ion suppression,1–5 especially using electrospray ionisation (ESI), which can give rise to incorrect data interpretation.6, 7 This effect can be enhanced by the use of ‘ultra-fast’ gradients, which allow many analytes to co-elute. In some cases, it may not be obvious that differing ion suppression may be occurring during an analytical run, further complicating the quantitative results. This is potentially exacerbated due to the variety of matrices used during analysis, which may give differing ion suppression depending on the application. This is further complicated when used in a discovery environment, where potentially hundreds of molecules with differing physicochemical properties (pKa, logD7.4) are analysed. These compounds may be differentially ion suppressed depending on their elution on a typical LC gradient, compared with the ion-suppressing agent, and their ability to compete with charge from the suppressing agent.
The studies described here concentrate on a particular known ion-suppressing agent (polyethylene glycol – PEG 400). This agent is often present in large quantities in DMPK samples, either from deliberate addition as an excipient in a dosing solution or as a contaminant from plastic-ware used in sample preparation. Ion suppression by PEG is well documented and strategies to minimise the effects have been published.8, 9 Identification of ion suppression due to PEG and effective removal of ion suppression are discussed along with the impact of PEG effects on typical PK parameters.
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Ion suppression effects in LC/MS/(MS) have been the subject of numerous publications in recent years.1–9 This has partly been due to the expanding use of this technique in the analysis of pharmaceutical, trace product and environmental samples. Ion suppression is known to have the effect of reducing overall sensitivity as well as giving rise to flawed analysis due to potentially differential suppression depending on matrix. The mechanism of ion suppression has been proposed to be due to the following:
Competition for charge between analyte and ion-suppressing agent leading to an overall reduced ionisation of analyte.
Large concentrations of ion-suppressing agent leading to increases in surface tension and viscosity of the droplets formed in atmospheric pressure chemical ionisation (APCI), ultimately leading to decreasing evaporation efficiency. This could certainly be a possibility for PEG, since it is often present in large concentrations and may well affect nebulisation and droplet formation due to changes in surface tension and conductivity of the liquid phase.
Gas-phase reactions between analyte ions and other sample components leading to an overall loss in charge from the analyte ions.
However, the latter reason has been disputed5 as the authors argue that gas-phase species should be present in APCI and ESI and the latter often shows more pronounced ion suppression. The authors suggested that changes to the solution properties due to high concentration of non-volatile solutes probably controlled ion suppression. The underlying reason why non-volatile species can give rise to ion suppression has not been clearly demonstrated to date, but possibilities include precipitation of analyte with the non-volatile species, which could limit transfer from the liquid to gas phase,2 or that the presence of non-volatile species changes the spray droplet solution properties.1, 5
The present study focused on the ion suppression caused by polyethylene glycol (PEG 400) in pharmacokinetic (PK) analysis of pre-clinical studies, which was either deliberately included in the dose formulation or extracted as part of sample workup from plastic-ware.
With regard to the use of PEG as part of a dosing vehicle, it was important to assess the concentration present in plasma samples from rats dosed either IV or PO in a typical protocol and whether the PEG interfered with the analytes of interest during LC/MS/MS analysis. The PEG PK profile was monitored following IV and PO administration of a typical NCE dosed in a PEG 400 formulation. For the compound studied, the initial PEG plasma concentrations following an IV dose were very high, but rapidly declined with a terminal half-life of 2 h. Following oral administration the Tmax was at ∼30 min with a rapid terminal half-life of 2 h. As initially the PEG concentrations are vastly in excess of analyte concentrations, one may assume that ion suppression could occur. In fact, the PEG concentrations are so high (100–1000 µg/mL) that overloading of the column stationary phase occurs, leading to the PEG eluting over a wide proportion of the chromatogram. In addition the retention time of the PEG oligomers increases with molecular weight, spreading out the elution even further. It was shown that, for the majority of analytes studied, co-elution occurred between PEG and analytes.
In order to investigate whether this co-elution could cause ion suppression, an equivalent in vivo PEG concentration was spiked into blank rat plasma along with ten project compounds. Ion suppression was observed for most analytes, which were subsequently shown to co-elute with PEG. In addition, the degree of ion suppression was variable depending on analyte (Fig. 5).
This unpredictable variability coupled with the ever-changing plasma concentrations of PEG (due to excretion) and subsequent reduction of ion suppression with time could potentially lead to incorrectly determined PK parameters including in vitro/in vivo scaling. This has indeed been shown to be the case with the effect of PEG in dosing vehicles.9 In this report, the authors had noted that clearance and volume of distribution could be overestimated by as much as six- and four-fold, respectively, due to ion suppression by PEG.
Attempts were made to modify the generic rapid LC gradient to shift the elution of PEG away from analytes. Partial success was achieved (Fig. 4), in which the bulk of the PEG oligomers eluted before 1 min, with the analytes generally eluting after 1.5 min. An alternative approach to eliminate PEG from plasma samples could be the use of strong anion/cation solid-phase extraction (SPE) for acid/basic chemistry prior to analysis.
Following the discovery of PEG contamination of blood from the blood collection tubes used for pre-clinical sampling, the impact of this potential ion suppression was investigated. A similar effect was seen to the investigations described earlier with PEG 400 in the dosing vehicle. Analyte response was suppressed to varying degrees up to a maximum of 88%. Clearly, with this example, incorrectly determined PK parameters could be obtained, which would be enhanced further if the standards and quality controls were prepared using plasma from an alternative source which may not have been contaminated with PEG to a similar extent.
To test this theory in practice, a test research compound that had been dosed to rats IV and PO and the blood collected in the PEG-contaminated blood tubes was investigated. The resultant PK parameters were rather unexpected and, at first, puzzling. Unusually, the compound had a high in vivo clearance that equated to a typical rat liver blood flow (70 mL/min/kg), which would not have been predicted from the in vitro rat hepatocyte screen. The volume of distribution was also higher than anticipated and, strangely, the bioavailability was good at 67%, which was not in keeping with a compound that should have undergone a near complete ‘first-pass effect’ by the liver following oral dosing.
Following some investigational LC/MS/MS work it was found that the analyte directly co-eluted with the PEG leached from the blood tubes. As the analyte was a basic compound (pKa ∼ 9), the mobile phase was changed from acidic to basic (pH 10.5) which had the effect of dramatically increasing the retention time of the analyte. The PEG peaks were unaffected by the mobile phase change due to their neutral nature. The result was chromatographic separation between analyte and PEG.
Following reanalysis of the samples by the modified HPLC method, the analyte plasma concentrations were determined to be much higher than the original analysis. This resulted in certain PK parameters changing from the original analysis.
Clearance was reduced from 70 to 14 mL/min/kg, which was in line with the value expected from the in vitro analysis. The volume of distribution was reduced from 23 to 10 L/kg, with the terminal half-life unchanged as expected. The bioavailability was also similar to the previous analysis as expected (similar effects on both IV and PO samples) and now was understandable due to the reduced clearance and lower first-pass effect. This effect of ion suppression from PEG in blood collection tubes culminated in similarly distorted PK parameters to those described previously with ion suppression due to PEG in the dosing vehicle.7, 9 Following this discovery, alternative blood collection tubes have been sourced which are found not to contain PEG and these are now used exclusively for rodent PK studies. In order to minimise ion suppression further, we avoid PEG 400 in dosing vehicles wherever possible, relying on alternative excipients, e.g. methyl cellulose, cyclodextran, etc. However, to ensure that these excipients do not cause ion suppression, two test solutions (±excipient) at nominal concentration are prepared in rat plasma, and following sample workup the two samples are injected onto the analytical HPLC gradient. The MS/MS peak responses are compared to check for ion suppression. If appreciable, then HPLC method development is applied until the analytes are chromatographically separated from the ion-suppressing agent.
The above rat pharmacokinetic example clearly demonstrates that the power of chromatographic resolution of analytes from PEG (or other ion-suppressing agents) should not be underestimated. Understanding the physicochemical properties of any particular analyte versus the analytical conditions used is essential to decide the best course of action for resolving analytes/ion-suppressing agent. Clearly, it is simpler to resolve the two via simple pH changes to the mobile phase when the analyte is either basic or acidic assuming the ion-suppressing agent is a neutral molecule. Ion suppression issues with neutral analytes can also be achieved relatively simply with changes in column chemistry, mobile phase changes or gradient modifications.
Ion suppression is an important consideration in DMPK analysis and should not be over-looked. It can lead to a reduction in analyte response and hence a higher limit of quatitation (LOQ), or more worryingly incorrect assignment and interpretation of PK parameters. We have shown that the source and degree of ion suppression due to PEG can be variable depending on the NCE analysed and the dosing vehicle/apparatus used during sample workup at the time. Although there have been accounts of PEG removal from biological samples,7, 9 at present there is no universal generic method of removing PEG from biological samples in a discovery environment, due to the range of NCE chemistries and their associated charge and polarities encountered on a daily basis.
However, the source and identification of ion suppression is crucial in order to address the phenomenon in each case. Relatively simple chromatographic modifications including changing the pH of the mobile phase or changes to overall best practise (e.g. alternative dosing vehicles, changes in blood collection tubes) can be applied to overcome this issue. It should be stressed that understanding the chemistry and chromatographic properties of the analytes is imperative, in order that appropriate chromatographic changes can be implemented to resolve them from PEG. This approach should be applied on a case-by-case basis, as it is unlikely that a generic solution can be found for the range of chemistries encountered in a typical research programme.