Bioanalytical challenges

Today, liquid chromatography–tandem mass spectrometry (LC–MS/MS) is established as the state-of-the-art methodology for quantitative bioanalytical assays in various matrices [1]. Chromatographic separation and MS/MS detection also play a pivotal role in providing satisfactory analytical selectivity, which is a basic criterion for ensuring the accuracy of a method [2]. Their applicability to a wide range of analytes and the optimal compatibility of the elution conditions with electrospray ionisation (ESI)—the mode of choice for charged polar solutes—have resulted the dominant use of reversed-phase (RP) stationary phases. However, for highly polar acidic compounds such as metabolites (glucuronides, sulfates, etc.), physiological intermediates (nucleotides, etc.) or drugs (peptides, antibiotics, etc.), one may encounter selectivity problems with RP-LC, especially when it comes to the analysis of biomatrices. The retentions of such solutes can usually only be achieved with strongly acidic eluents that contain a high fraction of water. Alternative approaches that have sometimes been pursued include the addition of ion-pair reagents to the eluent in RP-LC [3, 4], the use of polar phases in ion exchange LC [5] or hydrophilic interaction LC (HILIC) [6], as well as the use of porous graphitic carbon materials as stationary phase [7]. However, each of these valuable concepts suffers from its own shortcomings and limitations [6–11].

Sometimes analytical chemists simply neglect chromatographic selectivity issues, claiming “intuitively” that multistage MS operated in multiple reaction monitoring (MRM) mode is compound-specific. However, this is illusory [1, 11]. When analysing polar acids in a polar environment by RP-LC, ESI-MS/MS detection can be particularly prone to “matrix effects”, i.e. the coelution of matrix compounds with the target analytes, altering (usually reducing) the ionisation yield of the latter. Matrix effects can severely affect accurate quantification, and may even vary in magnitude between different batches of matrix [12–14]. The application of stable-isotope-labelled internal standards can in most cases adequately correct for matrix effects, but they are not always available, and in the worst case scenario may even have a different ionisation alteration susceptibility compared to the target analytes [15, 16].

Mass spectral interferences arising from the unintended in-source fragmentation of compounds, resulting in the release of a fragment that is identical to the target ion (as is often observed for instance for conjugated and unconjugated metabolites), or isobaric solutes with similar fragmentation properties may also seriously affect reliable determinations of target analytes in the case of coelution [17–20]. These problems may be amplified if inadequate sample preparation strategies are used to extract the target solute from the matrix or a direct injection strategy is chosen in order to minimise sample preparation expenditure and achieve a high sample throughput.

In general, one should bear in mind that the MRM mode does not guarantee satisfactory assay specificity and accuracy per se. Selective chromatography should not be overlooked as a crucial optimisation parameter for robust and accurate analytical conditions, and it is desirable to have a tool on hand that allows for flexible retention and selectivity adjustment.

Potential of mixed-mode reversed-phase/weak anion exchange stationary phases

Mixed-mode phases combining orthogonal separation principles, e.g. RP and ion exchange, offer much more optimisation potential than classical RP chromatography. Although well known from their use in solid-phase extraction materials [21] and capillary electrochromatography [22, 23], this approach has only rarely been exploited in LC [24] and has only become more popular very recently [13, 19, 22, 25–27]. One particularly useful variant is the mixed-mode reversed-phase/weak anion exchange (RP/WAX) material shown in Fig. 1 [13, 19, 25, 26], which is obtained by grafting N-(10-undecenoyl)-3-aminoquinuclidine onto thiol-functionalised silica particles [13, 19, 25] or monoliths [26]. It has distinct interaction sites on a single chromatographic ligand, such as a hydrophobic alkyl chain strand (hydrophobic domain), embedded polar functional groups (hydrophilic domains), and a terminal bicyclic quinuclidine ring which accommodates the anion exchange site (pK a ∼ 9.9 [28]) (ionic domain).

Fig. 1
figure 1

Structure of the mixed-mode reversed-phase/weak anion exchange (RP/WAX) material discussed in the text

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The separation material shown in Fig. 1 displays retention and selectivity patterns that are complementary to (polar-embedded) RP and classical amino phases. The strength of the interactions and thus the retentions at the individual interaction sites can be adjusted largely selectively by varying a broad range of parameters, such as the nature and amount of organic modifier in the eluent as well as the buffer type and concentration, additives, pH, and temperature. Various modes of gradient elution (positive and negative organic modifier gradients, mixed modifier/buffer gradients, pH gradients) add further flexibility during method development.

As a consequence of its distinct structural binding modules, the mixed-mode material of Fig. 1 is a true multi-modal phase that, depending on the elution conditions and the characteristics of the solutes used, can be operated in a variety of chromatographic modes, such as classical RP mode (e.g. for neutral molecules), WAX mode (for negatively charged solutes), ion exclusion chromatography mode (cationic solutes), HILIC mode (polar neutral, basic, amphoteric, and acidic compounds), and hydrophobic interaction chromatography mode (for e.g. larger peptides). The resulting versatility of this mixed-mode RP/WAX phase has already been demonstrated, as will be exemplified below for bioanalytical LC–MS/MS applications.

Bioanalytical applications of multi-modal chromatography using RP/WAX

Investigation of the metabolism of chlorpyrifos

Chlorpyrifos (CP) is a neutral lipophilic organophosphothioate-type insecticide which is rapidly metabolised to highly polar acidic O,O-diethyl phosphate (DEP; pK a ∼ 1.4) and O,O-diethyl thiophosphate (DETP; pK a ∼ 1.4) as well as the medium-polar acid 3,5,6-trichloro-2-pyridinol (TCP; pK a ∼ 5.9) in mammals (pK a values calculated with [28]). Due to the huge range of lipophilicities covered by the parent compound and the metabolites, for a long time a simultaneous separation proved elusive. However, the above RP/WAX phase made the separation of both parent compound and metabolites possible (Fig. 2), which allowed this assay to be used to determine urinary elimination kinetics [19, 29].

Fig. 2
figure 2

LC–ESI-MS/MS separation of CP, DEP, DETP, DBP, as well as TCP in RP/WAX mode, and pH-dependent log D profiles of analytes, as calculated with [28]. Reprinted in modified form from [19]

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CP was retained by hydrophobic interactions according to a typical RP mechanism, and despite being the most lipophilic solute it eluted first from the column. Under the given conditions (water–acetonitrile gradient, eluent pHa 6.45–7.45), the metabolites and the internal standard employed, O,O-dibutyl phosphate (DBP), were largely dissociated and thus followed an anion exchange retention mechanism. However, superimposed upon the latter was a hydrophobic partitioning mechanism, like that observed in RP-LC, and so the alkyl(thio)phosphates eluted in order of increasing hydrophobicity (i.e. increasing log D). TCP is the most retained compound even though it is the weakest acid because it has a significantly more lipophilic character than DEP, DETP, and DBP.

When real samples were analysed, two additional peaks showed up in the MRM traces of TCP, although they were well resolved from the target analyte. These peaks were later identified as arising from (partial) ESI in-source degradation of two distinct, formerly unknown, CP metabolites with a TCP moiety (namely TCP-O-glucuronide and mono-O-deethyl-CP). This finding illustrates the limitation of the MRM mode and reveals the absolute need for adequate chromatographic selectivity in order to preserve analytical assay selectivity.

Determination of ethanol consumption markers

Confirmation of ethanol intake is of great importance in forensic toxicology, psychiatry, and workplace monitoring. A number of different markers, from short term to long term, are available; each one with a specific window of detection [30]. Amongst these, the ethanol phase II metabolites ethyl glucuronide (EtG) and ethyl sulfate (EtS) have attracted particular attention lately as both are sensitive, specific, and stable intermediate-term markers of acute ethanol consumption in urine and other matrices [31].

We developed a LC–MS/MS assay for the simultaneous analysis of EtG and EtS as well as ethyl phosphate (EtP), a potential new marker with distinct nonspecific or nonenzymatic formation [32], in human urine [13]. Reliable quantification of these highly polar acidic metabolites (pK a of EtG ∼ 2.8; of EtP ∼ 1.9 and 6.4; of EtS ∼ −3.1 [28]) in the polar urinary matrix poses a unique challenge, particularly considering the direct injection methodology chosen for diluted urine. The optimised RP/WAX separation is shown in Fig. 3a.

Fig. 3
figure 3

a LC–ESI-MS/MS separation of EtG, EtP, and EtS in HILIC/WAX mode. Note that the elution of EtP was performed in back-flush mode. b Retention properties of EtG and EtS as a function of acetonitrile content and pHa of the hydroorganic eluent. Reprinted in modified form from [13]

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It is clear that the two singly charged solutes EtG and EtS are eluted before the doubly charged EtP (which was eluted in back-flush mode to speed up the analysis [13]), as expected from the primary anion exchange process. The elution order of EtG and EtS, in contrast, follows the order of increasing hydrophilicity (log D pH 7.4: EtS ∼ −3.4, EtG ∼ −5.9, EtP ∼ −5.4 [28]). Thus it may be concluded that in acetonitrile-rich eluents a HILIC mechanism is superimposed upon the primary anion exchange process, which is confirmed by the graph shown in Fig. 3b. As the acetonitrile fraction increases the retention factors are increased and the more hydrophilic EtG responds more strongly to the change in organic content, as is clear from the steeper curves, than the less hydrophilic EtS.

The RP/WAX column employed showed excellent stability, as it could withstand more than 600 matrix-matched validation runs and the analysis of about 70 real urine samples from a volunteer drinking study and from autopsied corpses tested positive for urinary ethanol without exhibiting any significant loss of performance [13].

Conclusions and outlook

The goal of this article was to document the great potential of mixed-mode stationary phases to serve as an alternative separation concept for bioanalytical investigations of polar analytes when RP chromatography experiences retention and selectivity problems. We focussed on discussing a RP/WAX separation material that is particularly well-suited to the analysis of very polar acids. Reciprocally, the concept may be adapted for the analysis of polar bases too, making use of mixed-mode cation exchangers [33]. As the selected examples demonstrated, the potential of mixed-mode phases for the convenient analysis of highly polar acidic compounds as well as for the simultaneous determination of lipophilic parent compounds and metabolites, and in particular their remarkable flexibility in terms of being able to adjust their retentions and selectivities, offer a great deal of benefits to users. In view of the above results, they undoubtedly show much promise in relation to the analysis of endogenous metabolites in fields like metabolomics and biomarker studies.

In general, with the introduction of modern, robust, sensitive, and user-friendly LC–MS/MS systems, a powerful analytical platform for bioanalytical assays has now been established in many laboratories. However, although sometimes blinded by the availability of high-end instrumentation, one should not overlook adequate chromatographic selectivity as an important requirement for valid, accurate and rugged analytical methods.