Matrix effect elimination during LC–MS/MS bioanalytical method development
Due to the presence of endogenous components in biofluids, ionization suppression or enhancement may occur for bioanalytical assays using LC–MS or LC–MS/MS technologies. The matrix effect may affect the precision and accuracy of a bioanalytical method and, therefore, compromise the quality of the results. Protein precipitation sample preparation along with LC–MS/MS is a high-throughput method most commonly used in bioana lysis and is largely affected by the matrix effect. In order to eliminate the matrix effect during the method development, some considerations may be used: cleaner sample preparations, more sensitive instruments, which allow less material to be injected, different chromatographic separations and much more must be investigated. More than giving tools to adequately assess the matrix effect during the method development, this review gives scientists numerous ways to eliminate or reduce the matrix effect based on novel sample-preparation techniques, new chromatographic optimization methods and new technologies.
LC coupled to atmospheric pressure ioniza-tion (API) MS/MS is presently the most com-mon technique used in bioanalysis. Indeed, LC–MS/MS is highly selective and a sensitive instrument successfully used to develop high-throughput bioanalytical assays. LC–MS/MS high-throughput method chromatograms rarely show interfering peaks with the analyte of inter-est, even if there are high levels of co-eluting compounds. However, co-eluting compounds may affect the ionization efficiency and repro-ducibility in the ionization source. This phe-nomenon, known as the matrix effect, was established by Kebarle et al [1,2] and was first reported in a bioanalytical assay by Buhrman et al. [3]. The matrix effect is also known to affect the precision and accuracy of bioanalyti-cal methods [3,4]. Consequently, this is a major concern for the development and validation of bioanalytical methods. For LC–MS- and LC–MS/MS-validated assays, evaluation of the matrix effect is required by the US FDA [101] and other regulatory agencies. Validation of the bioanalytical method must ensure that the accu-racy, precision, selectivity and sensitivity are not compromised by the matrix effect [101]. Other authors have already published excellent reviews, which may be consulted as a complement to the current paper [5,6]. The aim of this review is to highlight the recent developments published on the matrix effect, the procedures to assess the matrix effect and the strategies/solutions available to minimize or eliminate the matrix
effect in the course of method development. In addition to the theoretical review of causes and assessment methods of the matrix effect, this review includes information that may save the scientist’s time during method development for the elimination or reduction of the matrix effect. Indeed, different innovative sample preparations and chromatographic separation discoveries are presented, along with new technologies in ion-ization source design and sample-preparation techniques. All together, these more recent topics might reduce the bioanalyticalmethoddevelop-ment time and reduce matrix effect issues at the beginning of the development.
Type of ionization versus matrix effect Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources are the most commonly used ionization modes dur-ing the development of bioanalytical methods due to their reliability and ruggedness. The majority of the validated LC–MS/MS methods recently reported in the literature were developed using ESI or APCI interfaces [6]. The matrix effect ionizationsuppression mechanisms in ESI and APCI interfaces are related to their ion-formation mechanism. The ion-formation mechanisms for ESI and APCI are still under investigation today but the main hypotheses are reported in this review. Atmospheric pressure photoionization (APPI), preferred to ESI or APCI for normal-phase chiral ana lysis [7] and ana lysis of steroids, could also be subject to the matrix effect [8].
Bioanalysis
The process of analyzing drugs, drug metabolites or chemicals present in biological matrices in order to get more information about them and to enable drug discovery and development
lC–Ms/Ms
Analytical technique by which chemical substances are separated by high-performance LC and identified or quantified by sorting their respective gaseous ions by their mass to charge ratio (m/z) using electromagnetic fields
�� Electrospray ionizationElectrospray ionization mechanism can be described by two different models: the charged residue model [9] for large molecules (proteins) and the evaporation model [10] for small mole-cules. Based on the evaporation model, the ana-lyte is ionized in the mobile phase (liquid phase) and trapped in a charged droplet that is then vaporized into smaller droplets until the electri-cal field increase critically and releases the ions into the gas phase [2]. ESI shows a linear response from 10-8 to 10-5 M for a variety of organic bases. It was suggested early on that a co-eluting com-pound at a concentration higher than 10-5 M will affect the ionization efficiency of a quantified analyte under ESI [1]. These interfering com-pounds may be evaporated as neutral species into the gas phase. When the interferences show a higher affinity for proton (stronger gas-phase base) than the analyte, proton transfer occurs from the ionized analyte towards the interfer-ences. Therefore, the analyte ion is neutralized and its ion intensity decreases [1]. Others suggest that the liquid-phase part of the electrospray ion formation is implicated in the ion-suppression mechanism. The co-elution of an interfer-ing compound with a quantified analyte may change the droplet formation efficiency and/or the droplet evaporation, affecting the amount of charged droplets able to release ions into the gas phase and, consequently, the ion intensity of the analyte decreases [11].
�� Atmospheric pressure chemical ionization The atmospheric pressure chemical ionization mechanism is based on analyte vaporization into the gas-phase followed by its ionization by the corona discharge needle. Briefly, the corona dis-charge needle ionizes gas (air or nitrogen) to form a ‘primary ion’ that reacts with solvent molecules to produce ‘reagent ions’ that ultimately ionize the quantified analyte through proton transfer. A suggested theory of the APCI or ESI gas-phase ion-suppression mechanism is the formation of a solid, as a pure analyte or as a co-precipitate with other nonvolatile compounds [11].
�� Atmospheric pressure photoionizationIn APPI, more commonly used primary ioniza-tion modes are direct photoionization (PI) and dopant/solvent PI [12]. Direct PI is the excita-tion of a molecule by a photon, provided by a discharge lamp, followed by the direct ejection of an electron if the photon energy exceeds the ionization potential (IP) of the molecule [13–16].
In order to facilitate the PI in the case of a mol-ecule with a IP above the photon energy, an ioni-zable dopant is added [13,16]. Indeed, the presence of dopant photoions promotes the ionization of the molecule by collision in the ion source. This phenomenon is known as dopant/solvent PI. APPI is subject to the matrix effect since, if the matrix components have an equal or lower ionization potential as the analyte, competi-tion for charge in the gas phase may affect the analyte ionization [12].
Matrix effect assessment The variation of an analyte signal once extracted from a biological sample may be attributed to two processes: variation of the extraction reco-very and the matrix effect. The variation of the extraction recovery is due to the extraction efficiency, which varies from sample to sam-ple, whereas the matrix effect is the variation caused by the presence of matrix components in the extracted sample. In order to eliminate the matrix effect during early bioanalytical method development, the matrix effect must be assessed. The procedures described in this section are mainly focused on the assessment of the matrix effect alone. However, the standard line slope method and its simplified version are able to assess both the extraction recovery along with the matrix effect. Other useful tools for assessing the matrix effect, such as phospholipid monitoring, are described later.
�� Postextraction spike method The postextraction spike method is widely used to quantitatively evaluate the matrix effect. This method is often used to evaluate the absolute and/or relative matrix effect during the valida-tion of bioanalytical methods [6]. The absolute matrix effect is evaluated by comparing the analyte response in a neat solution against the analyte response of an extracted matrix blank reconstituted with a neat solution of analyte [3,4]. The absolute matrix effect is a useful tool in method development to determine if the varia-tion of a signal is caused by the matrix effect or by a variable extraction recovery. However, the relative matrix effect is more informative since it evaluates the variation of an analyte response spiked postextraction into different matrix lots (typically six to ten different matrix lots) [4]. A high variation of the relative matrix effect (%CV > 15% between the different matrix lots) indi-cates that the matrix effect is variable from lot to lot and could significantly affect the precision
ionizationsuppRession
Results from the presence of some compounds in a sample that can change the efficiency of droplet formation or droplet evaporation that impacts the amount of charged ion in the gaseous phase to reach the detector
MatRixeffeCt
The effect of the components of a sample other than the analytes that can have an impact on the way the ana lysis is conducted and the quality of the results obtained
BioanalytiCalMethoddevelopMent
The setting up of an analytical procedure that will be appropriate for the ana lysis of a biological sample. It includes optimization of extraction, chromatography and detection
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different matrix lots is used to confirm the pres-ence of the matrix effect and evaluate its impact on the precision and accuracy of the bioanalyti-cal method [4,18]. When the difference between the five slopes is above 3–4%, the matrix effect is considered significant, hence, it may impact the precision and accuracy of a bioanalytical method [18].
A simplified version of this method is to extract six to ten different lots of matrix spiked at low and high QC concentrations, to assess the precision and accuracy of the analyte response and to compare them together to verify the varia-tion between matrix lots [4]. The back-calculated concentrations should meet QCs acceptance cri-teria and the intermatrices variation should also be within acceptance criteria. As stated by the white paper published after the 2007 AAPS/FDA workshop [101], a high intermatrices varia-tion (%CV), such as 15% or above, indicates a significant matrix effect. The simplified ver-sion of the standard-line slope method is often used to evaluate the matrix effect during the validation of bioanalytical methods and during method development.
�� Phospholipid monitoring Phospholipids were reported as a main cause of the matrix effect in plasma and other biological samples [19]. Therefore, phospholipid monitoring during method development is a helpful tool to investigate the matrix effect. Positive ionization mode monitoring of the phospholipid’s typical mass transitions (taBle1) is the most commonly used phospholipid monitoring method. The in-source collision-induced dissociation (IS-CID) technique is another method that allows the monitoring of phospholipids. This method is based on the common ions obtained from the
and accuracy of a bioanalytical assay from sub-ject to subject. On the other hand, a given assay can lead to a high absolute matrix effect (i.e., a high difference between a neat solution and an extracted blank reconstituted with a neat solu-tion) but to a low relative matrix effect (i.e., a minimal variation of an analyte response spiked postextraction in different matrix lots). In this specific case, the different matrix lots show a similar extraction recovery for the suppressors (i.e., the same suppressors are present at similar concentrations in the different matrix lots).
�� Postcolumn infusion method The postcolumn infusion method is a procedure to evaluate the matrix effect over the course of a chromatographic run and was first described by Bonfiglio et al [17]. This qualitative method is a powerful tool used during method development. The matrix effect from co-eluting compounds cannot be directly observed on an LC–MS/MS chromatogram because they are nonvolatile compounds or because their mass transitions are not monitored. Postcolumn infusion method depicts, on a chromatogram, the region where ionization suppression or enhancement occurs. The experiment is designed by having an infu-sion pump deliver a constant amount of ana-lyte to the LC–MS/MS system via a ‘T-mixer’ installed between the analytical column and the mass analyzer (figuRe1). The ionization profile of the analyte (figuRe2) is obtained by injecting extracted blank sample and mobile phase on the postcolumn infusion set-up. The matrix effect using this method is evaluated by comparing the profile obtained between extracted blank sample and the mobile phase. The matrix effect is analyte dependent, therefore post column infusion experiments must be performed for all analyte(s) quantified [17]. Indeed, two analytes with different elution times experience differ-ent matrix effects, depending on where the sup-pression and/or enhancement regions are in the chromatogram.
�� Standard-line slope methodThe standard-line slope method is a quantita-tive method that evaluates the extraction effi-ciency and matrix effect together, but is generally referred to as matrix effect evaluation for sim-plicity. In order to assess the matrix effect, five calibration curves are usually spiked in five dif-ferent matrix lots and the slope of the standard line is determined for each matrix lot (curve). The variation of the slopes obtained in the
HPLC Analytical column
Infusionpump
Mass spectrometer
T-mixer
Figure 1. Postcolumn infusion set-up.
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fragmentation of the phospholipid polar head group (figuRe3). Indeed, high energy is used in the declustering region of the triple quadrupole mass spectrometer to induce the dissociation of the common ions (trimethyl ammonium ethyl-phosphate ion; m/z 184 or m/z 104). Moreover, the first (Q1) and third (Q3) quadrupoles are set at the same mass transition (184–184) and a low collision energy is selected for the second quadrupole to avoid further fragmentation [20]. Based on a recent study [21], a limitation of this technique is that it only allows the monitoring of the phosphatidylcholine (PC), lysophosphati-dylcholine (LPC), plasmalogen PC and sphingo-myelins (SM) phospholipids. Therefore, Xia and Jemal suggested a different technique for quali-tative assessment of all the phospholipids, the
so-called ‘all-inclusive’ technique [21]. Indeed, this technique monitors all classes of phospholipids, including LPC, PC, phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidyl-glycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS). This technique moni-tors the precursor ion m/z 184 and the neutral loss m/z 141, in positive mode, as well as the precursor ion m/z 153, in negative mode. Therefore, the ‘all-inclusive’ technique allows the detection of all classes of phospholipids by a single injection via both positive- and negative-ionization modes [21]. It is important to consider that monitoring of phospholipids can give precious information about the retention time of phospholipids, but it cannot be used during bioanalytical method development to directly assess all matrix effect,
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Figure 2. Effect of injecting blank plasma protein precipitation samples on a column with postcolumn infusion of each analyte. The infusion chromatogram from a mobile-phase injection is overlaid with an infusion chromatogram following each plasma extract injection. The difference between the two is due to the effect of endogenous plasma components eluting from the column. (A–C) Single reaction monitoring extracted ion-infusion chromatograms for the test compounds with a protein precipitation blank injected on column. (A) Merck compound, (B) phenacetin and (C) caffeine. Reproduced with permission from [17].
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since the phospholipids are not the only source of matrix effect in biological extracts. Moreover, it is not always true that the presence of phospho-lipids in an extract will automatically lead to the matrix effect since this will also depend on their concentrations and the analyte of interest (the matrix effect is analyte dependent) [21].
Early method‑development steps to avoid the matrix effect Four different approaches may be used during the development of bioanalytical methods to elimi-nate or minimize the impact of the matrix effect on the quantitation of an analyte: investigation of mass analyzer interfaces, chromato graphy optimization, sample-preparation optimization; and selection of suitable internal standards (IS).
�� Mass analyzer interfaces investigationIn bioana lysis, APCI interfaces were often found to be less susceptible to matrix effect than ESI [1,2,22–25]. The different behavior of the ESI and APCI interfaces could be explained by the different ionization mechanisms previously described. For instance, the quantification of finasteride in human plasma by LC–ESI–MS/MS indicated the presence of a matrix effect [22]: poor precision between standards spiked in five different plasma lots and the variable slope of the calibration curves (%CV = 23.4%), evaluated by using the standard line slope method. However, no matrix effect was observed when the inter-face was changed for APCI: higher precision and accuracy and similar slopes of the calibra-tion curves prepared in five different plasma lots (%CV = 2.4%).
Investigation between ESI and APCI inter-faces of three different instruments demonstrates that the matrix effect can also be source design
dependent. The investigation was performed using blank rat plasma spiked postextraction. No matrix effect was observed on the Applied Biosystems/MDS Sciex API 3000 system using ESI or APCI sources. The matrix effect was observed for the ESI source of a Finnigan TSQ 7000 system, but not when the APCI source was used. By contrast, the APCI source of a Waters Micromass Quattro Ultima system was more susceptible to the matrix effect than its ESI source [26].
Furthermore, by comparing the matrix effect observed using atmospheric pressure photoion-ization (APPI), ESI and APCI, Marchi et al. [13] demonstrated that the APPI is the least suscep-tible to the matrix effect (figuRe4). Moreover, according to Yang et al. [27], the use of APPI LC–MS for the ana lysis of idoxifene and its major metabolite SB245419 (SB19) in human plasma eliminates any detectable matrix effect (evaluate by postcolumn infusion) caused by the endogenous compounds.
It is also important to consider that the new generation of mass spectrometers, such as the
Table 1. Typical mass transitions used to monitor phospholipids during method development.
Phospholipids Mass transition (m/z)
Lyso-phosphatidyl choline 16:0
496–184
Lyso-phosphatidyl choline 18:0
524–184
Phosphatidyl choline 30:1 704–184
Phosphatidyl choline 34:2 758–184
Phosphatidyl choline 36:2 786–184
Phosphatidyl choline 38:6 806–184Data from [18,30,37,65].
NH3C
H3C
CH3
OP
O
O
O
O
R
O
R'
R,R´ = fatty acid
m/z = 184, +2H
m/z = 104, +H
NH3C
H3C
CH3
OP
O
O
O
O
R
O
H
R = fatty acid
m/z = 184, +2H
m/z = 104, +H
PC phospholipids LPC phospholipids
Figure 3. PC and LPC. Common ions obtained after the fragmentation of the polar head of phospholipids are indicated (trimethyl ammonium ethyl-phosphate ion; m/z 184 or m/z 104). LPC: Lysophosphatidylcholine; PC: Phosphatidylcholine.
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Applied Biosystems/MDS Sciex API5000 [102] or the Thermo TSQ Vantage [103], may produce less matrix effect when compared with older instruments due to their high sensitivity. These new technologies also provide the opportunity of using a higher dilution factor during the sam-ple preparation. Even if there are many advan-tages to changing from an older instrument to a newer one, as far as matrix effect is concerned, the obviously high cost of these changes may be a major drawback. Therefore, optimizations such as chromatographic, sample preparation and IS optimization are still the best options in the bioanalytical industry.
�� Chromatographic optimization Optimization of chromatography is one strat-egy to reduce or eliminate the matrix effect during bioanalytical method development. There are different ways to optimize the chro-matographic separation in order to avoid matrix effect: increase the capacity factor (k´), change column selectivity, mobile phase or column tem-perature, and improve resolution between ana-lytes and suppressors. The postcolumn infusion method and phospholipid monitoring are useful techniques to employ during chromatographic
optimization to avoid co-elution of the analyte with the endogenous interfering compounds.
As previously discussed, a HPLC method with a high capacity factor (k´) is able to reduce the matrix effect and increase the precision of bio-analytical methods [22,28]. Indeed, in reversed-phase chromatography, the solvent front con-tains a high concentration of un-retained polar compounds, which may cause the matrix effect. In general, the enhancement of the chromato-graphic separation efficiency results in a better resolution between the analyte and the solvent front and, therefore, it may decrease or elimi-nate the matrix effect. However, high separa-tion efficiency does not guarantee the absence of the matrix effect and the selection of a new chromatographic column may be required to eliminate the matrix effect. A better approach than blindly increasing the chromatographic resolution of the method is the use of postcol-umn infusion to reveal regions where the matrix effect will occur, helping resolve the analyte from these regions. As reported in the next example, the postcolumn infusion method pro-vides valuable information about the retention time of interfering compounds and facilitates column choice during method development.
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Figure 4. Comparison of matrix effects for (A) ESI, (B) APCI and (C) APPI, after on-line solid-phase extraction of human plasma. Chromatograms represent the total ion current of eight different compounds. APCI: Atmospheric pressure chemical ionization; APPI: Atmospheric pressure photoionization; ESI: Electrospray ionization. Reproduced with permission from [12].
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De Nardi et al. observed the ionization enhance-ment caused by the dosing vehicle PEG 400 by using post column infusion [29]. Afterwards, they performed the screening of HPLC columns by again using postcolumn infusion to select a column in which the quantified analyte was adequately chromatographically separated from PEG 400 and from any other region where the matrix effect may occur [29].
The postcolumn infusion method is also used to visualize how changes in chromato-graphic conditions may impact the separation between an analyte and a matrix effect region. Postcolumn infusion can assist in modifying chromatographic conditions to achieve chro-matographic separation between an analyte and the matrix effect region [30,31]. Moreover, using the IS–CID method to monitor the phospho-lipid, it is possible to identify the endogenous compounds that are the cause of the matrix effect in the region revealed by the postcolumn infusion method. Ismaiel et al. used these two methods to identify phospholipids as the cause of the matrix effect observed during method deve-lopment for the quantification of hydrocodone and pseudoephedrine in human plasma [31]. IS–CID monitoring of phospholipids was used to chromatographically resolve the analytes from phospholipids and, therefore, to successfully eliminate the matrix effect coming from these endogenous compounds [32].
Useful information to reduce or eliminate the matrix effect caused by phospholipids was obtained by studying phospholipid retention under reversed-phase chromatography and, more recently, under hydrophilic interaction chromato graphy (HILIC). Extensive work was performed by Little et al. and Chambers et al. [20,32]. It was discovered that, under reversed-phase chromatography, the LPCs elute before the PCs, which necessitates extensive column flushing with a high amount of organic solvent. Surprisingly, it was reported that metha-nol is more efficient than acetonitrile to elute phospholipids. This is principally observed for the PCs that elute in a broad peak from 7 to 16 min in pure acetonitrile against narrow peaks from 3 to 4 min in pure methanol. Conversely, the results of a recent study show that the most effective organic eluent to remove the phos-pholipids from the reversed-phase column used was isopropyl alcohol, followed by acetonitrile and methanol [21]. These opposite results led to the conclusion that the choice of the most effective eluent to remove the phospholipids in
reversed-phase chromatography is column-type dependent. Moreover, this study also demon-strates that a commonly used short flushing step (1–2 min) is not enough to wash out all the PCs retained and could result in worsening ionization suppression or enhancement [21]. The retention of phospholipids is also affected by the column temperature. As reported by Little et al., the PCs are less retained at 60°C compared with 30°C [20]. Finally, phospholipid retention is rela-tively independent of mobile-phase pH. In fact, the LPCs and PCs retention behavior is similar at low and high pH, but the retention of PCs slightly increases at high pH and needs a higher organic content to be eluted [32]. This last char-acteristic was used to achieve separation between phospholipids and two ionizable analytes: terfe-nadine and amitriptyline [32]. Basic analytes were less retained at low pH when compared with high pH, while the retention times of the phos-pholipids were only slightly affected under acidic or basic mobile-phase conditions (figuRe5).
It is important to notice that new reversed-phase columns (polymeric- or silica-based sor-bents of improved stability) allow the use of high-pH mobile phases. Hence, according to the outcome of a recent study, it is possible to obtain, by using a high-pH mobile phase in reversed-phase chromatography, comparable or even higher sensitivity of the basic analytes under ESI(+) mode than their sensitivity in low-pH mobile phases. However, under high-pH conditions, basic analyte retention will be improved [33], resulting in better separation from co-eluted interfering compounds, causing ionization suppression or enhancement.
Recent data from the well-established ultra-high-pressure LC (UHPLC) show that this technique, by providing better resolution and more narrow peaks, is able to almost elimi-nate the matrix effect by minimizing the co-eluted interferences during the ionization of the analyte [34]. Indeed, Gibb et al. demonstrated that UPLC (Waters UHPLC®) was able to separate endo genous components from terfena-dine, risperidone and verapamil by comparing extracted samples resulting from protein precipi-tation and SPE on both HPLC–MS/MS and UPLC–MS/MS [35].
Moreover, another well-established hyphen-ated technique, 2D-LC, is considered to be able to efficiently compensate for the signal suppression effect by increasing method selec-tivity and sensitivity. Indeed, this technique utilizes buffered solvents with reversed-phase
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chromatography as a first dimension to enhance selectivity by eliminating the presence of matrix interference and the second-dimension for sen-sitivity gains and selectivity [36,37]. Choi et al., using pesticides ana lysis, showed that the 2D LC technique allows the elimination of the matrix effect produced by the endogenous compounds along with sensitivity improvement [38].
Finally, HILIC could also be considered as a valuable option for achieving the selecti vity between an analyte and a suppressor when a lack of resolution is found in reversed-phase mode. In fact, under HILIC conditions, the elution order of the phospholipids is inverted: the more hydrophobic PCs elute before the LPCs. Under typical HILIC conditions, Mess et al. dem-onstrated the high retention of phospholipids (k´ approximately 40) on bare silica and diol bonded-phase columns [39]. Therefore, these columns should be used carefully to avoid the matrix effect on subsequent injections. Phospholipids show less retention on the cyano and amino bonded-phase columns under HILIC conditions when compared with the bare silica and diol columns. Indeed, the retention factor (k´) reported for the cyano and amino columns were 2.9 and 7.3, respectively, at pH 3.0 for the phospholipids [39]. As for reversed-phase chroma-tography, the retention of phospholipids is not affected by the mobile-phase pH under HILIC chromatography. In fact, it was found that the
retention of phospholipids was similar at pH 3.0 and 6.0 for all HILIC columns tested [39]. Therefore, a similar pH optimization may be used to resolve ionizable analytes and phos-pholipids using HILIC chromatography as for reversed-phase chromatography.
�� Sample-preparation optimizationChoice and optimization of sample preparation is an efficient way to reduce or eliminate the matrix effect from biological samples before their introduction into LC–MS and LC–MS/MS sys-tems. Sample preparation able to produce cleaner extracts greatly facilitates the development of high-throughput chromatography. Therefore, optimization of the sample preparation is an important part of the method development in order to benefit from the advantages of a cleaner extract.
Extraction procedures comparisonIon suppression (or enhancement) of different sample preparations is often evaluated using the post-extracted spiked method during method development and different sample preparations are compared with the percentage of enhance-ment or suppression on the analyte signal, to select the one that minimizes or eliminates the matrix effect. In the case of SR 27417 quantification in human plasma, a platelet-activating factor receptor antagonist stu died by
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472.1 > 436.16.07e5
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Figure 5. (i) Chromatogram showing in-source collision-induced dissociation of phospholipids from a plasma sample injected under (A) pH 2.7 and (B) pH 9 mobile-phase conditions. Multiple reaction monitoring transitions for (ii) 1 ng/ml terfenadine and (iii) amitriptyline under the same mobile-phase conditions.Reproduced with permission from [32].
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Buhrman et al. [3], the ion suppression deter-mined for liquid–liquid extraction (LLE), back-LLE and SPE were 26, 0 and 41%, respectively, suggesting that back-LLE lacks matrix effect and is a more suitable extraction method. Sample-preparation comparison between direct dilution and LLE for the quantification of indinavir in human urine shows, as expected, that ion sup-pression is more apparent for the diluted sample (60%) when compared with the LLE extracts sample (14%). This suggests that LLE produces a cleaner extract that contains a lower amount of compounds causing ion suppression [28]. In a recent publication [40], the relative matrix effect was determined for two potential ISs (analogue IS) in three different extractions: protein precipi-tation (PPT), LLE and SPE. The relative matrix effects were above 15% for the first potential IS extracted using PPT and LLE, but below 15% when extracted using the SPE procedure. Similar results were obtained for the second potential IS, except for the LLE, for which the relative matrix effect was below 15%. These results suggest that the SPE extract contains fewer suppressors than the LLE extract, followed by the PPT extract, which contains the highest amount of suppres-sors [38]. According to Matuszewski et al., LLE optimization from nonbuffered compared with buffered (pH 9.8) extraction was able to signifi-cantly reduce ion suppression for finasteride in human plasma (50% less suppression in some matrix lots using buffered LLE) [22]. It is a com-mon belief that the SPE has a better efficiency for phospholipid removal. However, from the examples reported above, it seems that it is really difficult to rank the extraction types in order of cleanliness since the matrix effect is compound dependent and the generic conditions used may have maximized matrix effect.
Phospholipid extraction Since for plasma samples phospholipids are the main cause of matrix effect and, consequently, of suppression/enhancement region detected using postcolumn infusion [19], a current strategy used to eliminate the matrix effect is to optimize sam-ple preparation in order to minimize phospho-lipid extraction [32,41,42]. For instance, Chambers et al. demonstrated that the methanol extract contains 40% more phospholipids than the ace-tonitrile (ACN) extract by monitoring the phos-pholipids in protein precipitation extracts using methanol or acetonitrile [32]. This result suggests that phospholipids are more soluble in methanol. However, the level of phospholipids was high in
both extracts and produced similar matrix effect of approximately -70% for various compounds tested [32]. According to Chambers et al., opti-mization of mix-mode cation-exchange (Waters Oasis MCX) SPE shows that methanol is more efficient for eluting phospholipids than acetoni-trile [32]. In fact, methanol removes 64% more phospholipids than acetonitrile when it is used to wash cartridges. The amount of phospholip-ids eluted from the sorbent was approximately 20-times lower in ACN when compared with methanol: for eight compounds, the matrix effect was evaluated to be between -6 and 14%, which corresponds to an insignificant matrix effect [32].
Based on an investigation Capka et al. of the Waters Oasis MAX polymer anion-exchange cartridges, the matrix effect is observed using 20–90% ACN as an elution solvent [41]. An investigation was carried out using MeOH:H
2O
or ACN:H2O containing 50–100% organic. A
maxi mum amount of phospho lipids are eluted from silica- and polymeric-based sorbents using 100% methanol. Below 50% methanol, no phos-pholipids elute from the silica and the polymeric-based sorbents. Similarly, Lahaie et al. reported that a high amount of phospholipids elutes from the polymeric sorbents Waters Oasis HLB, Biotage Evolute ABN and Varian Bond Elut Plexa, with 100% ACN (figuRe6B) [42]. However, no phospholipids elute from the silica-based sor-bent, Phenomenex Strata C18, Phenomenex Strata Phenyl, Varian Bond Elut C8 and Varian Bond Elut C18 using 100% ACN [42] (figuRe6a&B). Therefore, the elution of drug from the silica-based sorbent using pure acetonitrile is a valuable strategy to minimize phospholipids and, there-fore, to minimize the matrix effect. Moreover, according to Lahaie et al., another strategy to minimize matrix effect is to elute the drug from silica- and polymeric-based sorbents during SPE using a MeOH:H
2O solution containing less
then 50% methanol, which eliminates the pres-ence of the LPCs in the extract [42]. However, this may significantly affect drug recovery.
Selection of organic solvent and pH optimiza-tion of LLE is also a good strategy to minimize phospholipid extraction and the matrix effect. Recent work reported the optimal buffered con-ditions to reduce or eliminate the matrix effect coming from the LPCs and PCs in LLE by using chlorobutane or MTBE:chlorobutane 80:20% v/v as an extraction solvent [43]. The use of acidic (0.12 N HCl) and basic (0.1 N or 0.2 N NaOH) conditions instead of acidic- and basic-buffered
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conditions are recommended for acidic and basic compounds in LLE, respectively, using the afore-mentioned extraction solvent. To finish the LLE section, as extra information, we include results obtained with the supported liquid extraction (SLE) to remove the phospholipids [40].
It is mentioned in the literature that Turbof low™ technology, from Thermo-Scientific, can also eliminate the matrix effect from endogenous compounds, such as phos-pholipids [104]. The Turboflow technology is an on-line clean-up device based on the principle of size-exclusion chromatography. At a high flow rate, small molecules are retained in the pores of the stationary phase, whereas macromolecules are not. The analyte of interest is then eluted from the Turboflow column using a selective elu-ent. Evaluation over eleven compounds showed that the Turboflow on-line clean-up optimized method successfully excluded more than 99.9% of the phospholipids in plasma and serum [44]. Recent publications confirmed that Turboflow technology is able to significantly reduce the response variability over matrix species [44–46].
Tailor-made precolumn packing for HPLC is another technique described in the recent litera-ture that seems to be able to reduce the matrix effect. This technique is based on size-exclusion chromatographic principles and allows extractive clean-up of biofluids by using restricted access materials (RAMs). Due to the RAMs, only ana-lytes of low molecular mass have free access to the binding centers of the inner pore surface and, thus, can be retained and extracted selectively [47]. The Six-S ProcEdure (Six-SPE) is a multi-dimensional SPE sample-processing platform for complex fluids that consists of the combination
of two small LC columns: one packed with restricted access materials (RAMs) and the other with molecular imprinted polymers (MIPs). For instance, in the case of tramadol quantification, the authors used the Six-SPE to efficiently elimi-nate the matrix effect [48]. In this study, the RAM column removed interfering matrix components from complex aqueous samples, whereas the MIP column selectively extracted the imprinted target analyte(s) and eliminated potentially interfer-ing low-molecular-weight components from a c omplex sample [48].
New technologies New technologies are being increasingly devel-oped to allow the reduction of the matrix effect generated by phospholipids and the removal of other endogenous compounds. Recently, pro-mising supported protein precipitation plates appeared on the market. The Hybrid SPE™ protein precipitation (Hybrid SPE–PPT) technology from Sigma-Aldrich [104] and the Captiva ND™-lipids from Varian [105] are two examples. Furthermore, the use of colloidal silica (LUDOX® AS-40) along with a cation agent [49] or electrophoretic sample preparation [50] has been reported to remove phospholipids.
Sigma-Aldrich claims that the Hybrid SPE–PPT platform of zirconia-coated particles enable efficient extraction of phospho lipids while remaining nonselective towards a broad range of basic, neutral and acidic compounds. Indeed, using Hybrid SPE–PPT, rat plasma samples were depleted of greater than 99% of the phospholip-ids when compared with standard protein pre-cipitation [104]. Clonidine and protrytiline cases demonstrate that the severe ionization suppression
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Figure 6. Lyso-phosphatidylcholine extraction response obtained using silica (A: Strata C18, C8, phenyl and B: Bond Elut C18) versus polymeric (B: HLB, ABN and Plexa) solid-phase extraction with different percentages of acetonitrile.
Review| Côté, Bergeron, Mess, Furtado & Garofolo
Bioanalysis (2009) 1(7)1252 future science group
(>50% of the signal) obtained after standard pro-tein precipitation was eliminated using Hybrid SPE–PPT plates. According to the manufacturer evaluation [105], the Hybrid SPE–PPT capacity for phospholipid removal is not affected by the plasma volume extracted, the organic modifier used in the elution solvent or the optimal pH nec-essary for analyte extraction. Therefore, neutral, basic and acidic compounds could be efficiently separated from the phospholipids. Recently, Pucci et al. have compared the conventional pro-tein precipitation extractions using ACN with the novel Hybrid SPE protein precipitation pro-cedure on rat and human plasma. Hybrid SPE leads to significant reduction of the matrix effect by extensively reducing the phospholipid levels in the extracted biological samples [51].
Varian claims that the Captiva ND-lipid filtra-tion plates remove LPCs and PCs from biologi-cal fluids without retaining the analytes of inter-est [52,53]. An evaluation of the Captiva ND-lipids by Dicaire at al. using ACN and methanol in different ratios (1:3, 1:4 and 1:8 [plasma:organic solvent ratio]) demonstrates that the extraction efficiency of most of the fourteen different ana-lytes tested are similar to recoveries obtained with standard protein precipitation under the same conditions [54]. Captiva ND-lipid plates also remove the late eluted PCs under most of the conditions. However, in the case of the co-eluted phospholipids (LPCs) their elimination was not complete. In another study, a compari-son between the Captiva ND-lipids and standard filtration plates showed that the phospholipid extraction efficiency is slightly lower with the Captiva ND-lipids [55]. Moreover, the phospho-lipid removal was proven to be more complete using methanol than acetonitrile with the Captiva ND-lipids plates since ACN is only efficient at a ratio of 10:1 (plasma:ACN).
The direct addition of LUDOX AS-40 and manganese chloride (MnCL
2) to plasma sam-
ples was reported in 2006 by Shoener et al. as an approach to quantitatively remove the PC and LPC classes of phospholipids [49]. However, using MnCl
2 as a cation agent drastically reduces
the analyte extraction efficiency [56]. An evalua-tion of different cation agents added along with Ludox AS-40 was performed over ten different compounds by monitoring the PC, PE and SM phospholipid classes. Results revealed that the optimal conditions are the use of the lanthanium chloride (LaCl
3) with the colloidal silica [56] in
terms of extraction efficiency and phospholipid removal for ten tested analytes.
The 96-well plate electrophoretic sample-preparation procedure is a high-throughput approach for the depletion of phospholipids and salts. In this approach, in the 96-well plate, the analyte of interest will be attracted by the anode through a polyacrilamide gel to finally be cap-tured on the monolithic underlayer, whereas the phospholipids (anions) will be attracted by the cathode and kept in solution. The monolithic capture layer can then be removed and analyzed by MALDI–MS/MS without the presence of phospholipids. Recently, Grant et al. demon-strated that ana lysis of electrophoretic samples in cationic mode at pH 1.9 leads to phospholipid exclusion above 99.5% [50].
�� Stable isotopically labeled ISThe selection of an adequate IS is very impor-tant for the successful development of bioana-lytical methods. However, it does not reduce or eliminate the matrix effect even if it does increase the precision and accuracy, as it compensates for the loss or gain of analyte response by keeping the analyte:IS ratio constant. The co-elution of a structural analogue with a quantified analyte was reported to correct ionization saturation observed at the higher concentrations of the cali-bration curve [57]. Indeed, the chromatographic co-elution of the IS with the analyte resulted in a linear calibration curve.
Stable isotopically labeled ISDs (SIL-ISs) are believed to be the ideal IS to correct for the matrix effect because they share almost the same physicochemical properties as their unlabeled analogue. The SIL-IS and analytes that elute at or near the same retention time are simultane-ously ionized and undergo similar matrix effect. Comparison between the SIL-IS and analogue IS had demonstrated the superiority of the SIL-IS to correct the matrix effect [28,58,59]. The SIL-IS of angiotensin IV was compared with a structural analogue of angiotensin IV to correct the rela-tive matrix effect. The variation at the LLOQ concentration between six matrix lots was 5.2% for the SIL-IS when compared with 28.5% for the analogue IS [59]. A stable isotopically labeled IS was also reported to efficiently correct the irreproducible ionization of the product ion of ES-285, an anticancer agent. A single product ion was obtained from ES-285 fragmentation, corresponding to its dehydration [M+H-H
2O]+.
This type of product ion is not suitable for quan-tification purposes because premature in-source fragmentation into the dehydrated product ion is often observed and extremely variable. The
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in-source fragmentation of ES-285 was corrected using a SIL-IS that undergoes the same in-source fragmentation and, consequently, minimized the ionization process variation to 4.9% [59]. A stable isotopically labeled IS does not always correct the matrix effect; in fact, some cases of SIL-IS failing to correct the matrix effect have been reported [60–63]. In some cases, the chro-matographic separation between the SIL-IS and the analyte, known as the isotopic effect, may lead to a different ionization process between the two compounds [62,63]. The isotopic effect is due to the slight difference between deuterium and hydrogen properties, which may result in a slight chromatographic separation of the SIL-IS and its unlabeled analogue. This phenomenon is more pronounced for SIL-IS containing many deu-teriums and for those showing high separation efficiency (k´). Partial chiral chromatographic separation of racemic [2H
5]-carvediol and race-
mic carvediol and their elution into a large and sharp matrix effect region had led to a different ionization process between the two compounds and, therefore, the [2H
5]-carvediol was ineffi-
cient in correcting for the carvediol matrix effect (figuRe7) [62]. The use of SIL-IS may introduce an additional source of variation in a bioanalyti-cal method. The deuterium exchange between
[13CD3]-rofecoxib and rofecoxib was found to
decrease method precision and accuracy [64]. Therefore, it is important to note that deuterium must be added to a position where it is not acidic and thus will not exchange. Finally, SIL-IS con-taining 13C was found to be more stable than the deuterium form [64].
ConclusionThe matrix effect caused by phospholipids or other endogenous compounds in LC–MS/MS bioanalytical methods leads to ion suppres-sion or enhancement. Therefore, it is neces-sary to overcome this phenomenon at an early stage of method development. This review has listed different techniques for the matrix effect assessment. The postcolumn infusion and the phospholipid monitoring methods were the most useful tools for method development since these methods give an abundant amount of information in fewer experiments.
Throughout this review, method develop-ment scientists were able to find the most recent techniques in chromatographic optimization. In fact, the use of basic mobile phases, HILIC chromatography, UHPLC and 2D LC were dis-cussed regarding their capabilities of avoiding the ion suppression from interfering endoge-nous compounds. Furthermore, the optimiza-tion of sample extraction with common LLE and SPE was reviewed in terms of phospholipid removal. The ability of the new technologies, such as supported protein precipitation plates and the use of colloidal silica during protein precipitation, were also discussed, along with the Turboflow possibilities. Moreover, compari-son of different mass analyser and ionization modes and the importance of using a suitable IS were presented.
This article provided the bioanalytical scientists with an updated and exhaustive review of the different approaches to assess the matrix effect during their method development. More importantly, they now have a list of tools that can help them efficiently eliminate or minimize the matrix effect.
Future perspectiveSince the matrix effect is due to endogenous compounds present in the matrices that co-elute with the analyte of interest, injection volume optimization should become a part of the matrix effect evaluation during method development. Indeed, by injecting a higher volume into the LC–MS/MS, the suppression or enhancement
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[2H5]-carvediol
Carvediol
Figure 7. The postcolumn infusion of carvedilol (R and S mixture) with injection of an extract of the derivatized drug-free plasma lot was overlaid with the LC–MS/MS chromatograms of carvedilol and its stable isotopically labeled ISDs. A slight difference in retention time between carvedilol and [2H
5]-carvedilol was observed.
Reproduced with permission from [62].
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Bioanalysis (2009) 1(7)1254 future science group
Executive summary
�� Matrix effects involve the ionization mechanism of different atmospheric pressure ionization modes: electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization.
�� The matrix effect can be assessed in different ways, such as with the postextraction spike method, the postcolumn infusion method and the standard-line slope method.
�� Phospholipid monitoring gives useful information about matrix effect during method development.�� Chromatographic optimization offers the possibility of some control of the matrix effect by using a high pH mobile phase, hydrophilic
interaction LC and ultra-high-performance LC.�� New sample-preparation techniques, as HybridSPE™ and Captiva ND™-lipids plates, successfully reduce the matrix effect by eliminating
the presence of the phospholipids in the extracted samples.
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AcknowledgementsThe authors would like to thank Natasha Savoie of the Bioanalytical Services Department at Algorithme Pharma Inc. for her help and for offering valuable advice during the review of this manuscript.
Financial & competing interests disclosureThe authors have no relevant affiliations or financial involve-ment with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, con-sultancies, honoraria, stock ownership or options, expert tes-timony, grants or patents received or pending, or royalties.
No writing assistance was utilized in this manuscript.
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�� Websites101 AAPS workshop/conference report –
quantitative bioanalytical methods validation and implementation. Best practices for chromatographic and ligand binding assays 2007 www.aapsj.org/articles/aapsj0901/aapsj0901004/aapsj0901004.pdf
102 Applied Biosystems www.appliedbiosystems.com
103 Thermo Fisher Scientific Inc.www.thermo.com
104 Sigma-Aldrich Inc. www.sigma-aldrich.com
105 Varian Inc. www.varianinc.com
Matrix effect elimination during LC–MS/MS bioanalytical method development |Review
