Evaluation of an Accurate Mass Approach for the Simultaneous

Article pubs.acs.org/ac

Evaluation of an Accurate Mass Approach for the Simultaneous Detection of Drug and Metabolite Distributions via Whole-Body Mass Spectrometric Imaging Sheerin K. Shahidi-Latham,*,† Sucharita M. Dutta,‡ Maria C. Prieto Conaway,‡ and Patrick J. Rudewicz† †

Genentech, Inc., South San Francisco, California 94080, United States Thermo Fisher Scientific, San Jose, California 95134, United States

‡

ABSTRACT: In drug discovery and development, in vitro absorption and metabolism assays along with in vivo pharmacokinetic (PK), pharmacodynamic (PD), and toxicokinetic (TK) studies are used to evaluate a potential drug candidate. More recently, imaging mass spectrometry approaches have been successfully reported to aid in the preclinical assessment of drug candidates, resulting in the rapid and noteworthy acceptance of the technique in pharmaceutical research. Traditionally, drug distribution studies via mass spectrometric imaging (MSI) are performed as targeted MS/MS analyses, where the analytes of interest, drug and/or metabolite, are known before the imaging experiment is performed. The study presented here describes a whole-body mass spectrometric imaging (WB-MSI) approach using a hybrid MALDI-LTQ-OrbitrapMS to detect the distribution of reserpine at 2 h post a 20 mg/kg oral dose. This study effectively demonstrates the utility of obtaining accurate mass measurements across a wide mass range combined with postprocessing tools to efficiently identify drug and metabolite distributions without the need for any a priori knowledge.

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means to assess the intact tissue distribution of compounds earlier in the drug discovery phase. In most laboratories, MALDI-MSI experiments are performed as targeted analyses of known drug and metabolite using product ion scans.17,22−26 Although valuable information can be collected by this approach, one must optimize the parameters of the imaging experiment prior to analysis to ensure proper detection of the analytes of interest. More recently, several groups have shown the utility of accurate mass measurements for the simultaneous detection of drug, metabolites, and endogenous components (e.g., phospholipids, peptides, etc.) within a single imaging experiment.19,27−31 This serves as a major advantage over the, relatively speaking, more challenging and time-consuming targeted imaging experiments, since broadband spectra covering a wide mass-range can be obtained with no need for a priori knowledge about the molecular species of interest. Instead, the accurate mass imaging approach relies on postacquisition tools to mine the complex data sets based on multiple mass defect filters (MDF) that serve to “extract” ions that achieve a predefined mass tolerance (typically set to 5 ppm) centered on the exact mass for the compound of interest. Each mass defect filter sets a millidalton (mDa) mass defect window around a nominal mass window, which serves to simplify the complex data sets that are oftentimes overwhelmed with chemical noise from the matrix coating and/or endogenous species.

rug disposition studies are a hallmark of the drug discovery and development process. Gaining insight into the absorption, distribution, metabolism, and elimination (ADME) of a potential drug candidate is of great importance and can aid in the redesign for a superior molecule.2,3 Traditionally, compound optimization efforts are performed in the discovery phase, where newly synthesized compounds are screened in relatively quick and inexpensive in vitro and subsequent in vivo assays.4,5 In the case of compound distribution studies, in vivo experiments require the excision, homogenization, and extraction of the drug from tissue for subsequent analysis by liquid-chromatography tandem mass spectrometry (LC MS/MS).6−9 Although the absolute quantity of a compound in a target tissue or an organ can be determined from this approach, spatial information about the compound distribution across the tissue is lost. Conventionally, in order to obtain intact tissue distributions, expensive studies such as quantitative whole-body autoradiography (QWBA) using a radio-labeled drug would be reserved for only the most promising lead compounds in or beyond the preclinical development phase.10−12 A major drawback of this approach is a lack of mass specificity: radioactivity detection does not differentiate between drug and metabolites. In recent years, several groups have demonstrated the utility of mass spectrometric imaging (MSI) approaches to provide the ex vivo, label-free detection of drug-related compounds across thin tissue sections.13−21 MSI techniques, most notably imaging matrix-assisted laser desorption/ionization mass spectrometry (MALDI MSI), are now included in the pharmaceutical scientist’s tool box and serve as a less expensive yet robust © 2012 American Chemical Society

Received: June 3, 2012 Accepted: July 24, 2012 Published: July 24, 2012 7158

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power of ∼60 000 mass resolution (defined at m/z 400). Images were acquired at 400 μm pixel resolution, with each pixel consisting of up to 80 laser shots (1 microscan). Data were collected with automatic gain control on (AGC on) which does a prescan (3 laser shots) to determine the number of laser shots needed to optimally fill the ion trap and to avoid spacecharging effects. For each pixel, the final ion intensity is divided by the total number of laser shots determined by the AGC. Imaging data were processed using ImageQuest software for ion image extraction and with MetWorks software to filter the data for the discovery of unknown metabolites at 5 ppm mass tolerance.

Obtaining accurate mass measurements using a LTQOrbitrap MS for a single MSI experiment can be longer in duration than imaging experiments of similar pixel density performed on traditionally lower mass-resolution counterparts (e.g., TOF mass analyzers) due to the slow scan speeds used to obtain the high mass resolution data. However, a single, broadband accurate mass imaging experiment can provide a wealth of information without the need to reanalyze a tissue section (or serial section) to perform additional targeted imaging experiments and thus significantly decreases the overall sample burden and instrument time. Furthermore, with the hybrid LTQ-Orbitrap mass spectrometer, simultaneous collection of MS and MS/MS data have been demonstrated within a single imaging experiment by incorporating multiple ion trap scans during a single orbitrap scan event.32 This approach adds no additional acquisition time to the overall analysis and often eliminates the need to perform additional experiments for structural confirmation on analytes of interest. To demonstrate the utility of the accurate mass imaging approach for whole-body MSI (WB-MSI), reserpine was dosed at 20 mg/kg to male Sprague−Dawley rats and were euthanized 2 h postdose for imaging analysis on the MALDI-LTQOrbitrap mass spectrometer. Reserpine is a purified alkaloid drug extracted from the root of Rauwolf ia serpentine, a tropical woody plant indigenous to India, and used in the treatment of hypertension and psychosis.33 Few data are available on the pharmacokinetic (PK) properties of reserpine due to low concentrations of the drug and its metabolites; however, it is known that reserpine is extensively metabolized and that none of the parent drug is excreted unchanged,34−36 making this an ideal tool compound to assess the successful extraction of unknown metabolites by postprocessing software.

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RESULTS AND DISCUSSION

The ability to quickly obtain knowledge about the distribution of novel therapeutic compounds to a target organ is of great relevance in the pharmaceutical industry. Understanding drug disposition allows the pharmaceutical scientist to improve the metabolic, PK, and toxicological profiles of the compound. For the study described here, the distribution of 20 mg/kg reserpine at 2 h postdose was detected by accurate mass imaging MALDI MS across a broad mass range (m/z 275−1000) using an LTQOrbitrap mass spectrometer. Reserpine signal was detected from the tissue section with high mass accuracy (sub-1 ppm), while the previously determined metabolite, methyl reserpate,34 was detected at sub-3 ppm mass accuracy (Figure 1). Both

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EXPERIMENTAL SECTION Materials. 2,5-Dihydroxybenzoic acid (DHB) matrix and reserpine were purchased from Sigma Chemical Co. (St. Louis, MO). Filmolux tape was purchased from Neschen Americas (Elkridge, MD) and conductive double-sided tape was purchased from 3M (St. Paul, MN). Tissue Preparation. Reserpine was administered at 20 mg/ kg via oral gavage to 8-week old, male Sprague-dawley rats, which were fasted 16 h prior to the start of the study. At 2 h postdose, the rats were euthanized, while under isoflurane anesthesia, by exsanguination via cardiac puncture followed by a cardiac injection of euthasol. Rat carcasses were frozen in a hexane dry ice bath for 20 min and stored at −80 °C. Individual rat carcasses were embedded in water to form ice blocks in order to collect 20 μm thick whole-body sections (sagittal orientation) onto filmolux tape using a Leica CM3600 cryomacrocut (Leica Microsystems Inc., Bannockburn, IL) at −20 °C. Tissue sections were allowed to dehydrate overnight in the cryochamber, followed by mounting onto a MALDI target plate using conductive double-sided tape (tissue section was split down the middle in order to fit onto target plate). Tissue sections were coated using a Kontes glass spray nebulizer (Kimble Chase, Vineland, NJ) with 40 mg/mL DHB in 70% methanol until a uniform matrix coating was achieved (∼20 mL). Tissue Analysis. WB-MSI experiments were performed on an LTQ-Orbitrap XL mass spectrometer equipped with a 60 Hz N2 laser (337 nm) MALDI source (ThermoFisher Scientific, San Jose, CA). The mass spectrometer was operated in positive ion mode from m/z 275 to 1000 with a resolving

Figure 1. (A) Reserpine signal from stomach pixel. (B) Methyl reserpate metabolite signal from intestinal pixel. 7159

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Figure 2. Extracted ion images at 5 ppm mass tolerance. (A) Optical image of sagittal section of 20 mg/kg reserpine rat 2 h postdose; (B) reserpine (m/z 609.281); (C) methyl reserpate (m/z 415.223); (D) reserpic acid (m/z 401.207).

parent and metabolite signal were detected with sufficient sensitivity in order to obtain the appropriate isotopic distribution, thus further providing a means for the confident extraction of the distribution images (Figure 2). Reserpine distribution was detected in highly localized regions of the rat, including the stomach and testis. This observation is consistent with the knowledge that reserpine is extensively metabolized in vivo. Interestingly, detecting drug signal in the testis seems reasonable considering reserpine is known to cause sexual dysfunction side effects.37,38 Extraction of ion images corresponding to the known metabolites methyl reserpate and reserpic acid34,36,39 indicate intense signal in the intestines, as well as, the muscle, thoracic cavity, and liver. Detection of these two analytes in the intestines may be due to local metabolism or biliary elimination of metabolites that were formed in the liver. Although definitive ADME data is not easily obtained by this approach, detection of specific metabolites in the intestine suggests contributions of possible metabolic and elimination pathways and highlights the utility of a WB-MSI technique in the drug discovery setting. In this single experiment, WB-MSI provided a global-view of drug/ metabolite distribution and the potential elimination routes of reserpine. Although both reserpine drug and metabolite images were successfully extracted from the image data based on the known exact masses, reserpine can undergo in vivo biotransformations to form a multitude of metabolites, many of which have not been previously reported in the literature. The most common metabolic pathways include the Phase I (e.g., oxygenations and reductions) and Phase II conjugation (e.g., sulfation and glucuronidation) routes that can be used to predict the

expected metabolite masses; however, reserpine can also undergo metabolic pathways that generate uncommon or “unexpected” metabolites. An essential step in metabolite identification is the determination of the molecular ion. In the case of predictable metabolites, a number of techniques can be employed to help identify these ions including extracting the data for a particular molecular ion or isotopic pattern of interest. This often requires multiple extractions of the data since many metabolites can be produced from a single administered compound, as was demonstrated by the targeted extraction of methyl reserpate and reserpic acid images (Figure 2). To further complicate matters, image data sets collected from tissues are very complex and extraction of the data set across a nominal mass window (1 Da) can produce interfering images comprised of isobaric ions of endogenous or exogenous chemical (e.g., matrix) origin. Collecting image data using an accurate mass instrument allows for the exploitation of the high mass resolution measurements by filtering the data based on mass defects. The mass defect of a compound is the difference of the exact mass of the molecule based on the molecular formula and its nominal mass (e.g., reserpine: nominal mass = 608 Da, exact mass = 608.2728 Da, mass defect = 0.2728 Da or 273 mDa). Since the mass defect range of typical biotransformations can be predicted, one can leverage this knowledge to selectively detect metabolites in complex data sets using a MDF that is defined by a narrow mDa mass window rather than the nominal mass window used in rudimentary extractions. This approach allows for the facile, comprehensive identification of common and uncommon metabolites without the need for a priori knowledge. 7160

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Table 1. Multiple Mass Defect Filtersa

filter filter filter filter

#1 #2 #3 #4

biotransformation range

mass range lower limit (Da)

mass range upper limit (Da)

mass defect lower limit (mDa)

mass defect upper limit (mDa)

parent and Phase I Phase II: sulfate conjugates Phase II: glucuronide conjugates Phase II: glutathione conjugates

555 680 760 905

675 715 825 945

235.5 223.5 276.0 340.5

315.5 252.5 337.0 370.5

Four mass defect filters were used to deconvolute the complex image data set, providing filtered data displaying only the peaks that passed the predefined criteria. The filter range criteria were selected on the basis of the 50 most commonly known biotransformations.1 a

Figure 3. mMDF postprocessing simplifies data down to signals of interest. (A) Comparison of total ion current across entire whole-body image before and after filtering by mMDF demonstrates an abundance of unrelated signal convoluting the raw data set. (B) Comparison of raw unfiltered data to mMDF processed data from a single stomach pixel (#13183) demonstrates the advantage of the postprocessing tool to pull out drug-related signal that may not be easily visualized in the unprocessed data.

effects of the MDFs on the entire image data set, the total ion current was charted as a function of time before and after filtering (Figure 3A). As expected when collecting data in broadband mode, the raw unfiltered data appeared to have an abundance of ion current across the 750 min imaging experiment. After applying the multiple MDFs, the resulting simplified total ion current now represented only the signal satisfying the mass filter criteria. Unlike the unfiltered data, hotspots of likely reserpine-related signal were easily determined after filtering.

For the reserpine WB-MSI study described here, multiple MDF windows were applied for metabolites of differing mass defect similarities (Table 1). The four MDF windows were intentionally defined to encompass the 50 most common biotransformations, including Phase I and the various Phase II conjugates. In theory, the filters could have been selected to incorporate all possible biotransformations by defining overlapping MDF windows across a comprehensive mass range. However, in order to accommodate a reasonable processing time and manageable output for the large image data set, the abridged MDF windows were applied. To demonstrate the 7161

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Figure 4. Extracted metabolite ion images using mMDF: (A) desaturation m/z 607.267 (−2.016 Da); (B) demethylation m/z 595.267 (−14.016 Da); (C) hydroxymethylene loss m/z 579.269 (−30.011 Da); (D) oxidation m/z 625.275 (+15.995 Da); (E) demethylation plus glucuronidation m/ z 771.297 (+162.016 Da).

original imaging experiment. Three additional data-dependent ion trap scans were performed simultaneously with each orbitrap scan to provide structural confirmation. For example, the MS2 and MS3 product ion spectra of the methyl reserpate metabolite confirmed the fidelity of the approach to detect metabolites solely based on the accurate mass measurement (Figure 5). Furthermore, several of the metabolites identified in tissue by this high-resolution WB-MSI approach were confirmed in plasma by LC MS/MS.40 With sufficient sensitivity, one can use the isotopic distribution of the analyte, along with the accurate mass measurements, to determine the elemental composition of the small molecule without ever needing to perform product ion scans to obtain structural confirmation. However, in order to differentiate the distribution of specific isobaric metabolites sharing the same biotransformation (e.g., O-demethylation off the various locations on reserpine) or to identify the actual site of metabolism on the drug, product ion scans will be necessary but may still prove to be insufficient for definitive isomeric determination. The study presented here is the first example to demonstrate the utility of accurate mass imaging for the detection of drug distribution across a whole-animal tissue section and represents a significant advancement for the application of WB-MSI in

To further exhibit the effect of MDFs, a single time point representing a pixel from the stomach region was selected from the filtered total ion current (Figure 3B). Prior to filtering, the pixel contained tens of hundreds of peaks of varying intensity across a wide dynamic range. Identification of reserpine signal was not obvious, unless manual extraction of the signal was performed. In the case of metabolite identification, manual mining of all possible metabolites would have been even more cumbersome; however, application of the MDFs sieved and ultimately simplified the spectrum such that only the peaks that passed the filter criteria were displayed. Accordingly, lower intensity signals such as reserpine and all possible metabolites were automatically pulled from the data and easily visualized. Subsequently, images were extracted with 5 ppm mass tolerance by selecting peaks from the various spectra across the imaging experiment, representing the distributions of putative metabolites (Figure 4). Both Phase I and II pathways were represented in the metabolite images, each displaying a unique distribution across the rat. Despite the fact that the metabolite images represented in this paper could be selected with a high degree of confidence on the basis of accurate mass and isotopic patterns alone, additional LTQ product ion scans were incorporated into the 7162

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Figure 5. Simultaneous methyl reserpate metabolite confirmation in LTQ ion trap. (A) MS2 of m/z 415.2; (B) MS3 of m/z 382.2.

of interest, thus highlighting the powerful advantage of collecting image data across a wide mass range with high mass resolution and accuracy. Despite the clear advantages of whole-animal drug distribution studies using accurate mass WB-MSI, there are significant considerations that may currently limit the feasibility of such an approach for routine analysis. For example, since the data are collected in the fullscan mode, the mass analyzer can become overwhelmed with chemical noise originating from the ablated matrix, limiting the ion population available for

drug discovery. Utilizing the analytical approach as described in this paper can provide a means to correlate drug and metabolite distributions along with endogenous components, perhaps revealing contributions to observed efficacy and/or toxicological responses via a pharmacodynamic (PD) marker (i.e., approach can provide a global view of multiple exogenous and endogenous components from one single imaging experiment). Furthermore, the data can be archived for subsequent analyses occurring days, months, or even years after the original imaging experiment to mine and extract additional analyte distributions 7163

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Notes

detection and ultimately resulting in lower sensitivity. Several groups have demonstrated effective precoated matrix targets41 or matrix-free techniques42,43 that could significantly minimize or ameliorate the detectable chemical noise and should be further evaluated for potential improvement of the accurate mass WB-MSI approach described here. In addition, the overall analysis time and large data volumes that are obtained when imaging whole-animal tissue sections can further complicate factors for the accurate mass approach. Decreasing pixel resolution can help to reduce the overall pixel burden; however, in order to maintain useful resolution, the pixel volumes are generally >20 000 pixels per whole-animal section (e.g., 250 μm pixel resolution for 10 cm mouse or 500 μm pixel resolution for 20 cm rat, resulting in similar total image pixels). Total pixel numbers of this size are still significantly taxing for an accurate mass instrument, where each pixel can contain tens of megabytes of information, resulting in whole-body images of hundreds of gigabytes in size. The current solution of saving centroided data (used in this study) or peak lists can help to reduce the data burden but heavily rely on the vendor-supplied algorithms to confidently assign the measured accurate mass and, since the raw data is discarded, precludes the capacity to reprocess the data. Alternatively, adjusting to a lower mass resolution can also help to reduce the data load, but the mass resolution is generally maintained above 60 000 in order to achieve precise mass accuracy, thus resulting in scan speeds of >2 s/pixel or >10 h per whole-body section. All of these factors combined present the need for the development of solutions and/or instrumentation capable of augmenting the ascribed limitations of WB-MSI by an accurate mass approach. Fortunately, work is already underway to design top-quality instruments, such as the more recently described “mega” time-of-flight instruments that can provide superior duty-cycle and signal digitizing, all while maintaining high mass resolution and accuracy.44 It is anticipated that, with the current momentum of pharmaceutical applications using an MSI approach, advancements in sample preparation, instrument design, and computing power will continue and eventually lead toward a truly robust WB-MSI approach by accurate mass spectrometry.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Michael Riech and the IVS group at Genentech for coordinating and completing the in-life portion of this study. The authors also thank Marcel Hop, Brian Dean, and various members of the DMPK department for their thoughtful discussions.

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CONCLUSIONS Coupling imaging MALDI MS with accurate mass, high resolving power, and intelligent data mining allowed for direct tissue distribution analysis across a broad range of ions in the full scan mode. The study presented here exploited the advantages of an accurate mass imaging approach to determine drug distribution within a reserpine-treated rat and identified the distribution of known/unknown metabolites using multiple MDFs. WB-MSI data obtained by accurate mass MALDI MS successfully demonstrated the capacity to detect and determine the relative concentrations of drug in tissues and more importantly, and at the same time, the individual distributions of various “unknown” metabolites, valuable information that serves as an early tool for ex vivo evaluations of potential therapeutic compounds.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 7164

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