**3.2.1 Choice of matrix**

The choice of matrix used for MALDI-IMS depends on the mass range and chemical properties of the analytes. Among the many kinds of matrices, sinapinic acid (3,5-dimethoxy-4 hydroxycinnamic acid [SA]) is generally used for high-molecular-weight molecules, such as proteins, while α-cyano-4-hydroxycinnamic acid (CHCA) is often used for medium-molecularweight molecules, such as peptides. 2,6-dihydroxyacetophenone (DHA), DHB, or 9 aminoacridine (9-AA) is generally used for low-molecular-weight molecules, such as pharmaceutical compounds, lipids, or metabolites (Hattori et al., 2010; Hayasaka et al., 2009; Khatib-Shahidi et al., 2006; Sugiura et al., 2009; Woods & Jackson, 2006).

The development of new matrices is still being reported. We and other research groups recently reported the use of nanoparticles as new matrices (Hayasaka et al., 2010; McLean et al., 2005; Moritake et al., 2009; Su & Tseng, 2007; Sugiura & Setou, 2010). For example, iron oxide nanoparticles enable the visualization of sulfatide and phospholipid distribution (Ageta et al., 2009; Taira et al., 2008), silver nanoparticles can be used for the analysis of fatty acids (Hayasaka et al., 2010), and gold nanoparticles are appropriate for the sensitive detection of glycosphingolipids, such as sulfatides and gangliosides (Goto-Inoue et al., 2010a).

## **3.2.2 Matrix application**

There are various methods for applying the matrix onto the section, such as deposition, spraying, and sublimation. The matrix application method also influences analyte extraction efficiency. Compared to other methods, the deposition of matrix solution using automatic depositing robotic devices, such as a chemical inkjet printer (ChIP-1000; Shimadzu Corporation, Kyoto, Japan), increases signal sensitivity, but decreases spatial resolution (Aerni et al., 2006; Chansela et al., 2011; Morita et al., 2010). The other limitation of the inkjet printer is capillary clogging, which occurs when highly concentrated matrix solutions are used. Spraying is the most frequently used method in MALDI-IMS. Using this method, an entire tissue section can be homogeneously coated with relatively small crystals in a short time without special equipment. For its operation, several instruments, including Thin layer chromatography (TLC) sprayers and artistic airbrushes, are available; we use a metal airbrush with a 0.2-mm nozzle because of its simple and easy-to-handle design. This method requires skillful operation because some airbrush parameters are hand-operated. If there is an excess of matrix solution on the tissue, an inhomogeneous crystal can be formed with analytes that have migrated from their original location; on the other hand, if not enough matrix solution is sprayed and it evaporates without sufficiently moisturizing the tissue section, analytes cannot be adequately extracted from the tissue section. The operation should be performed at a constant room temperature and humidity. Beginners are recommended to practice spraying until homogeneous matrix spraying can be reproducibly achieved. Sublimation is a new method for applying matrix to tissue sections (Hankin et al., 2007). Using this technique, a matrix can be applied uniformly over a large sample plate in a few minutes without solvents. Additionally, previous reports demonstrated that this method increases analyte signal and that the fine microcrystals formed from the condensed vapor reduce the image resolution limitation caused by crystal size (Dekker et al., 2009; Vrkoslav et al., 2010).

#### **3.3 Measurement and data analysis**

#### **3.3.1 Measurement**

442 Pharmacology

The matrix plays a central role in MALDI-MS soft ionization (Karas & Hillenkamp, 1988; Karas & Kruger, 2003). Biomolecules are softly ionized in the cocrystal with the matrix, which absorbs the laser beam energy and protects biomolecules from the disruptive energy. Protonated ion ([M + H]+) or deprotonated ion ([M − H]−) molecules are generally detected. Sodium adduct ion ([M + Na]+) and potassium adduct ion ([M + K]+) are often observed by biological sample analysis. It is very important to choose appropriate matrices for obtaining meaningful biomolecular images. An overview of the matrices used for IMS can also be

The choice of matrix used for MALDI-IMS depends on the mass range and chemical properties of the analytes. Among the many kinds of matrices, sinapinic acid (3,5-dimethoxy-4 hydroxycinnamic acid [SA]) is generally used for high-molecular-weight molecules, such as proteins, while α-cyano-4-hydroxycinnamic acid (CHCA) is often used for medium-molecularweight molecules, such as peptides. 2,6-dihydroxyacetophenone (DHA), DHB, or 9 aminoacridine (9-AA) is generally used for low-molecular-weight molecules, such as pharmaceutical compounds, lipids, or metabolites (Hattori et al., 2010; Hayasaka et al., 2009;

The development of new matrices is still being reported. We and other research groups recently reported the use of nanoparticles as new matrices (Hayasaka et al., 2010; McLean et al., 2005; Moritake et al., 2009; Su & Tseng, 2007; Sugiura & Setou, 2010). For example, iron oxide nanoparticles enable the visualization of sulfatide and phospholipid distribution (Ageta et al., 2009; Taira et al., 2008), silver nanoparticles can be used for the analysis of fatty acids (Hayasaka et al., 2010), and gold nanoparticles are appropriate for the sensitive detection of

There are various methods for applying the matrix onto the section, such as deposition, spraying, and sublimation. The matrix application method also influences analyte extraction efficiency. Compared to other methods, the deposition of matrix solution using automatic depositing robotic devices, such as a chemical inkjet printer (ChIP-1000; Shimadzu Corporation, Kyoto, Japan), increases signal sensitivity, but decreases spatial resolution (Aerni et al., 2006; Chansela et al., 2011; Morita et al., 2010). The other limitation of the inkjet printer is capillary clogging, which occurs when highly concentrated matrix solutions are used. Spraying is the most frequently used method in MALDI-IMS. Using this method, an entire tissue section can be homogeneously coated with relatively small crystals in a short time without special equipment. For its operation, several instruments, including Thin layer chromatography (TLC) sprayers and artistic airbrushes, are available; we use a metal airbrush with a 0.2-mm nozzle because of its simple and easy-to-handle design. This method requires skillful operation because some airbrush parameters are hand-operated. If there is an excess of matrix solution on the tissue, an inhomogeneous crystal can be formed with analytes that have migrated from their original location; on the other hand, if not enough matrix solution is sprayed and it evaporates without sufficiently moisturizing the tissue section, analytes cannot

found in other reviews (Chughtai & Heeren, 2010; Kaletas et al., 2009).

Khatib-Shahidi et al., 2006; Sugiura et al., 2009; Woods & Jackson, 2006).

glycosphingolipids, such as sulfatides and gangliosides (Goto-Inoue et al., 2010a).

**3.2 Matrix application** 

**3.2.1 Choice of matrix** 

**3.2.2 Matrix application** 

MALDI-IMS should be performed as soon as possible after matrix application, regardless of the coating method. The procedure to obtain a good spectrum in MALDI-IMS is almost the same as that for traditional MALDI-MS; mass range, detector gain, and laser power must be optimized. From the mechanical setting perspective, there are 3 differences between MALDI-MS and MALDI-IMS. The first difference is the above-mentioned matrix application. The second difference is the need for focusing of the laser beam. To obtain meaningful biological images by MALDI-IMS, the laser spot size should be reduced to 10–50 μm. The third difference is that a two-dimensional region must be set for analyses. The scan pitch, which signifies the distance between laser irradiation spots, must be fixed. The limitation of the scan pitch, which decides the spatial resolution of the image, depends on the laser spot size and mechanical movement control of the mass spectrometer sample stage. We have developed a new instrument (Mass Microscope) that can move the sample stage by 1 μm, and in which the finest size of the laser diameter is approximately 10 μm (Harada et al., 2009). The measurement time depends on the number of data spots, the frequency of the laser, the number of shots per spot, and the time required to move the sample stage. For example, when researchers select the region of interest as a 1 × 1 mm2 area with a 10-μm scan pitch (10,000 data points), it takes about 1 h to complete the measurement using a mass microscope equipped with a 1000-Hz laser (100 shots/data point).

MALDI-IMS ionizes numerous compounds in a tissue at the same time. Sometimes, we detect multiple molecules with the same *m/z* value. In such cases, a new imaging technique, "MS/MS imaging," is effective. Using this technique, we can separate each ion derived from their specific fragment ions. Some reports have described the use of MS/MS imaging for IMS of endogenous metabolites and an exogenous drug (Khatib-Shahidi et al., 2006; Porta et al., 2011). Additionally, the combination of ion-mobility separation with MALDI-IMS provides a unique separation dimension to further enhance the capabilities of IMS (Jackson et al., 2007; McLean et al., 2007; Stauber et al., 2010). It can be used to produce images without interference from background ions of similar mass, and this can remove ambiguity from imaging experiments and lead to a more precise localization of the compound of interest.

#### **3.3.2 Data analysis**

A large amount of data (a few gigabytes) is obtained from MALDI-IMS; therefore, visualization software packages that can rapidly and efficiently analyze enormous spectra have been developed. BioMap (a free software; Novartis, Basel, Switzerland), FlexImaging

Application of Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry 445

18:2) (*m/z* 671.6) and cholesterol oleate (CE 18:1) were characteristically distributed in lipid-rich regions, and the ion at *m/z* 566.9 was localized in the calcified region. These biomolecules were hardly detected in the normal aortic roots of ApoE-deficient mice. We applied this method to other vascular diseases, such as varicose veins, arteriovenous fistulae, abdominal aortic aneurysm, and triglyceride deposit cardiomyovasculopathy, and observed the characteristic distribution of biomolecules (Tanaka et al., 2010; Tanaka et al., 2011). In the analysis of several vascular diseases with atherosclerotic lesions, we often observed ectopic TG distribution. Although the role of TG in the evolution of atherosclerosis remains unknown, there is a possibility that TG plays an important role in the evolution of some kinds of atherosclerosis, as we previously found that characteristic atherosclerosis accumulated TG in aortic lesions, while the accumulated cholesterol was normal (Hirano et al., 2008). The reexamination of vascular diseases by IbHE may result in new findings, because IbHE can visualize the localization of low-molecular-weight molecules, which are rarely visualized by other techniques. We believe IbHE is of considerable value as a new histopathological examination because IbHE can

Fig. 4. Representative molecular images of specific ions in a mouse atherosclerotic lesion.

Visualization of biomolecules in atherosclerotic roots (a-j). Scale bar, 100 μm. Specific ion images of region 1 (a and b) and the combined image of *m/z* 671.6 and 673.6 (c). Specific ion images of region 2 (d and e) and the combined image of *m/z* 804.5 and 832.5 (f). Specific ion images of region 3 (g) and the monochrome image of *m/z* 566.9 (h). Comparison of HE staining (i) and the merge images of regions 1, 2, and 3 (j). An image of non-atherosclerotic aortic roots of mice at 12 weeks of age (k-m). Scale bar, 200 μm. HE staining after IMS (k). Oil red O staining (l). Immunostaining of α-actin, which is a marker for smooth muscle cells (m). Merge image of CE (18:2) and CE (18:1) (n). Merge image of PC (diacyl 16:0/20:4) and PC (diacyl 18:0/20:4) (o). Ion image of *m/z* 566.9 (p). "Reprinted from Atherosclerosis, 217. 2, Zaima et al., Imaging mass spectrometry-based histopathologic examination of atherosclerotic lesions, 430.,

visualize metabolic abnormalities in disease.

Copyright (2011), with permission from Elsevier."

(Bruker Daltonics, Bremen, Germany), and ImageQuest (Thermo Fisher Scientific, CA, USA) are generally used for visualization. For biomarker analysis of the MALDI-IMS dataset, data mining should be used (Hayasaka et al., 2011; Zaima et al., 2011b; Zhang et al., 2004). Data mining software effectively reduce the number of biomarker candidates (Hayasaka et al., 2011; Zhang et al., 2004). We previously reported the use of principal component analysis (PCA) to discover different biomolecules in starvation-induced fatty livers and normal livers (Zaima et al., 2009). Hierarchical clustering was also used to analyze the data obtained from gastric cancer and non-neoplastic mucosa tissue sections (Deininger et al., 2008). Several studies have reported the discovery of biomarkers using MALDI-IMS (Bakry et al., 2011; Ducret et al., 2006; Hong & Zhang, 2011; Solassol et al., 2009; Zaima et al., 2011b).

#### **4. Instruments**

The requirement for performing IMS is the availability of an *x-axis-y-axis* moving stage with electronic controls. Most modern MS instruments produced by major MS hardware companies (*i.e.*, Shimadzu, ThermoFisher Scientific, Bruker Daltonics, Applied Biosystems, Waters) can be adapted for MALDI-IMS. Time of flight (TOF) is the most widely used technology. TOF analyzers allow the separation of ionized accelerated molecules according to their *m/z* ratio. TOF-MS offers suitable performance for MALDI-IMS, namely, good transmission ratio (50– 100%), sensitivity, mass range, and repetition rate. However, TOF-MS lacks the capability to perform effective tandem MS experiments. This disadvantage of TOF-MS was overcome with the introduction of hybrid analyzers, such as a combination of quadrupole mass analyzer and TOF (so-called qTOF), combination of quadrupole ion trap (QIT) and TOF (so-called QIT-TOF), combination of ion mobility spectrometry (IMS) and TOF (so-called IMS-TOF), or a combination of two TOF mass spectrometers (so-called TOF-TOF). These combination systems revolutionized the application of TOF-MS systems for structural analysis with tandem MS. In general, the first system is used to select a precursor ion for fragmentation, while the second TOF system is employed for fragment analysis. Other mass analyzers (and their combinations), such as linear ion trap (LIT) (Landgraf et al., 2009; Wiseman et al., 2006; Zaima et al., 2010a), triple quadrupole (QqQ) (Hopfgartner et al., 2009; Porta et al., 2011), and Fourier transform ion cyclotron resonance (FTICR) (Taban et al., 2007), are used for MALDI-IMS. The advantages of commercially available LIT instruments are miniaturization, capability of sample analysis on nonconductive glass slides, MALDI performance at intermediate pressure, and superior performance on multistage MS. The QqQ system allows quantitative analysis and single or multiple reaction monitoring (SRM/MRM). The FTICR system offers very high mass resolving power and high mass measurement accuracy.

#### **5. Applications of MALDI-IMS**

#### **5.1 Imaging mass spectrometry-based histopathologic examination**

Recently, we applied MALDI-IMS for pathologic examination of atherosclerotic aorta (Fig. 4). We named it imaging mass spectrometry-based histopathologic examination (IbHE) (Zaima et al., 2011c). IbHE revealed the characteristic distribution of biomolecules in smooth muscle cells, lipid-rich regions, and calcified regions of an atherosclerotic lesion obtained from aortic roots of apolipoprotein E (ApoE)-deficient mice. We found that phosphatidylcholine (PC), which contains arachidonic acid (20:4) (*m/z* 804.5), was distributed in the smooth muscle cells of the atherosclerotic lesion. Cholesterol linoleate (CE

(Bruker Daltonics, Bremen, Germany), and ImageQuest (Thermo Fisher Scientific, CA, USA) are generally used for visualization. For biomarker analysis of the MALDI-IMS dataset, data mining should be used (Hayasaka et al., 2011; Zaima et al., 2011b; Zhang et al., 2004). Data mining software effectively reduce the number of biomarker candidates (Hayasaka et al., 2011; Zhang et al., 2004). We previously reported the use of principal component analysis (PCA) to discover different biomolecules in starvation-induced fatty livers and normal livers (Zaima et al., 2009). Hierarchical clustering was also used to analyze the data obtained from gastric cancer and non-neoplastic mucosa tissue sections (Deininger et al., 2008). Several studies have reported the discovery of biomarkers using MALDI-IMS (Bakry et al., 2011;

The requirement for performing IMS is the availability of an *x-axis-y-axis* moving stage with electronic controls. Most modern MS instruments produced by major MS hardware companies (*i.e.*, Shimadzu, ThermoFisher Scientific, Bruker Daltonics, Applied Biosystems, Waters) can be adapted for MALDI-IMS. Time of flight (TOF) is the most widely used technology. TOF analyzers allow the separation of ionized accelerated molecules according to their *m/z* ratio. TOF-MS offers suitable performance for MALDI-IMS, namely, good transmission ratio (50– 100%), sensitivity, mass range, and repetition rate. However, TOF-MS lacks the capability to perform effective tandem MS experiments. This disadvantage of TOF-MS was overcome with the introduction of hybrid analyzers, such as a combination of quadrupole mass analyzer and TOF (so-called qTOF), combination of quadrupole ion trap (QIT) and TOF (so-called QIT-TOF), combination of ion mobility spectrometry (IMS) and TOF (so-called IMS-TOF), or a combination of two TOF mass spectrometers (so-called TOF-TOF). These combination systems revolutionized the application of TOF-MS systems for structural analysis with tandem MS. In general, the first system is used to select a precursor ion for fragmentation, while the second TOF system is employed for fragment analysis. Other mass analyzers (and their combinations), such as linear ion trap (LIT) (Landgraf et al., 2009; Wiseman et al., 2006; Zaima et al., 2010a), triple quadrupole (QqQ) (Hopfgartner et al., 2009; Porta et al., 2011), and Fourier transform ion cyclotron resonance (FTICR) (Taban et al., 2007), are used for MALDI-IMS. The advantages of commercially available LIT instruments are miniaturization, capability of sample analysis on nonconductive glass slides, MALDI performance at intermediate pressure, and superior performance on multistage MS. The QqQ system allows quantitative analysis and single or multiple reaction monitoring (SRM/MRM). The FTICR system offers very high

Ducret et al., 2006; Hong & Zhang, 2011; Solassol et al., 2009; Zaima et al., 2011b).

mass resolving power and high mass measurement accuracy.

**5.1 Imaging mass spectrometry-based histopathologic examination** 

Recently, we applied MALDI-IMS for pathologic examination of atherosclerotic aorta (Fig. 4). We named it imaging mass spectrometry-based histopathologic examination (IbHE) (Zaima et al., 2011c). IbHE revealed the characteristic distribution of biomolecules in smooth muscle cells, lipid-rich regions, and calcified regions of an atherosclerotic lesion obtained from aortic roots of apolipoprotein E (ApoE)-deficient mice. We found that phosphatidylcholine (PC), which contains arachidonic acid (20:4) (*m/z* 804.5), was distributed in the smooth muscle cells of the atherosclerotic lesion. Cholesterol linoleate (CE

**5. Applications of MALDI-IMS** 

**4. Instruments** 

18:2) (*m/z* 671.6) and cholesterol oleate (CE 18:1) were characteristically distributed in lipid-rich regions, and the ion at *m/z* 566.9 was localized in the calcified region. These biomolecules were hardly detected in the normal aortic roots of ApoE-deficient mice. We applied this method to other vascular diseases, such as varicose veins, arteriovenous fistulae, abdominal aortic aneurysm, and triglyceride deposit cardiomyovasculopathy, and observed the characteristic distribution of biomolecules (Tanaka et al., 2010; Tanaka et al., 2011). In the analysis of several vascular diseases with atherosclerotic lesions, we often observed ectopic TG distribution. Although the role of TG in the evolution of atherosclerosis remains unknown, there is a possibility that TG plays an important role in the evolution of some kinds of atherosclerosis, as we previously found that characteristic atherosclerosis accumulated TG in aortic lesions, while the accumulated cholesterol was normal (Hirano et al., 2008). The reexamination of vascular diseases by IbHE may result in new findings, because IbHE can visualize the localization of low-molecular-weight molecules, which are rarely visualized by other techniques. We believe IbHE is of considerable value as a new histopathological examination because IbHE can visualize metabolic abnormalities in disease.

Fig. 4. Representative molecular images of specific ions in a mouse atherosclerotic lesion.

Visualization of biomolecules in atherosclerotic roots (a-j). Scale bar, 100 μm. Specific ion images of region 1 (a and b) and the combined image of *m/z* 671.6 and 673.6 (c). Specific ion images of region 2 (d and e) and the combined image of *m/z* 804.5 and 832.5 (f). Specific ion images of region 3 (g) and the monochrome image of *m/z* 566.9 (h). Comparison of HE staining (i) and the merge images of regions 1, 2, and 3 (j). An image of non-atherosclerotic aortic roots of mice at 12 weeks of age (k-m). Scale bar, 200 μm. HE staining after IMS (k). Oil red O staining (l). Immunostaining of α-actin, which is a marker for smooth muscle cells (m). Merge image of CE (18:2) and CE (18:1) (n). Merge image of PC (diacyl 16:0/20:4) and PC (diacyl 18:0/20:4) (o). Ion image of *m/z* 566.9 (p). "Reprinted from Atherosclerosis, 217. 2, Zaima et al., Imaging mass spectrometry-based histopathologic examination of atherosclerotic lesions, 430., Copyright (2011), with permission from Elsevier."

Application of Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry 447

WBA makes it possible to obtain more reliable data for absorption, distribution, metabolism, and excretion of drugs (Atkinson et al., 2007; Caprioli et al., 2008; Clench et al., 2008; Stoeckli et al., 2006). The application of MALDI-IMS to pharmacokinetics in a whole-body mouse section was first reported by Rohner et al. in 2005 (Rohner et al., 2005). In this study, they showed a good correlation between WBA and MALDI-IMS data. Figure 5 shows the simultaneous visualization of drug and metabolites in a whole-rat sagittal tissue section (Khatib-Shahidi et al., 2006). Khatib-Shahidi et al. visualized the temporal distribution of dosed olanzapine (brand name Zyprexa) (8 mg/kg) and its metabolites. In this study, MALDI-IMS was further

Fig. 6. Detection of drug and metabolite distribution at 6 h post-dose in a whole-rat sagittal

Optical images of a 6 h post-olanzapine (OLZ)-dosed rat tissue section across 4 gold MALDI target plates (A). Organs are outlined in red. MS/MS ion image of OLZ (m/z 256) (B). MS/MS ion image of N-desmethyl metabolite (m/z 256) (C). MS/MS ion image of 2 hydroxymethyl metabolite (m/z 272) (D). Scale bar, 1 cm. "Reprinted with permission from

Khatib-Shahidi et al., 2006. Copyright 2006 American Chemical Society."

tissue section by a single IMS analysis.

extended to detect proteins from organs present in a whole-body section.

#### **5.2 IMS for exogenous drugs**

MALDI-IMS is a powerful tool for visualizing the distribution of exogenous drugs and their metabolites. Porta et al. reported the visualization of the distribution of cocaine and its metabolites down to a concentration of 5 ng/mg in intact single hair samples from chronic users (Porta et al., 2011) (Fig. 5).

Fig. 5. Imaging of cocaine in hair samples H7 and H5. (H7 and H5 are sample names used in this article)

Optical image of hair sample H7 (a). MALDI-SRM/MS image based on the SRM trace of COC (m/z 305 > m/z 182) for five replicates of single hair samples from individual H7 (b) and single hair analysis from individual H5 (c). The quantitative results from LC-SRM/MS routine analysis were as follows: 130 ng/mg (H7, whole sample); 4.9 ng/mg (H5, segment 0–10 mm), and 8.5 ng/mg (H5, segment 10–50 mm). SRM; selected reaction monitoring. "Reprinted with permission from Porta et al., 2011. Copyright 2011 American Chemical Society."

MALDI-IMS is also applicable to pharmacokinetic analysis. As a Food and Drug Administration (FDA)-mandated pharmacokinetic test, whole-body autoradiography (WBA) is widely performed to determine spatial and quantitative information about a drug compound. Although much information can be acquired by WBA, it has several limitations. First, WBA requires the compound of interest to be radioactively labeled. Furthermore, the detected signal does not distinguish between the original radiolabeled compound and its metabolites that have retained the radiolabel. To complement the disadvantage of WBA, MALDI-IMS and WBA have recently been used together. The combination of MALDI-IMS and

MALDI-IMS is a powerful tool for visualizing the distribution of exogenous drugs and their metabolites. Porta et al. reported the visualization of the distribution of cocaine and its metabolites down to a concentration of 5 ng/mg in intact single hair samples from chronic

Fig. 5. Imaging of cocaine in hair samples H7 and H5. (H7 and H5 are sample names used in

Optical image of hair sample H7 (a). MALDI-SRM/MS image based on the SRM trace of COC (m/z 305 > m/z 182) for five replicates of single hair samples from individual H7 (b) and single hair analysis from individual H5 (c). The quantitative results from LC-SRM/MS routine analysis were as follows: 130 ng/mg (H7, whole sample); 4.9 ng/mg (H5, segment 0–10 mm), and 8.5 ng/mg (H5, segment 10–50 mm). SRM; selected reaction monitoring. "Reprinted with

MALDI-IMS is also applicable to pharmacokinetic analysis. As a Food and Drug Administration (FDA)-mandated pharmacokinetic test, whole-body autoradiography (WBA) is widely performed to determine spatial and quantitative information about a drug compound. Although much information can be acquired by WBA, it has several limitations. First, WBA requires the compound of interest to be radioactively labeled. Furthermore, the detected signal does not distinguish between the original radiolabeled compound and its metabolites that have retained the radiolabel. To complement the disadvantage of WBA, MALDI-IMS and WBA have recently been used together. The combination of MALDI-IMS and

permission from Porta et al., 2011. Copyright 2011 American Chemical Society."

**5.2 IMS for exogenous drugs** 

users (Porta et al., 2011) (Fig. 5).

this article)

WBA makes it possible to obtain more reliable data for absorption, distribution, metabolism, and excretion of drugs (Atkinson et al., 2007; Caprioli et al., 2008; Clench et al., 2008; Stoeckli et al., 2006). The application of MALDI-IMS to pharmacokinetics in a whole-body mouse section was first reported by Rohner et al. in 2005 (Rohner et al., 2005). In this study, they showed a good correlation between WBA and MALDI-IMS data. Figure 5 shows the simultaneous visualization of drug and metabolites in a whole-rat sagittal tissue section (Khatib-Shahidi et al., 2006). Khatib-Shahidi et al. visualized the temporal distribution of dosed olanzapine (brand name Zyprexa) (8 mg/kg) and its metabolites. In this study, MALDI-IMS was further extended to detect proteins from organs present in a whole-body section.

Fig. 6. Detection of drug and metabolite distribution at 6 h post-dose in a whole-rat sagittal tissue section by a single IMS analysis.

Optical images of a 6 h post-olanzapine (OLZ)-dosed rat tissue section across 4 gold MALDI target plates (A). Organs are outlined in red. MS/MS ion image of OLZ (m/z 256) (B). MS/MS ion image of N-desmethyl metabolite (m/z 256) (C). MS/MS ion image of 2 hydroxymethyl metabolite (m/z 272) (D). Scale bar, 1 cm. "Reprinted with permission from Khatib-Shahidi et al., 2006. Copyright 2006 American Chemical Society."

Application of Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry 449

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#### **6. Conclusions**

MALDI-IMS can be applied to pathological examinations leading to the discovery of potential targets for new drugs, and for the distributional analysis of exogenous drugs in animal and human tissues. We recently used MALDI-IMS in the discovery of metabolites that have pharmacological effects on natural resources. MALDI-IMS will become an essential tool for molecular imaging in pharmacology in the near future.

#### **7. Acknowledgement**

This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry (BRAIN).

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**21** 

*Spain* 

**Closed-Loop Control of Anaesthetic Effect** 

The interest in automation technologies applied to anaesthesia has been grown exponentially in last decade. The main difference with other fields of automation is that the presence of a human supervisor has been never questioned. In spite of this, the use of automation tools to monitor and control the main variables during surgery notably helps the anaesthetist during surgery. The basic functions of the automation systems in anaesthesia are monitoring and control of the main variables of the process. This leads to two expected benefits. First, the anaesthetist will be freed of some routinary tasks so that he can concentrate more on the state of the patient. On the other hand, using these tools contributes to improve the global performance of the process in terms of safety, costs

During the surgery operation three main variables have to be regulated: hypnosis, analgesia and muscle relaxation. To achieve this, drugs have to be properly administered to the patient. In recent years many efforts have been made in the development of new drug delivery technologies (Bressan et al., 2009). Most of the difficulties to calculate the proper drug rate to each patient were the inexistence of precise methods to monitor the anaesthetic state of the patient. In the past, patient monitoring was performed just by observing several patient signs (sweat, head lifting, movement, etc.). Nowadays the way that anaesthesia is

Concerning hypnosis regulation, many efforts have been made to provide the anaesthesiologist with reliable methods for monitoring. In particular, the introduction of the Bispectral Index (BIS) to measure the depth of anaesthesia was one of the key elements in

The other main problem in designing control algorithms to regulate hypnosis arises from the complexity of the patient response to drug infusion. This response can be divided in two subsystems. One is the Pharmacokinetics (PK) that refers to the adsorption, distribution, biotransformation and excretion of the drug. And the other is the Pharmacodynamics (PD) that describes the equilibrium relationship between concentration in the body and visible effect produced in the patient. In practice a linear model has been accepted to describe the

the development of new ways of drug administration (Sigl and Chamoun, 94).

**1. Introduction** 

reduction and patient comfort.

monitored has changed considerably.

PK and a nonlinear model for the PD part.

Santiago Torres, Juan A. Méndez,

*Universidad de La Laguna* 

Héctor Reboso, José A. Reboso and Ana León

