Spectrometry of Medicines and Sugars
3-1 Introduction
Smectite group, known as the clay with a thousand uses, is used in many industrial fields and many areas of everyday life, for example, pharmaceutics and iatrology [1], geology and meteorology [2], architecture and engineering [3]. In chemical field, many catalysts and high polymer composites are made by smectite and other materials (such as metals and polymers).
Garade et al. proposed that Co-saponite (a type of smectite group) could be used as the synthetic catalyst in liquid phase oxidation reactions [4]. Zhen et al. investigated the structure and properties of thermoplastic smectite / poly nanocomposites catalyst and made use to some biochemical reactions [5]. Nikolopoulou et al. developed Solvothermal preparation method to produce TiO2 / smectite nanocomposites, in addition, they verified remarkable photocatalytic activity in the reaction of decomposing NOx gas [6].
Some medicines and sugars, such as acetylsalicylic acid (ASA or Aspirin) and glucose, are incontestable the well-known pharmaceutical products in the world. ASA, which considered as the oldest, the most widely-used and the widespread medicine, is often used as an analgesic to relieve minor aches and pains, as an antipyretic to reduce fever, and as
83
an anti-inflammatory medication [7]. Glucose plays a vital role in metabolomics and nutrition, as it is the ubiquitous key energy source in most living organisms for regular mental effort [8]. Hence, it is no doubt that a rapid, accurate and easy-operated measurement should be developed to detecting medicines and sugars directly.
It is reported that numerous analytical techniques and methods have been applied to determine medicines and sugars in both chemicals and real human plasma, including gas chromatography mass spectrometry (GC-MS) [9], high performance liquid chromatography (HPLC) [10], desorption electrospray ionization mass spectrometry (DESI-MS) [11]. Conventional MALDI-MS is not a suitable tool because these analytes are quite hard to be protonated by only using conventional matrices, like 2, 4, 6-trihydroxyacetophenone, THAP, however, they are primarily ionized by attachment of alkali metal ions, mainly sodium [12]. Unfortunately, alkali metal ion peaks, e.g. ([M+Na] + and [M+K] +) are present in MALDI-MS spectrometry only at low level. In order to overcome these drawbacks, several methods have been made up to now, including some matrix-free techniques, such as silicon nanowire (SiNW) [13], desorption/ionization on Silicon (DIOS) [14, 15], and nanostructure-initiator mass spectrometry (NiMS) [16]. The application of nano-particles (NPs) instead of organic matrix, for example, gold (AuNPs) [17], silver (AuNPs) [18], Silicon (SiNPs) [19] and platinum (PtNPs)
[20] have also been proposed. Besides these, making co-matrix which is consisted of ammonium salts [21], phosphoric acid [22], serine [23], saccharides [24], proteins [25] and
84
traditional organic matrix molecules for conventional MALDI-MS analysis is considered as an effective way to upgrade the quality of mass spectra as well.
To our knowledge, smectite group have not been applied to MALDI analysis. So here, we used a synthetic inorganic polymer with a saponite structure, called sumecton SA, as the matrix additive to make co-matrix with conventional matrix THAP for the mass spectral measurement of sugars and medicines which are mentioned above. In our study, we chose these co-matrices since THAP itself was not a good matrix for measuring sugars and medicines under our experimental condition, therefore we expected that the advantage by making matrix with smectite and THAP could be clearly shown. It was found that the co-matrix consisting of organic matrix and cation-exchanged smectite effectively enhanced the peak intensities of analyte-related ions. In order to refrain from the occasionality happened during our tests, all the detections had been repeated three times under same factors, comprising laser power, sample plate conditions which are considered as the main determinant for the reproducibility of MALDI-MS [26].
85
3-2 Experiment 3-2-1 Chemicals
2, 4, 6-trihydroxyacetophenone (THAP, 168Da) and 2, 5-Dihydroxybenzoic acid (DHBA, 154Da) and all the medicines and sugars were purchased from Wako Chemical (Osaka, Japan).
Smectite (Sme) was supplied by Kunimine Material Industries (Tokyo, Japan). Peptides, substance P (SubP, 1347Da) and AngiotensinII (AngII, 1046Da) were purchased from Sigma-Aldrich (Tokyo, Japan).
3-2-2 Cation-exchange
For the preparation of NaSme, 8.2g CH3COONa dissolved in 50ml distilled water was mixed with 500mg smectite in advance, and then the suspension were heated at 70 degree centigrade with a magnetic stirring. After stirring for 1h, the solution was separated with the precipitate through filter. Then the same amount of CH3COONa dissolved in 50ml distilled water was added into the precipitate. After implementing the same treatment 3 times, all the precipitate were acquired and then dried through freeze drying to get NaSme. For the preparation of KSme, other steps were same except the amount of CH3COOK was 9.8g.
3-2-3 MALDI-MS analysis
The organic matrix and smectite particles were dispersed in a mixture of acetonitrile and
86
distilled water (4:1 in volume) using ultrasonic bath and the concentration of organic matrix molecules in the solution was kept at 4 mg/mL. For THAP, a series of mixtures of organic matrix and smectite with weight ratios of 4:0.25, 4:0.5, 4:0.75, 4:1 and 4:2 were prepared. For comparison, pure matrix solutions (4mg/mL) were prepared with the same method of preparation mentioned above. All the analyte, ASA, for example, was dissolved in the mixed solvent of acetonitrile and deionized water (4:1 in volume) and then the concentration was adjusted to 1mg/mL. One microliter each of the matrix-smectite mixture and the analyte was pipetted onto a stainless steel sample plate and the plate was left in air for several minutes to evaporate the solvent. Then, the plate was put inside a commercial MALDI-TOF mass spectrometer (Waters) equipped with an N2 laser (337nm). The laser power for excitation was typically 8μJ. The laser firing rate was 10Hz, and 100 laser shots were summed to generate each spectrum. The instrument was operated in the positive ion reflection mode with the accelerating voltage of 17kV, and the delay time was set to 500ns.
3-2-4 Characterization
The morphology of SmFeN MPs was monitored with a scanning electron microscope (SEM, JSM-6100, JEOL) and the spectroscopic change of the organic matrix molecules by mixing with SmFeN MPs was detected by diffuse reflectance spectrum. X-ray diffraction patterns (XRD) of the samples were recorded on a RIGAKUD/MAX 2550/PC diffractometer using Cu Kα1 radiation (λ = 1.540 56Å) at 40 kV and 40 mA, over a 2θ range of 4-70o with a
87
resolution of 0.02 o at a scanning speed of 6 o /min. The phases were identified using the power diffraction file (PDF) database (JCPDS, International Centre for Diffraction Data).
88
3-3 Results and discussions
3-3-1 X-ray diffraction patterns of original and Na-exchanged smectite
Figure 3-1 X-ray diffraction patterns of Na-exchanged smectite and untreated smectite XRD is one of the most popular techniques used to demonstrate the nano-sized composite structure due to its availability and accuracy. Fig 3-1 shows the XRD patterns of Na-exchanged smectite and the original smectite. As showed in the figure, the reflection peak at 2θ=60° is observed in both samples indicated the formation of trioctahedral clay structure which is the characteristic peak of smectite [27, 28]. After cation exchange, the framework of smectite had not been destroyed. The first peak (001) declared the reflections of smectite. The basal layer spacing of smectite (001) plane determined by Bragg’s formula (nλ=2dsinθ) was 1.20nm. After Na-exchange, the diffraction peak shifted toward small angle (1.25nm), indicating the increase in layer spacing of smectite.
89
3-3-2 Co-matrix of THAP and smectite
Figure 3-2 (a) Mass spectrum of acetylsalicylic acid (ASA) measured with 2, 4, 6-trihydroxyacetophenone (THAP) as the matrix. Mass spectra of ASA measured with the co-matrix of THAP and smectite (Sme) with weight ratio of (b) 4:0.25 (c) 4:0.5 (d) 4:0.75 and (e) 4:1. Spectra were obtained with 7μJ laser excitation at 337nm.
90
Figure 3-2 shows the mass spectrum of the model medicine, acetylsalicylic acid (ASA), measured with 2, 4, 6-trihydroxyacetophenone (THAP) and original smectite as the matrix for laser desorption/ionization. The intensity of analyte-related peak was invisible. Therefore, it is understood that THAP only was not an efficient matrix for the measurement of ASA under our experimental conditions. When smectite was mixed with THAP to form co-matrix, this phenomenon hadn’t transformed distinctly. It is probably because that first, ASA is quite difficult to be protonated which leads to the practical impossibility of forming [ASA+H] +, second 337nm wavelength of laser is away from the ultraviolet-visible (UV-Vis) absorption region of THAP which means the power cannot be absorbed well for the desorption/ionization, moreover, those dissociative and exchangeable cations distribution in the layer spacing of smectite may prevent the ionization process of ASA. Consequently, it is no doubt that a cation exchange procedure is necessary for our analysis.
91
3-3-3 Co-matrix of THAP and cation-exchanged smectite
Figure 3-3-1. (a) Mass spectrum of ASA measured with THAP as the matrix. Mass spectra of ASA measured with the co-matrix of THAP and sodium-exchanged smectite (NaSme) with weight ratio of (b) 4:0.25 (c) 4:0.5 (d) 4:0.75 and (e) 4:1. Spectra were obtained with 7μJ laser excitation at 337nm.
Alkali metal salts (LiAc, NaAc, KAc, RbAc and CsAc) were used for cation exchang.
Finally we selected Na+ and K+ for ion exchange since that Rb+ and Cs+ are too different to
92
exchange with the cations in the layer spacing of smectite, while Li+ is less effective than Na+ and K+ under our conditions. Figure 3-3-1 shows the mass spectrum of ASA detected with co-matrices of THAP and Na+-exchanged smectite (NaSme) in different mass ratio. An enhancement of the analyte-related peak, [ASA+Na] + was clearly observed when NaSm were put into THAP as the matrix additive. Figure 3-3-1 (b) shows the mass spectrum of ASA measured by using the co-matrix of THAP and NaSme with the weight ratio of 4:0.25 (THAP4NaSme0.25). The peak intensity of the analyte-related ion was clearly increased in comparison with that displayed in Figure 3-3-1 (a). Almost 20-fold increase in peak intensity was observed for [ASA+Na] +. The function of NaSme is not only to provide Na+ ion source but so to accelerate the desorption/ionization process. In addition, a new peak appeared at m/z
= 225, which was assignable to [ASA-H+2Na] +. By increasing the weight ratio of NaSme to 4:0.5 (THAP4NaSme0.5), the peak intensities of the analyte-related ion as well as [ASA-H+2Na] + also increased, as shown in Figure 3-3-1 (c). Almost 40-fold increase in peak intensity was observed for [ASA+Na] +. However, the peak intensities of most of the species decreased when co-matrices with the weight ratios of THAP4NaSme0.75 and THAP4NaSme1
was used in Figure 3-3-1 (d) and (e). This could be easily understood as that the total numbers of THAP molecules in the excitation area decreased by mixing a large amount of NaSme since the sizes of NaSme are microns, comparable to the size of excitation area. Therefore, we chose the weight ratio of THAP4NaSme0.5 for further mass spectrometry.
93
Figure 3-3-2. (a) Mass spectrum of ASA measured with THAP as the matrix. Mass spectra of ASA measured with the co-matrix of THAP and potassium-exchanged smectite (KSme) with weight ratio of (b) 4:0.25 (c) 4:0.5 and (d) 4:0.75. Spectra were obtained with 7μJ laser excitation at 337nm.
Figure 3-3-2 shows the shows the mass spectrum of ASA detected with co-matrices of THAP and K+-exchanged smectite (KSme). Similar with Figure 3-3-1, the analyte-related ion peak, [ASA+K] + had an explicitly augment, the additional peak, [ASA-H+2K] + also emerged at the same time. But the prominent distinction presented in the best ratio between THAP and KSme, differentiate from THAP4NaSme0.5, THAP4KSme0.25 was the most resultful co-matrix in detecting ASA. This dissimilarity may come from the different physicochemical properties between NaSme and KSme, including particle sizes and specific surface areas. In hence, THAP4KSm0.25 was picked for next analysis.
94
3-3-4 Co-matrix of THAP and inorganic salts
Figure 3-4 Mass spectrum of ASA measured with co-matrix of THAP and (a) NaCl (b) NaSme (c) KCl and (d) KSme. The best ratios were selected for each co-matrix respectively.
Spectra were obtained with 7μJ laser excitation at 337nm.
95
To ensure the peak intensity enhancement observed in Figure 3-3 authentically due to the feature of cation-exchanged smectite, mass spectrometry of ASA was executed by using conventional alkali metal salt instead of smectite under the same experimental conditions. As shown in Figure 3-4 (a) and (c), very minimal enhancement of ASA-related peaks was observed compared with that in Figure 4 (b) and (d). As a result, it was speculated that the function of cation-exchanged smectite was not only the Na+ and K+ source supplier but also the promoter of the desorption/ionization process of the matrix and the analyte owing to the particular physicochemical properties. Meanwhile, it demonstrated that directly use high concentrations of sodium and potassium salts is not an appropriate way because they can inhibit the formation of crystalline MALDI matrix crystals and form adducts with analyte molecules during desorption/ionization process.
96
3-3-5 Various methods of sample preparation
Figure 3-5-1 Mass spectrum of ASA measured with co-matrix of THAP and NaSme with (a) mixing method (b) stirring method (c) sonication method. The best and same ratios were selected. Spectra were obtained with 7μJ laser excitation at 337nm.
In order to verify the significance of sample preparation method, additional two methods of making co-matrix were carried out besides sonication method in advance. One was THAP and NaSme were directly dripped by the solution after mixing in mortar (mixing method) while another was THAP and NaSme were stirred for 3h instead of mixing in mortar (stirring method).
Figure 3-5-1 shows the mass spectrum of ASA measured with co-matrix of THAP and NaSme with (a) mixing method (b) stirring method (c) sonication method. It clearly displayed that
97
mixing method was the least effective. The intensities of matrix- and analyte-related peak were lowest. Stirring method had a little superiority than mixing method because the intensity of analyte-related peak was almost 3-folds, while it was easy to find the advantage of dispersing method, whether the peak intensity of [ASA+Na] + or the ratio of analyte-related peak was the optimal. Based on the results observed above, we proposed a hypothesis, as showed in figure 3-5-2. The extent of THAP embedding into the layer of Smectite played a vital role. It was easily to understand that only physical mixing didn’t help to this process, while comparing the solid phase and liquid phase, it was apparently that THAP in liquid phase was much simpler to insert the smectite layer. According to the structure of smectite, it indicated that co-matrix dispersed in the solution in order to facilitate THAP embedding into Smectite layer completely played a vital role in detecting nice spectrum.
Figure 3-5-2 hypothesis of intercalation process
98
3-3-6 Peptide measured with co-matrices
Figure 3-6 (a) Mass spectrum of AngII measured with THAP as the matrix. Mass spectra of AngII measured with the co-matrix of THAP and (b) NaCl (c) NaSme (d) KCl and (e) KSme.
Spectra were obtained with 7μJ laser excitation at 337nm.
99
Some model peptide samples were singled out as the analyte to be tested. Figure 3-6 shows one of the model peptide AngII measured with different co-matrices. As a classical and typical matrix, THAP has been widely applied to MALDI analysis of macromolecular compounds and model peptides for a long time, so an intense peak of the analyte-related ion which was singularly higher than the intensity of matrix-related ion peaks was observed even if without any adduct. However, the appearance of so many fragments which was the one of the significant problems of conventional MALDI analysis could not be suppressed. After employing co-matrix made by THAP and cation-exchanged smectite, the intensities of analyte-related ions didn’t enhance any more, on the contrary, it had a tendency of decrease, and the fragmentation during detection wasn’t restrained as well. As we know, co-matrix consisted of organic matrix and salt prejudiced the crystallization process as well as the desorption/ionization process, so it wasn’t strange that the intensities of analyte-related peaks were lower than without any adduct which showed in figure 3-6(b) and (d). However, in figure 3-6(c), the intensity of analyte-related peak was only half of that in figure 3-6(a), while in figure 3-6(e), the phenomenon was almost the same. These results manifested that THAP and cation-exchanged smectite matrix didn’t have any superiority for peptide measurement compared with conventional organic matrix because of the distinction of sample size between bigger peptides and smaller medicines, which declared appropriate sample size was necessary for micro-particle supported matrix.
100
3-3-7 Analyte size dependence of co-matrix made by THAP and NaSme
Figure 3-7-1 Mass spectrum of sugars measured with co-matrix of THAP and NaSme with (a) monosaccharide (b) disaccharide (c) tri-saccharide (d) hexaose (e) heptose and (f) octose.
Spectra were obtained with 7μJ laser excitation at 337nm.
According to the discovery above, we proposed that co-matrix of THAP and NaSme was analyte size dependence. Six kinds of sugars, galactose(mono-saccharide), cellobiose(di-saccharides), raffinose(tri-saccharides), maltohexaose(hexose or six-saccharides), maltoheptaose(heptose or seven-saccharides) and γ-cyclodextrin(octose or eight- saccharides) were selected as analytes for MALDI measurement. Figure 3-7-1 shows
101 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
3 3.5 4 4.5 5 5.5 6 6.5 7
Ratio (analyte/matrix)
Size (A)
the mass spectrum of sugars as analytes measured with co-matrix of THAP and NaSme. The intensity of matrix-related peak was almost the same; however, the tendency of intensity of analyte-related peak was obviously decreasing. When the polymerization reached octose, whose molecular size approached to the layer size of smectite, the analyte-related peak disappeared, indicating that octose could hardly enter into the layer to meet matrix. The trend line was showed in Figure 3-7-2, where X axis was the particle size of these sugars; Y axis was the ratio of analyte-related peak / matrix-related peak. From the figure, we can clearly find out the decline of ratio without any doubt. It expounded that co-matrix made by THAP and NaSme was only appropriate for smaller size samples, preventing larger ones to contact with matrix only when another supporter was applied to this system instead of smectite.
Figure 3-7-2 Trend line of analyte size and intensity ratio
102
In order to exclude the interference of sensitivity of mass spectrometry, MCP correction was selected to investigate the influence factors between sensitivity and analyte size dependence. At the present stage, the arguments of the determinant of MCP correction are two factors, one is related to momentum and another depends on kinetic energy. Here, we assumed that the sensitivity was due to momentum. According to the formula of time of flight, the signal intensity ∝ 1
𝑚𝑣 ∝ √𝑚1 . The influence of response intensity was showed in the following figure 3-7-3. It was easily to understand that the decreasing tendence of experimental curve was much stronger than theoretical curve, which indicanted the influence of analyte size dependence was much higher than sensitivity. So we considered that analyte size dependence was reasonable.
[mono+Na]+ [di+Na]+ [tri+Na]+ [hex+Na]+ [hep+Na]+ [oct+Na]+
Mass 203 365 527 1013 1175 1320
Signal(assumed) 4.81 3.59 2.99 2.15 2 1.89
Intensity(real) 7901 4401 2661 566 228 108
Intensity(Nor.) 138.27 77.02 46.57 9.90 3.99 1.89
Figure 3-7-3 Influence of response intensity
0 20 40 60 80 100 120 140 160
0 200 400 600 800 1000 1200 1400
Normalized intensity(a.u.)
mass (m/z)
experimental curve (normalized) theoretical curve
103
3-3-8 Possible mechanism
Figure 3-8-1. Diffuse reflectance spectra of the co-matrix THAP4NaSme1. Spectra of THAP (only), NaSme (only), and NaCl (only) were shown for comparison.
Figure 3-8-2. Diffuse reflectance spectra of the co-matrix THAP4KSme1. Spectra of THAP (only), KSme (only), and KCl (only) were shown for comparison.
104
Figure 3-8-3. Transform diffuse reflectance spectra between THAP4NaSme1 and THAP.
Figure 3-8-1 shows the diffuse reflectance spectra of co-matrix of THAP and NaSme (Figure 3-8-2 is THAP and KSme), THAP4NaSme1 was adopted for the measurement to observe the intuitive spectroscopic changes of THAP clearly by mixing NaSme instead of the co-matrix of THAP4NaSme0.5, which was used for mass spectrometry of medicines. The diffuse reflectance spectra of THAP only and NaSme only as well as THAP4NaCl1 matrix were also described for comparison. It was assuredly observed that the band intensity around 350 nm was increased for THAP4NaSme1 and THAP4NaCl1, the band intensity around 300 nm was enhancive for THAP4NaSme1 whereas decreased for THAP4NaCl1, comparing for THAP only. The peak around 300nm was the characteristic peak of THAP, the salts blocked the crystallization of THAP was the primary cause which resulted in the decreasing for THAP4NaCl1, and it was easily understood that NaCl played an important role for the
1.5
1.0
0.5
0.0
Reflectance (KM)
900 800
700 600
500 400
300 200
wavenum ber
THAP4NaSm e1-THAP only
105
increasing around 350nm. The transform spectra of THAP4NaSme1 minus THAP (only) showed in Figure 3-8-3 indicated the significant variation after adding NaSme. Since the spectrum of NaSme did not show any characteristic structure, the increase of the band intensity at 350 nm was attributable to the spectroscopic change of THAP by mixing NaSme.
Three reasons can be ascribed to this important transformation. First is the change of the rate constant of the vibrational energy relaxation of a guest molecule in a host. The vibrational relaxation rate is [29]
γ = π ∑(𝜈)׀𝐺(𝜈)׀2 𝑒𝑥𝑝(𝛽ħ𝜔)−1
∏ [exp(𝛽ħων)−1]𝜈 ρν (1)
where γ is rate constant of vibrational relaxation of molecule, ρν is the compound many-phonon density of status. From Equation (1), it is easily understood that the rate constant of the vibrational relaxation, γ, is proportional to the phonon density of states, ρν. The rate constant of vibrational relaxation of THAP was weakened due to the existence of NaSme. According to Born–Oppenheimer approximation, ΔEtotal = ΔEelectronic + ΔEvibrational + ΔErotational, after absorbing energy from external, the change of vibrational energy will simultaneously affect the total energy of molecules and the interaction Hamiltonian, which is
Ηe(r,R)χ(r,R) = Eχ(r,R). (2)
After the process of intra-molecular vibrational-energy redistribution in THAP molecule in the electronic ground state, the vibrational energy that dissipated efficiently to the
106
surrounding media was used to ionize the analyte. Second is the different arriving procedure of the molecule from electronic ground states (S0) to electronic excited states (Sn). THAP only is straightway from S0 to Sn while in THAP with NaSme, this procedure is divided to several steps, which can be described as
ΔE = ΔE(S1-S0) + ΔE(S2-S1) + … + ΔE(Sn-Sn-1) (3)
It will unambiguously make THAP simply to be excited. The absorbance of THAP at 337 nm, which is the excitation wavelength for mass spectrometry, was increased 1.5 times by making co-matrix. Therefore, it is no doubt that an enhanced absorption by THAP in the presence of NaSme can be observed, which leads to large peaks of analyte- and matrix-related ions on the mass spectra. Third is the change of the interaction Hamiltonian, which was caused by the cation-π interactions effect, especially Na+ and K+. In general case, the value of interaction Hamiltonian (H2) which depended on the interactions of cation-π in matrix system was very tiny so it could be ignored compared with the value of electric filed Hamiltonian (H1). But after using NaSme, the value of H2 cannot be neglectful because it should be much larger than usual resulted by the enrichment of Na ion. At last, the transition moment value is higher than the situation without NaSme. Of course, another possible reason is NaSme can definitely promote desorption / ionization process which lead to the enhancement of peak intensity in mass spectrum because desorption / ionization process is also significant beside energy
107
absorption by matrix.
The morphology of NaSme used in medicine analysis was confirmed by Scanning Electron Microscope (SEM), Figure 3-8-4 shows the SEM images of NaSme and THAP4NaSme1. It was clearly observed that THAP aggregated on the surface of NaSme homogeneously, same as patterning obtained though the camera built in the MADLI instrument. This distribution had a great advantage for THAP catching energy adequately from laser and delivering to analyte most. At the same time, there were huge waves of irregularities on the NaSme surface, which may be the active sites that enhance the desorption/ionization process of matrix and analyte. Therefore, it was understood that existence of these sites on NaSme surface promoted the desorption/ionization process of matrix- and analyte- related ions, whereas the light absorption by matrix molecules was enhanced by the spectroscopic interaction with NaSme.
Figure 3-8-4. SEM images of (a) NaSme and (b) THAP4NaSme1.
108
3-3-9 Another advantage of cation-exchanged smectite
Another apparent superiority of cation-exchanged smectite is the improvement of reproducibility, which is also one of the serious problems of MALDI. Through cation exchange, the reproducibility of MALDI had been increased dramatically. As showed in Table 3-1, inner-spot RSD values as well as inter-spot RSD values were significantly decreased, which indicated the THAP crystals distributed on the Na-exchanged smectite homogeneously, which led to the higher reproducibility and less errors.
Table 3-1 Peak intensities and relative standard deviation (RSD) value of [THAP+Na] + Sample
Spot
Peak Intensity (*103) Average of Intensity(*103)
Inner-spot RSD (%)
Inter-spot RSD (%) Seq.1 Seq.2 Seq.3
THAP (only)
Spot 1 9.31 7.43 4.57 7.10 33.60
35.11 Spot 2 6.37 5.28 4.42 5.36 18.24
Spot 3 4.56 2.82 4.70 4.03 26.01 THAP-
NaSme
Spot 1 10.84 10.79 10.17 10.60 3.52
9.90 Spot 2 10.09 10.06 9.75 9.97 1.89
Spot 3 9.03 8.64 8.02 8.56 5.95
109
3-3-10 Other medicines and sugars
Figure 3-9-1 Mass spectrum of D-glucose measured with co-matrix.
110
Figure 3-9-2 Mass spectrum of Ibuprofen measured with co-matrix.
Figure 3-9-3 Mass spectrum of D-galactose measured with co-matrix.
111
The benefit of cation-exchanged smectite was applied to analyze other medicines and sugars as the analyte. Figure 3-9-1 shows the mass spectrum of D-glucose measured with co-matrix of THAP and cation-exchanged smectite. THAP without any particle was used for comparison. An intense peak of the analyte-related ion, which was almost half of the intensity of matrix-related ion peaks, was observed in Figure 3-9(c). An enhancement of the peak intensity of analyte-related ions was also observed when KSme was used. Although the peak intensity of the analyte-related ion was lower than that obtained using NaSme, an enhancement was clearly observed compared with the peaks in Figure 3-9(a), which was obtained by using only THAP. Figure 3-9-2 and 3-9-3 show the mass spectrum of ibuprofen (Ibu) and D-galactose (Gala) as the analyte with co-matrix. The results were similar with Figure 3-9-1, which indicated that co-matrix made by THAP and cation-exchanged smectite, a fine supporter and guest molecular compound, was a promising and highly efficient new material for MALDI mass spectrometry analysis.