Article pubs.acs.org/ac
Analyte-Size-Dependent Ionization and Quantification of Monosaccharides in Human Plasma Using Cation-Exchanged Smectite Layers Yuqi Ding,†,‡,§ Kento Kawakita,‡,§ Jiawei Xu,‡ Kazuhiko Akiyama,‡ and Tatsuya Fujino*,‡ †
Department of Food Inspection, Zhejiang Institute for Food and Drug Control, No. 86, Lane 1, Jichang Road, Jianggan District, Hangzhou, Zhejiang, China ‡ Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan S Supporting Information *
ABSTRACT: Smectite, a synthetic inorganic polymer with a saponite structure, was subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Typical organic matrix molecules 2,4,6-trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHBA) were intercalated into the layer spacing of cation-exchanged smectite, and the complex was used as a new matrix for laser desorption/ ionization mass spectrometry. Because of layer spacing limitations, only a small analyte that could enter the layer and bind to THAP or DHBA could be ionized. This was confirmed by examining different analyte/matrix preparation methods and by measuring saccharides with different molecular sizes. Because of the homogeneous distribution of THAP molecules in the smectite layer spacing, high reproducibility of the analyte peak intensity was achieved. By using isotope-labeled 13C6-D-glucose as the internal standard, quantitative analysis of monosaccharides in pretreated human plasma sample was performed, and the value of 8.6 ± 0.3 μg/mg was estimated.
S
determination as saccharides are difficult to protonate by using only conventional matrices, such as α-cyano-4-hydroxycinnamic acid (CHCA) and 2,4,6-trihydroxyacetophenone (THAP). However, saccharides could be ionized by attaching alkali metal ions, such as Na+ and K+.7,8 Unfortunately, the peak intensity of an analyte with alkali metal ion adduction is low. In addition, MALDI MS has other disadvantages; for instance, it has limited application in the low-molecular-weight region because of the fragmentation of matrix molecules in the desorption/ionization process, which makes the analysis of small molecules difficult. Several matrix-free techniques have been developed to overcome those drawbacks, including silicon nanowire (SiNW), 9 desorption/ionization on silicon (DIOS),10,11 and nanostructure-initiator mass spectrometry (NiMS).12 The application of nanometer-sized particles, for example, Au,13 Ag,14 Pt,15 Si,16 and CdTe,17 instead of an organic matrix has also been proposed. In addition, the use of a comatrix consisting of ammonium salts,18 phosphoric acid,19 serine,20 saccharides,21 and proteins22 with conventional organic matrix molecules is considered an effective way to
accharides are undoubtedly the most well-known pharmaceutical product in the world. They play vital roles in metabolomics and nutrition as a ubiquitous key energy source in most living organisms.1 However, prolonged high blood sugar levels after food intake cause diabetes mellitus, one of the most common metabolic diseases. It is widely known that high blood sugar levels induce such symptoms as thirst, urination, and hunger and eventually seriously damage vital parts of the human body, such as heart and eyes. In order to diagnose this disease, it is important to accurately determine the levels of sugars, particularly glucose, in blood or plasma in both fasting and nonfasting conditions. So far, a lot of references have been reported on the mass spectrometry analysis of saccharides.2,3 For examples, Liu et al. applied hydrophilic interaction liquid chromatography (HILC) to separate nine chosen saccharides in crude and processed rehmanniae radix.4 Zhang et al. employed desorption electrospray ionization (DESI) to detect saccharides by using phenylboronic acids.5 Füzfai et al. used gas chromatography (GC) coupled to ion-trap mass spectrometry to obtain characteristic fragmentation patterns of the trimethylsilyl (TMS) and its oxime derivatives of various saccharides.6 In contrast to the above-mentioned techniques, matrixassisted laser desorption/ionization mass spectrometry (MALDI MS) is not a suitable tool for saccharide © 2015 American Chemical Society
Received: May 12, 2015 Accepted: July 7, 2015 Published: July 7, 2015 7944
DOI: 10.1021/acs.analchem.5b01770 Anal. Chem. 2015, 87, 7944−7950
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improve the mass spectra as well. In our previous work, we found that some zeolite materials, such as mordenite (M), Y, and ZSM5, were applicable adducts as they could prevent the dissociation of matrix molecules and enhance the intensity of the protonated analyte peak. We concluded that the advantages using zeolite were attributable to (1) the high phonon density of states in zeolite for releasing excess vibrational energy stored in matrix molecules23 and (2) the efficient proton supply from Brönsted acid sites on the zeolite surface to the analyte via matrix molecules.24 Smectite has similar structure and chemical composition to mica and zeolite.25 The schematic diagram of smectite is shown in Figure 1. The interaction force between the silicate layers is
Article
EXPERIMENTAL SECTION
Materials. Smectite (Sumecton SA) was supplied by Kunimine Material Industries. Typical MALDI matrices, THAP and 2,5-dihydroxybenzoic acid (DHBA), were purchased from Wako Chemical. Acetylsalicylic acid (ASA) and ibuprofen (Ibu) were also purchased from Wako Chemical. DGlucose (Glu), D-galactose (Gal), D-fructose (Fru), and isotope-labeled 13C6-D-glucose (13C6-Glu) were purchased from Sigma-Aldrich. Substance P (SubP), a model peptide, and human plasma (solid) were obtained from Sigma-Aldrich. Preparation of Matrices and Samples. For the preparation of Na+-exchanged smectite (NaSm), 8.2 g of CH3COONa dissolved in 50 mL of distilled water was mixed with 500 mg of smectite, and the suspension was heated at 70 °C with magnetic stirring. After stirring for 1 h, the solution was separated from the precipitate by passing through a sheet of 0.2 μm filter paper. Then, the same amount of CH3COONa dissolved in 50 mL of distilled water was added to the precipitate. After implementing the same treatment three times, all the precipitates were combined and dried by freeze-drying to obtain NaSm. For the preparation of KSm, the same steps were taken except that the amount of CH3COOK used was 9.8 g. Mass Spectrometry. An organic matrix (THAP) and a cation-exchanged smectite (NaSm) were dispersed in a mixture of H2O/acetonitrile (ACN) (3:7 v/v) by ultrasonication to make THAP-intercalated Na+-smectite (THAPNaSm). A series of mixtures of THAP and NaSm with weight ratios of 4:0.25, 4:0.5, 4:0.75, 4:1, and 4:2 were prepared, and the concentration of organic matrix molecules in the solution was kept at 4 mg/ mL. For comparison, pure matrix solutions (4 mg/mL) were prepared with the same method as that mentioned above. The analyte was dissolved in H2O/ACN (3:7 v/v) and the concentration was adjusted to 1 mg/mL. The same volume of THAPNaSm and analyte solutions was introduced into a test tube, and the mixture was sonicated. A volume of 2 μL of the mixture (THAPNaSm and analyte solutions) was pipetted onto a stainless steel sample plate, and the plate was left in the air for several minutes to evaporate the solvent. Then, the plate was placed inside a commercial matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (Waters) equipped with an N2 laser (337 nm). The laser power for excitation was typically 8.0 μJ except for the quantitative analysis of saccharides (10.1 μJ). The laser firing rate was 10 Hz, and 100 laser shots were summed to generate a spectrum. The instrument was operated in the positive ion reflection mode. Quantitative Analysis. For the quantitative analysis of monosaccharides in human plasma, 5 mg of human plasma sample was diluted with 2.5 mL of H2O/ACN (3:7 v/v). After being shaken by a vortex mixer for 10 min, the solution was filtered through a sheet of 0.2 μm filter paper in order to separate proteins. Then, the filtrate was freeze-dried overnight and the residue was dissolved again in 100 μL of H2O/ACN. Standard solutions of D-glucose spiked with 1.0 mg of 13C6-Glu were prepared separately. A volume of 20 μL of the treated plasma sample was taken and mixed with 25 μL of 13C6-Glu solution with a concentration of 1 mg/mL. In total 5 μL of H2O/ACN was added to this solution to make the final volume of 50 μL. The standard solutions and the plasma sample were measured by MALDI MS as described above. Powder XRD and TG-DTA. Powder XRD (X-ray diffraction) patterns were recorded from 2θ = 4−70° (0.02°
Figure 1. Schematic diagram of smectite.
somewhat weak as the number of isomorphous substitutions in a layer, which determines layer charge, is small. Therefore, smectite easily swells in water and forms sol−gel in the presence of a large amount of water. In addition, smectite shows cation exchange ability and intercalation ability with other molecules. Because of those unique properties, smectite is used in many fields.26−28 In chemistry fields, for example, Cosaponite was used as the catalyst in the liquid-phase oxidation of p-vanillyl alcohol.29 Nanocomposites of saponite and poly(vinyl alcohol) exhibit remarkable improvement in mechanical property, such as thermal stability and water resistance.30 TiO2/smectite nanocomposites were produced and their photocatalytic activity in the decomposition of NOx gas was verified.31 To our best knowledge, however, smectites have not been used in MALDI MS. In this study, one kind of smectite, a synthetic inorganic polymer with a saponite structure, was used. THAP, a MALDI matrix, was intercalated into the smectite layer spacing in order to ionize small analytes that enter the layer spacing. It was found that the comatrix of THAP and smectite can ionize only small saccharides and is therefore a “low-molecular-weight mass filter.” It was also observed that cation-exchanged smectite enhanced the peak intensities of analyte-related ions as in the case of zeolite.8,24 Moreover, because of the homogeneous distribution of THAP in the smectite layers, high reproducibility of the analyte peak intensity could be achieved, which enabled us to quantitatively analyze monosaccharides in human plasma. 7945
DOI: 10.1021/acs.analchem.5b01770 Anal. Chem. 2015, 87, 7944−7950
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Analytical Chemistry intervals, 5° min−1) using a commercial XRD system (Rigaku). Cu−Kα X-ray (λ = 0.1541 nm) generated under the tube voltage of 50 kV and the current of 300 mA was used for the study. Thermogravimetry-differential thermal analysis (TGDTA) measurements (Rigaku) were carried out from RT to 500 °C under the heating rate of 10 °C min−1. α-Al2O3 was used as the reference.
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RESULTS AND DISCUSSION Smectite. In order to evaluate the properties of smectite, powder XRD patterns of untreated smectite (Sm), Na+- and K+-exchanged smectite (NaSm and KSm), and THAPNaSm were measured. The basal layer spacing of each sample calculated from (001) reflection is summarized in Table 1. Table 1. Summary of XRD and TG measurements for Sm, NaSm, KSm, and THAPSm sample
2θ (deg)
layer spacing (nm)
weight loss (%) 120 °C
smectite (Sm) NaSm KSm THAP/NaSm
6.91 6.62 7.13 6.39
1.28 1.34 1.24 1.38
9.58 11.7
The layer spacing of NaSm was larger than that of untreated Sm; however, the layer spacing of KSm was smaller than that of NaSm even though the ionic radius of K+ is larger than that of Na+. The same tendency was also found in the previous work.32 To understand the reason for those changes, TG-DTA of Sm and NaSm was carried out and the results are shown in Figure 2. The TG curves in the temperature region of 25 to 120 °C revealed that the weights of both Sm and NaSm decreased very rapidly; weight loss at 120 °C was estimated to be 9.58% for Sm and 11.7% for NaSm. In addition, it was found from the DTA curves that the weight loss in TG was accompanied by endothermic peaks at around 55−60 °C. As the desorption of molecules from the surface is an endothermic reaction, the weight loss in the TG curves is attributable to the desorption of water molecules from the smectite surface. From the finding that the weight loss of NaSm was larger than that of Sm, we confirmed that more water molecules were adsorbed to the NaSm surface than the Sm surface. It is known that Na+, Ca2+, Mg2+, and K+ cations exist on untreated Sm surface as exchangeable cations. Among them, divalent cations Ca2+ and Mg2+ induce a relatively strong interaction between the silicate layers, leading to small layer spacing and restriction of the intercalation of water molecules. In the case of NaSm, however, Na+ induces only a weak interaction between the silicate layers, which leads to large layer spacing and the intercalation of many water molecules in the layer spacing compared with Sm. In addition, the difference in layer spacing between NaSm and KSm could be explained by cation solvation; solvation enthalpy is −420.8 kJ/mol for Na+ and −337.1 kJ/mol for K+.33 This means that NaSm has more water molecules than KSm, which leads to the larger layer spacing for NaSm than KSm. For THAPNaSm, its layer spacing was larger than that of NaSm, although the number of water molecules must be the same as that of NaSm. Therefore, this difference was due to the intercalation of THAP; THAP molecules enlarged the layer spacing of NaSm. Figure 2b shows the diffuse reflectance spectrum of THAPNaSm and that of solid-state THAP. The UV−vis absorption spectrum of THAP in ACN (5.5 × 10−5 M) was
Figure 2. (a) TG and DTA spectra of untreated smectite (Sm) and Na+-exchanged smectite (NaSm). (b) Diffuse reflectance spectra of THAPNaSm (blue) and THAP solid (red). The UV−vis absorption spectra in ACN (black broken) and ACN/H2O (green broken) were also shown for comparison.
also depicted for comparison (black broken line). The UV−vis absorption spectrum of THAP was measured in another solution. THAP was dissolved in acetonitrile to which H2O containing the same molar amount of THAP was added. The observed spectrum was also shown in Figure 2b as a green broken line. The intensity of the shoulder peak around 330 nm (blue belt in the Figure) was increased, whereas the main peak at 280 nm was unchanged. Therefore, it was understood that the growth of the shoulder peak was caused by the THAP molecules interacting with water molecules. From Figure 2b, it may be possible to consider that the diffuse reflectance spectrum of THAPNaSm somewhat resembles the UV−vis absorption spectrum of THAP in solution rather than the spectrum of THAP in the solid state. However, the wavelength of the main peak was blue-shifted from 280 to 265 nm and the intensity of the shoulder peak around 330 nm was increased, and furthermore the bandwidth of each peak was broadened. Judging from these results, following things might be suggested; (1) bond length of THAP became slightly shorter since THAP molecules existed in narrow layer spacing of NaSm (as a reason for the blue shift), (2) THAP molecule is interacting with many water molecules (increase of peak intensity around 330 nm), (3) there are no specific sites in smectite for adsorption of THAP and many adsorption structures are possible in the layer spacing (broadening of bandwidth), and (4) THAP is fairly monodispersed in the layer spacing of NaSm (similarity to the spectrum obtained in solution phase). Laser Desorption Ionization. Then, THAPNaSm was applied to MALDI MS. Figure 3a shows the mass spectrum of acetylsalicylic acid (ASA) measured with THAP only as in conventional MALDI MS. ASA is difficult to ionize by proton 7946
DOI: 10.1021/acs.analchem.5b01770 Anal. Chem. 2015, 87, 7944−7950
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interference by NaCl. Therefore, we confirmed that NaSm is an efficient Na+ ion source in our present studies. In addition, judging from the peak intensity enhancement of [ASA + Na]+ (approximately 6-fold), the desorption/ionization process of ASA might be also accelerated by the presence of NaSm. Then, the model peptide, SubP, was singled out as the analyte. As a classical and typical matrix, THAP has been widely used in the MALDI MS analysis of macromolecular compounds and model peptides for a long time, and therefore, an intense peak of the analyte-related ion could be observed even if there were no adducts. After employing the comatrix as shown in Figure 3e, however, the intensities of the analyte-related ions were not enhanced; on the contrary, they tended to decrease (S/N = 11 for [M + Na]+, R = 0.02). This result manifested that THAPNaSm did not present any advantage for peptide measurement compared with the conventional organic matrix. As shown in Table 1, the size of the layer spacing of NaSm was 1.34 nm; therefore, it was assumed that a peptide whose size is larger than that of ASA could not access THAP existing in the smectite layer spacing, which was why the ionization of SubP ended in failure. The above assumption was verified by (1) examining the analyte/matrix preparation method and (2) changing analyte size. For the former, two methods were carried out instead of the method described in the Experimental Section. In one method, both solutions of THAPNaSm and analyte (ASA) were simply mixed on a sample plate. In the other method, a mixture of THAPNaSm and ASA solutions was stirred for 1 h using a vortex mixer, and then it was dropped on a sample plate. The mass spectra of the samples prepared by those two methods are shown in parts a and b in Figure 4, respectively. It
Figure 3. Mass spectra of ASA (acetylsalicylic acid) measured with (a) THAP (only), (b) THAPNaSm (mass ratio = 4:0.5), (c) THAPNaSm (4:1), and (d) THAP with NaCl. (e) Mass spectrum of SubP measured with THAPNaSm (mass ratio = 4:0.5).
adduction. In fact, the peak of [ASA + H]+ could not be observed and only a small peak of [ASA + Na]+ was visible at m/z = 203 (the signal-to-noise ratio; S/N = 106, the peak intensity ratio of [ASA + Na]+/[THAP + Na]+; R = 0.45). Figure 3b,c shows the mass spectra of ASA measured with THAPNaSm at different weight ratios. An enhancement of the peak intensity of [ASA + Na]+ was observed when THAPNaSm was used as the comatrix. When THAPNaSm with the weight ratio of 4:0.5 (THAP4NaSm0.5) was used, the peak intensity of [ASA + Na]+ (S/N = 590, R = 0.83) showed an almost 6-fold increase compared with that in Figure 3a. In addition, the peak assignable to [ASA − H + 2Na]+ also had a high intensity. In contrast, the peak intensities of most of the species decreased when THAPNaSm with the weight ratio of 4:1 (THAP4NaSm1) was used, as shown in Figure 3c. This could be easily understood as that the total number of THAP molecules in the excitation area was decreased by mixing a large amount of NaSm. We also examined comatrixes with weight ratios of 4:0.25 and 4:0.75 and confirmed that 4:0.5 was the best weight ratio to yield an intense analyte peak. Data obtained with these weight ratios were shown in Figure S1 in the Supporting Information. Thus, we chose THAP4NaSm0.5 for further mass spectrometric measurements. Hereinafter, we use the abbreviation THAPNaSm to represent THAP4NaSm0.5 in the studies discussed below. In order to confirm that the Na ion for the ionization of ASA originated from NaSm and that the enhancement of the peak intensity of [ASA + Na]+ was due to NaSm, the comatrix of THAP and NaCl was examined, and the results are shown in Figure 3d. The weight ratio of THAP to NaCl was also set to 4:0.5. Contrary to Figure 3a−c, the peak intensity of [ASA + Na]+ became very weak (S/N = 7, R = 0.02) because of the
Figure 4. Mass spectra of ASA subjected to different analyte/matrix preparation methods: (a) simple mixing on sample plate and (b) stirring.
was found that the mixing method was less effective than the method that used THAPNaSm with the weight ratio of 4:0.5 (Figure 4a vs Figure 3b). The peak intensities of all the ion species shown in this mass region including [ASA + Na]+ (S/N = 117) were almost the same as those in Figure 3a, which was measured with THAP only (without smectite). On the other hand, the stirring method showed little superiority over the mixing method as the peak intensity of [ASA + Na]+ was only approximately 2-fold of that in Figure 4a (S/N = 216). Comparing those results with Figure 3b, we found that the peak intensity of [ASA + Na]+ was dependent on the extent of access of ASA to THAP embedded in the NaSm layer; physical mixing alone did not help promotion of this process. Then, the influence of analyte size on the peak intensity was verified. Six kinds of saccharides: galactose (one glucose unit), 7947
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results were plotted relative to the molecular diameter in Figure 5g, which was estimated from the van der Waals volume assuming a sphere.35 The fitting result with an exponential function was also shown with a solid line. Therefore, THAPNaSm was appropriate for only small analytes. The advantages of smectite for MALDI MS were also confirmed for DHBA, another organic matrix. The mass spectrum of ASA, which was measured in the presence of the comatrix between DHBA and NaSm (DHBANaSm), is shown in Figure 6a. As was noted in Figure 3b, the peak of [ASA +
cellobiose (two glucose units), raffinose (three glucose units), maltohexaose (six glucose units), maltoheptaose (seven glucose units), and γ-cyclodextrin (eight glucose units) were selected as analytes. Figure 5 shows the mass spectra of those sugars
Figure 6. Mass spectra of (a) ASA measured with DHBANaSm, (b) Ibu measured with THAPKSm, and (c) Glu measured with THAPKSm.
Na]+ was observed at m/z = 203 (S/N = 853); therefore, it was confirmed that NaSm could be used in combination with other matrix molecules. Spectrum of ASA measured with DHBA only was shown in Figure S2a in the Supporting Information. It was found that the spectrum was very noisy although the peak of [ASA + Na]+ was observed. Then, K+-exchanged smectite (KSm) was applied to the ionization of small molecules. Figure 6b shows the mass spectrum of ibuprofen (Ibu). The mass peak of K+-adducted Ibu was observed at m/z = 245 (S/N = 403), proving that the ionization of analyte by the alkali cation on smectite surface was possible. D-Glucose (Glu) could be also ionized by forming an adduction with K+ and the peak of [Glu + K]+ was observed at m/z = 219 (S/N = 230), as shown in Figure 6c. For comparison, the spectra of Ibu and Glu measured with THAP only were shown in Figure S2b,c in the Supporting Information, and only the small peaks of analyte were observed. Therefore, it was found that smectite is beneficial to reduce peaks in the low-molecular-weight region and to enhance the peak intensity of analyte-related ions.
Figure 5. Mass spectra of various saccharides measured with THAPNaSm: (a) galactose, (b) cellobiose, (c) raffinose, (d) maltohexaose, (e) maltoheptaose, and (f) γ-cyclodextrin. Numbers in parentheses are the polymerization numbers of the saccharides. (g) Relationship between peak intensity ratio ([M, analyte + Na]+/[matrix + Na]+) and diameter of each molecule assuming a sphere. Solid curve shows fitting result by an exponential function.
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measured with THAPNaSm. The intensity of the matrix-related peak was almost the same; however, the intensity of the analyte-related peak showed an obvious decrease with increasing polymerization number of the saccharides. In the case of γ-cyclodextrin that has eight glucose units, the intensity of the analyte-related peak was barely visible. Of course these sugars have inherently different ionization yields. However, in our previous reports, maltohexaose and maltoheptaose, for example, showed the peak intensities as high as the intensity of THAP when Na+ or Li+ substituted zeolite was used.8,34 Therefore, the result in Figure 5 might indicate that such a large molecule could hardly enter the layer spacing, and thus, it could not bind to THAP molecules embedded in the NaSm layer and would not be ionized. For Figure 5a−f, the [analyte(M) + Na]+/[THAP + Na]+ intensity ratios were calculated, and the
QUANTITATIVE ANALYSIS OF MONOSACCHARIDES IN HUMAN PLASMA The peak intensities of Na+-adducted monosaccharides measured with THAPNaSm are summarized in Table 2. Glu, Gal, and Fru were used as the representative monosaccharides. For every monosaccharide, the peak intensities obtained from different spots (no. 1−3) and measurements (Seq 1−3) are summarized. A comparison of the average intensities revealed that the peak intensities of the monosaccharides could be regarded as the same within the standard deviation (SD). This means that the ionization efficiency of the three monosaccharides by forming adducts with Na+ can be regarded as almost the same. In addition, the relative standard deviation 7948
DOI: 10.1021/acs.analchem.5b01770 Anal. Chem. 2015, 87, 7944−7950
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Analytical Chemistry Table 2. Comparison of Ionization Efficiencies among Glu, Gal, and Fru peak intensity (a.u.) sample spot no. Glu
Gal
Fru
1 2 3 1 2 3 1 2 3
SD (a.u.)
Seq 1
Seq 2
Seq 3
intra
4.3 4.0 4.0 4.4 4.7 4.2 4.3 4.4 4.6
3.8 4.5 3.8 3.9 4.4 4.6 4.3 4.6 3.9
3.6 4.2 3.6 3.8 4.2 4.1 4.5 3.8 4.6
3.9 4.2 3.8 4.0 4.4 4.3 4.4 4.3 4.4
inter
intra 0.36 0.25 0.20 0.32 0.25 0.26 0.12 0.42 0.40
4.0
4.3
4.3
RSD (%) inter 0.31
0.30
0.30
intra
inter
9.2 5.9 5.3 8.0 5.7 6.2 2.6 9.8 9.3
overall
7.8 (34.2) 7.1
7.9
6.9
observed at m/z = 207, and no peaks due to monosaccharides in the human plasma sample were observed. Human plasma is complicated because it contains a large number of compounds, and it is assumed that those compounds hinder the ionization of monosaccharides. Therefore, purification pretreatment was carried out, and the treated plasma sample was subjected to the quantitative analysis of monosaccharides. The mass spectrum of treated human plasma sample containing 13C6-Glu as the internal standard is shown in Figure 7b. The peak of nonlabeled Glu, [Glu + Na]+, was observed at m/z = 203, and that of 13C6Glu was observed at m/z = 209, as shown in Figure 7b. Then, a regression line for the plot of the peak intensity ratio vs the amount ratio of nonlabeled Glu to 13C6-Glu was generated, as shown in Figure 7c. Excellent linearity was obtained with a correlation coefficient of approximately 1. By using Figures 7b,c, the value of monosaccharides in the human plasma sample measured in this study was estimated to be 8.6 ± 0.3 μg/mg. Then, the results of quantitative analysis (3 spots and 3 sequences) are summarized in Table 3. It is known that
(RSD) for Glu was as small as 7.8%, whereas RSD was 34.2% when THAP alone was used as the matrix (without using NaSm). The RSD values for Gal and Fru were 7.1% and 6.9%, respectively. The RSD for the overall measurements of 7.9% indicated that THAP molecules were distributed homogeneously in the smectite layers, which led to the high reproducibility of the mass spectra. Therefore, THAPNaSm is applicable to quantitative analysis, and in this study, monosaccharides in human plasma were examined. Glu was used as the representative monosaccharide. Isotope dilution was carried out as described in the Experimental Section as 13 C6-Glu was expected to exhibit the same properties, including crystal formation and desorption/ionization efficiency, as conventional Glu. Figure 7a shows the mass spectrum in the mass region from m/z = 202−210 for human plasma (as received) sample that was not subjected to any of the pretreatments described in the Experimental Section. Only the peak of [THAP + K]+ was
Table 3. Results of Quantitative Analysis of Monosaccharides in Human Plasma Samples sample
estimated values (μg/mg)
spot no. plasma
1 2 3
Seq 1
Seq 2
Seq 3
intra
9.11 8.46 8.48
8.37 8.91 8.30
9.04 8.31 8.60
8.84 8.56 8.46
SD (μg/mg) inter
intra
inter
8.62
0.41 0.31 0.15
0.32
saccharide content in human plasma is approximately 0.1%. Considering that 90% of human plasma is water, almost 1% in dried human plasma corresponds to saccharides. Of course, saccharide content in human plasma would vary according to the health condition of the subject. Nevertheless, our obtained value (0.86%) is in fairly good agreement with the previous study.36 Therefore, we understood that the method presented in this study were also applicable to the quantitative analysis by MALDI MS. By using our method, the levels of saccharides in complex biological samples can be detected accurately and rapidly by MALDI MS without complicated pretreatments. It opens a new domain for saccharides quantitative detection. We believe our discoveries are helpful not only in precise detection and statistical analysis for the type and content of saccharides in various biological samples but also in monitoring the blood sugar level which is the major index for diabetes diagnosis.
Figure 7. (a) Mass spectrum of untreated human plasma sample. (b) Mass spectrum of treated human plasma sample containing 13C6-Glu as the internal standard. (c) Regression line for the plot of the peak intensity ratio vs the amount ratio of nonlabeled Glu to 13C6-Glu. 7949
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CONCLUSION Smectite, a synthetic inorganic polymer with a saponite structure, was subjected to MALDI MS. THAP was intercalated into cation-exchanged smectite (NaSm) layer spacing, and the complex (THAPNaSm) was used for the laser ionization of small molecules. It was found that THAPNaSm ionized only small molecules that entered the layer spacing; this was confirmed by examining different analyte/matrix preparation methods and by measuring saccharides with different molecular sizes. It was also found that NaSm was a good dispersant for THAP, and high reproducibility of the analyte peak intensity was achieved. Quantitative analysis of monosaccharides in human plasma was performed by using THAPNaSm, and the value of 8.6 ± 0.3 μg/mg was estimated for the human plasma sample used in this study.
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ASSOCIATED CONTENT
S Supporting Information *
Mass spectra with different mixing ratio; mass spectra by conventional MALDI. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01770.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone:+81-42-677-2531. Fax: +8142-677-2525. Author Contributions §
Y.D. and K.K. had equal contribution to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Y.D. acknowledges the Asian Human Resources Fund (International Student Special Selection at Tokyo Metropolitan University) from the Tokyo Metropolitan Government. T.F. acknowledges a Grant-in-Aid for Scientific Research (B) (Grant No. 15H03772) from JSPS.
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DOI: 10.1021/acs.analchem.5b01770 Anal. Chem. 2015, 87, 7944−7950
