Determination of Neutral Monosaccharides as Per-O-methylated

Anal. Chem. , 2015, 87 (21), pp 10856–10861. DOI: 10.1021/acs.analchem.5b02252. Publication Date (Web): October 7, 2015. Copyright © 2015 American ...
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Determination of Neutral Monosaccharides as Per-O-methylated Derivatives Directly from a Drop of Whole Blood by Gas Chromatography−Mass Spectrometry Ionel Ciucanu,*,† Luminiţa Pilat,† Cristian Ionuţ Ciucanu,‡ and Eugen Şişu*,‡ †

Department of Chemistry, West University of Timisoara, Strada Pestalozzi 16, RO-300115 Timisoara, Romania Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy of Timisoara, Piaţa Eftimie Murgu 2, RO-300041 Timisoara, Romania

‡

ABSTRACT: A new analytical procedure was developed for the simultaneous quantification of neutral monosaccharides from a drop of whole blood using gas chromatography−mass spectrometry analysis (GC−MS) of their per-O-methylated derivatives. The perO-methylation reaction with methyl iodide and solid sodium hydroxide in methyl sulfoxide was used for the first time for analysis of blood monosaccharides. A blood drop volume of 0.6 μL was used without special purification. The elimination of the undesirable components was carried out during methylation in the presence of a strong base and by liquid extraction of the per-O-methylated monosaccharides. The neutral monosaccharides with an anomeric center gave four per-O-methylated isomers, which were wellseparated using a capillary column. Identification was done by electron impact mass spectrometry fragmentation, retention times, and library searching. The limits of detection were determined for standards and varied from 2.0 to 2.3 ng mL−1. Recoveries for human blood samples varied from 99.22% to 99.65%. The RSD values ranged from 1.92 to 2.37. The method is fast, sensitive, reproducible, and an alternative to current methods for quantitative analysis of blood monosaccharides.

T

measurement. With an enzymatic reagent, the method is specific for one sugar, and it cannot give information about the concentration of the other sugars. The chromatographic analysis can give information about all the monosaccharides from blood. The most often used methods are gas chromatography (GC) and liquid chromatography (LC). High-performance liquid chromatography (HPLC) can be done with a great variety of columns and detectors, with their advantages and disadvantages. GC is a good alternative for analysis of sugars because the separation power is good and the retention time is relatively short. Due to their high polarity and low volatility, all sugars need to be converted into volatile and stable derivatives for GC−MS analysis. Classical derivatization methods consist of substitution of the hydroxyl groups of neutral carbohydrates in order to increase their volatility. Methyl ethers, acetates, trifluoroacetates, and trimethylsilyl ethers are the most common derivatives used for the determination of carbohydrates.8,9 Trimethylsilyl (TMS) ethers are the most widely used derivatives10 because they are appropriate for a wide range of functional groups and have a good volatility. However, the TMS ethers are sensitive to decomposition even in the presence of air moisture and some

he human body regulates blood sugar concentration, maintaining a condition of equilibrium that depends on every individual alone. The changes in plasma monosaccharide concentrations above or below the normal range are the echo of the changes in cellular sugar and indicate the presence of some diseases.1 Elevated glucose levels are present in diabetes mellitus, Cushing’s syndrome, liver disease, and hyperthyroidism, while decreased glucose levels are present in Addison’s disease, hyperinsulinism, and hypothyroidism.2 The most prevalent of these diseases is diabetes mellitus. Left untreated, the diabetes can lead to complications affecting heart, kidney, teeth, eyes, and nerves. Mannose is a metabolic product in candidasis caused by infection with species of the genus Candida. A high concentration of mannose in body fluids is an indicator of the disseminated form of the Candida infection.3 Galactose is recognized for its effect in galactosemia, which is a severe, hereditary disease resulting from the inability to metabolize galactose.4 1,5-Anhydroglucitol (1,5-AG) levels could allow differentiation between subtypes of diabetes.5 1,5AG is a marker that responds to changes in glycemia over the course of weeks that may aid in the modification of therapy.6 Fructose is a blood monosaccharide that is connected with obesity.7 Current analysis of blood sugars involves a chemical or enzymatic reaction between blood sugars and a specific reagent followed by spectroscopic, electrochemical, or chromatographic © XXXX American Chemical Society

Received: June 15, 2015 Accepted: October 7, 2015

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DOI: 10.1021/acs.analchem.5b02252 Anal. Chem. XXXX, XXX, XXX−XXX

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from JEOL (Tokyo, Japan) with AMDIS_32 software for data acquisition and processing. The GC was equipped with an injector in splitless mode and with an HP-1 fused silica capillary column (30 m × 0.25 mm i.d.) with 0.25-μm polydimethylsiloxane cross-bonded film from Hewlett-Packard (Avondale, PA). High-purity helium was used as carrier gas at a constant flow rate of 1 mL/min. The GC oven temperature was maintained at 70 °C for 3 min after sample injection, increased to 280 °C at 10 °C/min, and maintained 5 min at 280 °C. The temperature of the injector was 280 °C. The mass spectra were recorded in the positive-ion electron ionization (EI) mode over the scan range m/z 40−600. The ion chamber was set to 210 °C. The filament emission was set to 70 eV and was turned off for the first 3 min to avoid filament damage during sample analysis. The transfer line temperature was set to 280 °C. The electron multiplier voltage and automatic gain control target were set automatically. Total ion current was used for qualitative and quantitative analysis. The identification of the peaks was performed by the interpretation of the fragmentation patterns and by the comparison of the retention times and mass spectra with those of standard per-O-methylated monosaccharides. Methods. Collection of Blood Samples. The blood samples were obtained from the Clinical Laboratory of Arad County Hospital and received without patient identifier and with institutional approval. The experiments were performed in accordance with local laws, as well as instrumental regulations. The samples were collected from healthy adult male after obtaining informal consent by finger pricks technique. A volume of 24 μL of blood was collected with an HPLC syringe and was introduced into a 12 mL glass vial containing 2.4 mL of DMSO. Methylation Method with Solid Sodium Hydroxide and Methyl Iodide in Dimethyl Sulfoxide. The per-O-methylation reactions were performed at 25 °C in an electrically thermostated module using 2 mL glass vials having a V shape and with silicone septa lined screw caps. The agitation was performed with a magnetic stirrer or a vortex shaker. The procedure for the per-O-methylation of monosaccharides was modified from a standard procedure.18−20 An aliquot of collected blood sample dissolved in DMSO (0.6 mL) was introduced with an HPLC syringe into a conical glass vial with 0.4 mL of DMSO. For quantitative analysis, xylitol dissolved in DMSO was added to the sample as internal standard at a concentration of 140.00 ± 2.56 μg mL−1. The sodium hydroxide pellets were ground in a dry mortar with a pestle to obtain a fine powder. The powder was made in bulk for multiple experiments and stored in an airtight system to prevent the contact of the powder with moisture. An amount of 50−60 mg of powdered sodium hydroxide was added to the sample solution, which was vortexed at room temperature for a while to get a suspension. Methyl iodide (0.3−0.4 mL) was added with a syringe, and the mixture was stirred vigorously for 10−15 min. Methylation was performed without special drying conditions or inert gas atmosphere. In each case, the methylation reaction was quenched with a few drops of water and the reaction mixture was heated at 35−40 °C for 5 min. The per-O-methylated products were extracted three times by adding dichloromethane (1.0 mL), shaking the mixture, and separating the layers by centrifugation. The combined organic layers were washed with water (3 × 3 mL) and dried with nitrogen flow in a heating module. Once the vial was cooled, the dried residue containing the per-O-methylated sugars was

care should be done prior to analysis. Methyl ethers are the most stable derivatives in acid and alkaline media11 and have an acceptable volatility for GC. The derivatization step can be seen as a drawback, but it can be an advantage in the mass spectrometry (MS) process by improving the efficiency of the MS ionization and the mass spectra interpretation. A major problem in the application of GC to sugars is that monosaccharides generate multiple peaks because, in aqueous solutions, monosaccharides predominantly exists in two cyclic (pyranose and furanose) forms and each of them has two anomeric (α and β) forms. This may interfere in qualitative identification and in quantitative measurement of complex sugar mixtures. The most fruitful approach to diminishing the number of peaks from each sugar has been to prepare acyclic derivatives. Reduction to alditols, followed by acetylation and GC, has developed into a standard technique for aldoses.12 The reduction creates new problems. Different aldoses can yield the same alditol upon reduction. The reduction of ketoses yields a mixture of two alcohols. Other approaches that eliminate the anomeric center include the formation of aldonitrile, oxime, and O-methyloxime.9 The formation of oxime derivatives is not entirely satisfactory, because the oxime can exist as GC separable syn- and anti-isomers.13 The transformation of aldoses into acyclic aldononitrile acetates14 was used for methylated aldoses in combination with characterization by MS.15 Each aldose gives a single unique derivative, but the ketones do not give aldonitrile derivatives. The solution of the problem of multiple derivatives could be the separation of all these multiple derivatives by high-resolution capillary GC and MS with detection by selective ion monitoring (SIM).16,17 This is possible because the GC−MS instruments have been greatly improved, resulting in higher separation efficiencies of mixtures, higher sensitivity of ionization and detection, faster scan rates, and high-speed data processing. The blood is a very complex sample and the GC analysis of blood sugars requires a few sample preparation steps consisting of the separation of hemoglobin by centrifugation, deproteinization with different reagents followed by centrifugation, and derivatization.1,17 Supplementary, the derivatization step needs the drying of the samples prior to derivatization and a final cleanup of the derivatives. All these sample preparation steps need reagents and equipment and are relatively timeconsuming. This study reports a technique that is based on the per-Omethylation of neutral monosaccharides in a drop of blood without removal of hemoglobin, proteins, water, or other components from blood prior to derivatization. The process was optimized, and per-O-methylated monosaccharides were separated by capillary GC and identified by MS.

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EXPERIMENTAL SECTION Reagents and Materials. D-glucose, D-fructose, D-mannose, D-galactose, 1,5 anhydro-D-glucitol (1,5-AG), myoinositol, D-xylitol, and 1,2,3,4,6-penta-O-methyl-β-D-glucopyranose were from Sigma (St. Louis, MO). Pellets of sodium hydroxide (NaOH), methyl iodide (MeI), dimethyl sulfoxide (DMSO) with 0.2% water, chloroform, and molecular sieves were purchased from Merck (Darmstadt, Germany). DMSO was kept in a reagent glass bottle with molecular sieves to ensure dryness. Instrumentation. GC−MS analysis was performed with an HP-6890N gas chromatograph from Hewlett-Packard (Avondale, PA) coupled with a JEOL MS-600 mass spectrometer B

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Figure 1. GC−MS total ion chromatogram of the per-O-methylated neutral monosaccharides from blood. The numbered peaks are identified in Table 1. I.S. is internal standard. Chromatographic and mass spectrometric conditions are outlined in the Experimental Section.

hydroxide ion consumed in the reaction with sugar. The solubility of solid NaOH was improved by agitation and using finely crushed NaOH pellets. However, the rate of methylation did not depend on the rate of stirring. These facts indicate that eq 2 does not occur on the surface of solid NaOH by a heterogeneous pathway but rather by a homogeneous one. The term “solid-phase permethylation” used by some authors for this process22 is not adequate because the solid NaOH is partially soluble in DMSO.21 An approach to the concept of “solid-phase organic reaction”, in which the molecules of reactant are chemically bonded to polymeric beads23,24 is not also adequate, because the sugar molecules interact first with OH− groups generated by NaOH solubilized in DMSO. The amount of monosaccharides, including internal standard, is in our blood sample around 0.65 μg, which for per-O-methylation in anhydrous conditions requires 2.16 μg of NaOH. The amount of NaOH solubilized in 1 mL of DMSO is 350 μg, which is 162 times more solubilized NaOH as needed for permethylation. The equilibrium in eq 2 is pushed over to the alkoxide side if the water is eliminated from the reaction system. Complete conversion of sugar hydroxide to the corresponding alkoxide requires anhydrous conditions. These anhydrous conditions were performed with solid NaOH, which is very hygroscopic and can withhold the reaction water by adsorption or absorption (eq 3). This is an important role of the solid NaOH in this system. The blood has water, but the property of the solid NaOH to scavenge the water made possible the per-O-methylation of sugars in the presence of the trace of water in the initial sample. The amount of water that can be present in the sample without a negative influence on the per-O-methylation yield is dependent upon the presence of an additional excess of solid

redissolved in dichloromethane and an aliquot was used for GC−MS analyses. This approach eliminates the need for sample cleanup prior to GC derivatization.

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RESUTS AND DISCUSSION Sample Derivatization. The per-O-methylation of monosaccharides is based on the substitution of the proton from sugar hydroxyl with a methyl group in stepwise reactions.18 Briefly, the first step (eq 2) consists of the formation of alkoxide ions. The second step in the methylation process is the reaction of the alkoxide with methyl iodide (eq 4), producing methyl ether. The above steps are continued for each hydroxyl group from sugar in their order of reactivity until the per-Omethylation (eq 5) was achieved. NaOH(solid) + solv ⇔ Na +(solv) + OH−(solv)

(1)

R(OH)n (solv) + OH−(solv) ⇔ R(OH)n − 1O−(solv) + HOH(solv)

(2)

HOH(solv) + NaOH(solid) ⇒ HOH· NaOH(solid) + solv

(3)

R(OH)n − 1O−(solv) + MeI(solv) ⇒ R(OH)n − 1OMe(solv) + I−(solv) (4) .................................................................................................................. R(OME)n − 1O−(solv) + MeI(solv) ⇒ R(OME)n (solv) + I−(solv)

(5)

The solubility of solid NaOH in DMSO (eq 1) is low (0.35 g/L DMSO at 25 °C),21 but sufficient to replace quickly the C

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Analytical Chemistry Table 1. Partial EI Mass Spectra Data for Per-O-methylated Monosaccharides Identified by GC−MS in Figure 1 peak no.

identified monosaccharide

retention time, min 11.63

2

1,5-anhydro-Dgulcitol D-glucopyranose

3

D-fructofuranose

12.66

4

D-mannofuranose

12.78

5

D-fructofuranose

12.86

6

D-mannopyranose

13.01

7

D-fructopyranose

13.03

8

D-glucopyranose

13.10

9

D-mannopyranose

13.18

10

D-glucofuranose

13.21

11

myo-inositol

13.36

12

D-glucofuranose

13.41

13

D-fructopyranose

13.53

14

D-mannofuranose

13.62

1

12.61

m/z (% rel abundance) for per-O-methylated monosaccharide 71 (98.2), 75 (61.2), 88 (35.4), 101 (100), 111 (30.9), 115 (41.1), 125 (13.9), 129 (4.6), 143 (36.1), 157 (8.3), 158 (11.3), 175 (54.9), 188 (26.4), 220 (2.4) 71 (12.6), 73 (16.1), 75 (52.9), 88 (100), 101 (49.7), 111 (3.0), 127 (2.7), 131 (2.5), 145 (4.2), 149 (7.2), 155 (0.9), 159 (1.9), 173 (1.1), 176 (3.5), 187 (2.4), 205 (0.7) 71 (27.7), 73 (18.8), 75 (30.3), 88 (18.3), 89 (12.4), 101 (100), 111 (13.1), 115 (19.0), 119 (32.7), 131 (18.0), 141 (28.4), 145 (16.4), 173 (21.6), 187 (7.3), 205 (73.2), 219 (6.6) 71 (8.3), 73 (7.8), 75 (19.4), 88 (5.0), 89 (5.1), 101 (100), 115 (6.3), 119 (19.7), 129 (2.8), 131 (3.7), 141 (17.1), 145 (9.1), 155 (1.7), 173 (8.3), 187 3.6), 205 (36.1), 219 (2.6) 71 (8.3), 73 (6.6), 75 (18.6), 88 (3.6), 89 (6.6), 101 (100), 111 (5.3), 115 (13.3), 119 (19.2), 131 (2.6), 141 (20.2), 145 (11.3), 155 (3.1), 173 (12.9), 187 (8.3), 205 (59.3), 219 (3.1) 71 (11.1), 73 (11.4), 75 (54.5), 88 (100), 89 (14.7), 101 (53.0), 111 (2.6), 117 (3.4), 127 (3.4), 129 (1.3), 131 (3.0), 145 (4.3), 149 (7.9), 155 (1.9), 173 (1.5), 176 (3.4), 187 (3.8), 205 (1.5), 219 (1.9) 71 (23.1), 73 (32.8), 75 (38.6), 88 (100), 89 (9.3), 101 (81.8), 115 (29.5), 119 (48.8), 126 (20.3), 129 (4.7), 141 (20.5), 145 (23.1), 149 (7.7), 173 (42.6), 187 (9.3), 205 (96.0) 71 (13.9), 73 (14.6), 75 (47.2), 88 (100), 101 (57.7), 111 (9.4), 119 (6.9), 127 (7.2), 131 (4.6), 145 (6.8), 149 (10.5), 155 (4.2), 159 (3.7), 173 (3.3), 176 (8.4), 187 (10.8), 205 (5.9), 219 (0.6) 71 (8.6), 73 (11.3), 75 (51.2), 88 (100), 89 (13.5), 101 (85.2), 111 (2.0), 113 (2.6), 117 (2.7), 119 (2.4), 127 (4.7), 131 (4.7), 145 (7.0), 149 (19.8), 155 (1.0), 173 (4.3), 176 (4.6), 187 (3.3), 205 (1.3), 219 (1.0) 71 (7.0), 73 (11.4), 75 (73.9), 88 (24.8), 89 (24.5), 101 (100), 115 (4.3), 117 (15.0), 129 (12.3), 131 (2.9), 139 (2.8), 145 (13.7), 155 (12.7), 161 (34.8), 173 (7.0), 205 (0.4) 71 (4.3), 73 (24.5), 75 (61.4), 88 (56.7), 89 (5.3), 101 (100), 111 (1.0), 114 (40.9), 119 (4.0), 129 (15.0), 131 (28.1), 141 (2.0), 144 (54.6), 145 (29.4), 157 (12.1), 161 (1.3), 162 (1.9), 264 (6.4) 71 (3.3), 73 (3.4), 75 (45.5), 88 (15.7), 89 (23.8), 101 (100), 115 (2.3), 117 (15.1), 127 (2.0), 129 (4.0), 145 (21.8), 155 (2.0), 161 (31.9), 173 (3.0) 71 (19.7), 73 (51.1), 75 (82.6), 88 (100), 89 (37.7), 101 (69.8), 115 (31.4), 119 (68.5), 141 (49.4), 149 (7.2), 159 (15.0), 173 (45.7), 187 (11.5), 205 (98.4) 71 (7.2), 73 (4.7), 75 (56.8), 88 (9.4), 89 (15.3), 101 (100), 111 (2.0), 117 (16.9), 129 (3.7), 130 (5.7), 131 (4.7), 145 (13.2), 155 (1.9), 161 (16.8), 173 (7.8), 187 (3.6), 205 (5.8), 219 (1.6)

Table 2. Relative Concentration of Anomers for Three Standards of Per-O-methylated Monosaccharides monosaccarides D-glucose

D-mannose

D-fructose

% relative concentration peak 2 72.20 ± 0.83 peak 4 4.04 ± 0.27 peak 3 10.72 ± 0.41

peak 8 23.55 ± 0.57 peak 6 41.23 ± 0.73 peak 5 84.37 ± 0.73

peak 10 1.93 ± 0.21 peak 9 48.86 ± 0.82 peak 7 2.23 ± 0.19

peak 12 2.32 ± 0.22 peak 14 5.86 ± 0.25 peak 13 2.68 ± 0.18

Under anhydrous conditions, the per-O-methylation was achieved with an excess of 3 mmol of solid NaOH and 3 mmol of MeI per mmol of replaceable H.18 In the presence of a small trace of water, an additional amount of at least 4 mg of solid NaOH for each 1 μL of water must be added for a complete per-O-methylation.19,20 In our case, each sample has approximately 6 μL of blood. If we assume that this 6 μL of blood is equivalent with 6 μL of water, the amount of solid NaOH added into the vial must be at least 24 mg. Practically, we used a larger excess, because 50−60 mg of solid NaOH was added. Under the above methylation conditions, the fatty acids (free and bounded) will be transformed to their corresponding methyl esters.25 By addition of water to the reaction mixture at the end of the methylation process, the methyl esters of fatty acids and other organic acids are hydrolyzed to their corresponding anions25,26 in an irreversible reaction. This saponification process is accelerated by increasing the amount of base and by heating. The fatty acid anions are polar compounds and are not extracted with organic solvents. Under these conditions, the methyl ethers of monosaccharides were stable and were extracted with dichloromethane. GC−MS Analysis. Figure 1 shows a typical GC−MS total ion chromatogram that illustrates the separation of per-Omethylated monosaccharides from a drop of blood. The

NaOH, which should be added to the reaction vial to retain this water.19,20 The derivatization reaction for the per-O-methylation of monosaccharide standards and blood samples was performed in a vial with magnetic or vortex agitation and was compared with spin column and capillary reactors.22 It was noted that with spin and capillary reactors, the sample passed through the reactor very slowly and finally the reactor tube clogged. The clogging effect was generated by a NaOH paste as a result of the retention of water by the solid NaOH at the top of the reactor tube. In the standard technique using a vial with agitation, the excess of water is distributed uniformly on the whole amount of solid NaOH, and this side effect was avoided. Blood is a complex mixture that contains also different amounts of other compounds, such as erythrocytes, leukocytes, platelets, and plasma. Many of these blood components are precipitated in the presence of solid NaOH, making impossible the derivatization process in the spin column and capillary reactor. Some difficulties may also occur with magnetic stirring, because the rotation of the magnetic bar can be hindered when the volume of DMSO is small and the amount of NaOH is high. For this reason, a vial with vortex agitation was used further in these experiments. D

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of per-O-methylated D-mannofuranose because the base peak is at m/z at 101. Mass spectra of peaks 6 and 9 have the intensity of m/z 88 being higher than that of m/z 101, showing the presence of α and β anomers of per-O-methylated Dmannopyranose. Peak 6 is the α anomer because the intesity of m/z 187 is higher than that of m/z 176, and peak 9 is β anomer because the intensity of m/z 176 is higher. Peaks 3, 5, 7, and 13 were assigned to per-O-methylated D-fructose due to the intense characteristic fragments in the mass spectra at m/z 205, 173, 141, and 119. From the relative intensities of m/z 101 and 88, peaks 3 and 5 are generated by α and β anomers of furanose rings and peaks 7 and 13 by α and β anomers of pyranose rings. Peak 11 was assigned to per-O-methylated-myoinositol and was identified by mass spectra due to its molecular ion at m/z 264 and characteristic fragments at m/z 201 and 200. The fragment at m/z 201 is formed from the molecular ion by losses of CH3−OH and •OCH3 and the fragment at m/z 200 by elimination of two methanol molecules. Quantitative evaluation of the proposed method was carried out by the internal standard method using xylitol. In the case of monosaccharides with four isomers, the correlation between concentration and peak area was done using the peak area of the major isomer and its relative contribution (Table 2) to the total area of the evaluated monosaccharide. Using this constant ratio between isomers, the quantitative evaluation was improved for small peaks and overlapped peaks. The linearity, the limit of detection (LOD), the limit of quantification (LOQ), and the precision of the MS detector were calculated for all monosaccharides. The calibration curves and the test of linearity were done by plotting the peak area of the major peak versus the concentration of the standard monosaccharides that were per-O-methylated under optimal conditions described above and then analyzed by GC−MS. Linearity ranging between 0.9982 and 0.9991 was very good up to a concentration of 1 μg mL−1 for each analyte. The repeatability of the calibration curves expressed as the relative standard deviation (RSD) for interday analysis of four replicates ranged from 1.46 to 1.98%. LOD and LOQ depend first on the sensitivity of the MS detector and were experimentally determined from the injection of progressive dilutions of each standard of per-O-methylated monosaccharide until the signal-to-noise ratio for any major peak of monosaccharide reached a value of 3 for LOD and 10 for LOQ.30 The detection limit and limit of quantification (expressed in ng mL−1) for perO-methylated monosaccharides were 2.1 and 7.0 for D-glucose, 2.1 and 7.0 for D-mannose, 2.3 and 7.6 for D-fructose, 2.2 and 7.3 for 1,5-anhydro-D-glucitol, and 2.0 and 6.6 for myo-inositol. The accuracy of the method was estimated by recoveries calculated as the ratio of the spiked amount of monosaccharide in the blood sample to the found amount of monosaccharide by GC−MS analysis. The average recovery values (n = 4) summarized in Table 3 varied from 99.22 to 99.67% and certify a very good accuracy of the method. The RSD values ranging from 1.92 to 2.37 and having generally higher values at lower concentrations show the good reproducibility of the method. The monosaccharide concentrations (μg mL−1) in the blood of healthy male are 890.42 ± 20.56 for glucose, 28.85 ± 0.66 for mannose, 7.34 ± 0.17 for fructose, 19.54 ± 0.38 for 1,5-anhydro-D-glucitol, and 8.05 ± 0.16 for myo-inositol. The results are expressed as mean ± standard deviation.

baseline of the chromatogram is stable and the shape of the peaks is very good. The chromatogram peaks generated by perO-methylation of monosaccharides were identified primarily from EI mass spectra, which are summarized in Table 1. All identifications were verified by the comparison of the retention time of the sample peaks with those of standard per-Omethylated monosaccharides and by library searching. Selective ion monitoring was very useful for identification of the minor peaks. The per-O-methylation of the standard monosaccharides with hemiacetal and hemiketal groups gave four isomers generated by cyclic forms with five and six atoms and by the presence of anomeric centers. These isomers were wellseparated and that is the reason why there are more peaks for one compound in the chromatogram. It was noticed that each monosaccharides with anomeric center gave two relative major peaks and two minor peaks. The relative proportions of these four peaks depend first on the type of solvent, on the alkalinity of the solution, and on the temperature.27 Table 2 gives the relative concentration of anomers for standards of perO-methylated glucose, mannose, and fructose. With DMSO as solvent and with a high excess of NaOH at room temperature, the ratio of anomers had a very good reproducibility and can be also used for identification. myo-Inositol and 1,5-anhydroglucitol have no anomeric center and gave only one peak. Disaccharides can also be per-O-methylated and can be detected by this method, but in our samples, their amount was not enough for identification. The mass spectra of the per-O-methylated monosaccharides investigated are very similar and contain fragment ions at the same m/z value, but their different intensities allow identification of the compounds. Peaks in the high mass range allow the nature of the monosaccharides to be established. The recognition of the monosaccharides’ ring size can be done as a function of the relative intensities of fragments m/z 101 and 88. Furanose spectra are characterized by an intense peak at m/z 101 and pyranose at m/z 88.28,29 The use of mass spectra to determine anomers in these compounds may be subject to ambiguity, because their mass spectra are very similar. However, the different ratio between ions of m/z 187 and 176 for pyranose hemiacetals can provide a reasonable prediction of the conformation.28 The identity of these isomers in the chromatogram peaks was also determined by comparison with mass spectra of standard isomers, where possible. Peak 1 was assigned to per-O-methylated 1,5-anhydro-D-glucitol and was easily identified by mass spectra because of the molecular ion at m/z 220 and characteristic peaks at m/z 188 by elimination of methanol and at m/z 175 by loss of •CH2− OCH3. The ion m/z 175 gave further m/z 143 and 111 by successive losses of methanol molecules. Peaks 2, 8, 10, and 12 were assigned to per-O-methylated D-glucose. Mass spectra of peaks 2 and 8 have m/z 88 with higher intensity than m/z 101 and consequently are α and β anomers of pyranose forms. Peak 2 was identified as β anomer by comparison with the retention time of per-O-methyl-β-D-glucopyranose and from the intensity of m/z 187 being higher than that of m/z 176. Consequently, peak 8, with the intensity of m/z 187 being less than that of m/ z 176, is corresponding to α anomer. Peaks 10 and 12 were assigned to α and β anomers of furanose ring because they have a base peak at m/z 100 and a characteristic peak at m/z 161. Peaks 4, 6, 9, and 14 were assigned to per-O-methylated Dmannose. They were mainly identified by retention time, relative intensity of the peaks, and comparison of mass spectra of standards. Peaks 4 and 14 are generated by α and β anomers E

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Article

Analytical Chemistry

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Table 3. Recovery of Monosaccharides by Analyzing Spiked Blood Samples monosaccharide

amount added (μg mL−1)

amount found (μg mL−1)

recovery (%)

RDS (%)

0 100.56 0 20.25 0 40.57 0

890.42 990.98 28.85 48.72 7.34 47.56 19.54

− 99.65 − 99.22 − 99.27 −

2.31 2.32 2.29 2.26 2.37 2.33 1.98

30.18 0 40.89

49.40 8.05 48.94

99.35 − 99.30

1.93 1.99 1.92

D-glucose

D-mannose

D-fructose

1,5-anhydro-Dglucitol myo-inositol

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CONCLUSION The results of this work show that the neutral monosaccharides can be simultaneously analyzed by GC−MS as per-Omethylated derivatives directly from a drop of blood. The per-O-methylation was performed with methyl iodide and solid sodium hydroxide in DMSO from a drop of whole blood without purification steps of the blood. Erythrocytes, leukocytes, platelets, and some plasma elements were eliminated during the methylation and extraction of the perO-methylated monosaccharides. The developed method allows the determination of low concentrations of monosaccharides in the presence of a high concentration of glucose. The limits of detection are in the same range with other chromatographic methods using MS as detector. Quantitative evaluation can be done both with calibration curves and internal standard method. The time required for sample preparation and for GC−MS analysis is 35−40 min for one blood sample and is less than other similar GC or LC analysis of blood monosaccharides. Taking into account the duration necessary for analysis of these five monosaccharides by commercial enzymatic analysis, the method is proven to be very fast and highly reproducible. The method represents an alternative to current methods for quantitative analysis of blood monosaccharides from both adults and children.

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AUTHOR INFORMATION

Corresponding Authors

*I.C.: e-mail, [email protected]; phone, +40-256-592636; fax, +40-256-592620. *E.S.: e-mail,[email protected]; phone/fax, +40-256-220482. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Romanian National Authority for Scientific Research (CNCS-UEFISCDI) through project PN-II-PCCA-2011-142. Part of research was done at the Center of Genomic Medicine of the “Victor Babes” University of Medicine and Pharmacy of Timisoara, POSCCE 185/48749, contract 677/09.04.2015. GC−MS analysis by Tsukuba University (Tsukuba, Japan), Department of Biochemistry, is gratefully acknowledged and was supported by the Japan Society for the Promotion of Science.

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REFERENCES

(1) Pitkänen, E. Clin. Chim. Acta 1996, 251, 91−103. F

DOI: 10.1021/acs.analchem.5b02252 Anal. Chem. XXXX, XXX, XXX−XXX