Mass Spectrometry Approach for Differentiation of Positional Isomers of Saccharides: Toward Direct Analysis of Rare Sugars

Abstract

Rare sugars have gained popularity in recent years due to their use in anti-aging treatments, their ability to sweeten with few calories, and their ability to heal infections. Rare sugars are found in small quantities in nature, and they exist typically as isomeric forms of traditional sugars, rendering some challenges in their isolation, synthesis, and characterization. In this work, we present the first direct mass spectrometric approach for differentiating structural isomers of sucrose that differ only by their glycosidic linkages. The method employed a non-contact nano-electrospray (nESI) platform capable of analyzing minuscule volumes (5 μL) of saccharides via the formation of halide adducts ([M+X]⁻; X = Cl and Br). Tandem mass spectrometry analysis of the five structural isomers of sucrose afforded diagnostic fragment ions that can be used to distinguish each isomer. Detailed mechanisms showcasing the distinct fragmentation pattern for each isomer are discussed. The method was applied to characterize and confirm the presence of all five selected rare sugars in raw honey complex samples. Aside from the five natural alpha isomers of sucrose, the method was also suitable for differentiating some beta isomers of the same glycosidic linkages, provided the monomeric sugar units are different. The halide adduct formation via the non-contact nESI source was also proven to be effective for oligosaccharides such as raffinose, β-cyclodextrin, and maltoheptaose. The results from this study encourage the future development of methods, which function with simple operation to enable straightforward characterization of small quantities of rare sugars.

Graphical Abstract:

INTRODUCTION

Saccharides are one of the most important, abundant, and heterogeneous biological molecules. Their roles in biological systems range from being major sources of energy, providing structural solidity as well as their roles in cell membrane. Saccharides also find several applications in pharmaceutical, textile, food, and chemical industries^1–4. In addition, there has recently been an increased interest in “rare” sugars regarding their large-scale synthesis, characterization, and application^5–8. That is, although there are abundant natural sources for commonly used sugars (e.g., D-glucose, D-fructose, and sucrose, just to name a few),^5,9,10 most other sugars are naturally rare, of which there are hundreds of them. Due to their limited natural sources, the rare sugars must be prepared from their biomass precursors through multistep chemical or enzymatic syntheses.

The recent interest in rare sugars is rekindled by their observed superior biological activity, which has been demonstrated in a wide range of areas including their use in anti-aging,^11–13 low-caloric sweeteners,^7,14,15 and non-steroidal anti-inflammatory drugs for arthritis and renal inflammatory disorders.¹⁶ Often, the synthetic methods to prepare some rare sugars simply involve an isomerization process,^5–8 thus making their characterization to be very challenging. Take sucrose as an example. Sucrose has five structural isomers with distinct glycosidic linkages (Scheme 1): trehalulose, turanose, maltulose, leucrose, and palatinose. While excessive sucrose intake is associated with health problems, such as dental caries, obesity, type 2 diabetes, and cardiovascular disease, the sucrose isomers are nutritional sugars with about 50% sweetness of sucrose, and do not cause gastrointestinal discomfort and aftertaste^17–21. Some of these isomers (e.g., turanose and leucrose) can inhibit lipid accumulation, something that is good for obesity control^22,23. Similarly, palatinose is less hygroscopic, less soluble, and more stable to acidity than sucrose, and has non-cariogenic, low glycemic index properties, and reduced insulinemic response¹⁹. These nutritional and medicinal benefits of isomers of sucrose make their fermentation chemistry and structural characterization to be important.

Scheme 1:

Structures of positional isomers of sucrose

Among the many methods employed for characterization of saccharides, nuclear magnetic resonance (NMR) spectroscopy has been shown to be efficient for structural elucidation and isomer differentiation but requires relatively larger quantities of sample²⁴. Mass spectrometry (MS) has proven to be an important technique for saccharide analysis^{1–4, 24–34}. But major challenges persist for MS analysis of saccharide isomers. For example, the highly hydrophilic nature and less surface activity present oligosaccharides with reduced ionization efficiency when analyzed by electrospray ionization (ESI) MS³⁵. The structural complexity of saccharides resulting in linkage, position, and stereo isomers make it insufficient to use MS alone for their characterization, and thus requiring additional complicated instrument modifications. While several studies have reported the use of various ionization methods for sugar analysis by MS^1,35,36, very few experiments involve direct isomer characterization without ion mobility spectrometry (IMS) or a front-end chromatography^36–42. Other analytical procedures utilize chemical derivatization^43,44. While ensuing reactions provide enhanced sensitivity, they rarely afford opportunities for positional isomer differentiation without IMS (Scheme 2). The ability to sensitively analyze and differentiate positional isomers of saccharides using collision-induced dissociation (CID) tandem MS (MS/MS), which is readily available on almost all commercial mass spectrometers, has potential to transform rare sugar characterization.

Scheme 2:

Comparison of this work (negative-ion mode MS/MS analysis) with commonly used analytical approaches (positive- and negative-ion mode IMS-MS and IMS-MS/MS) for saccharide analysis.

Herein, we present the direct differentiation of structural isomers of sucrose via negative-ion mode ESI MS/MS enabled by halide (chloride and bromide) anion attachment. The negative-ion mode adduct formation was achieved using a non-contact nano-electrospray ionization (nESI) process⁴⁵, which uses minuscule volumes of analyte solution (down to picoliter quantities)^46,47 while also minimizing joule heating⁴⁸ that is common in traditional contact-mode nESI. It is known that chloride anions (Cl⁻) can be generated from a reaction between a base (e.g., alkoxide from alcohol) and alkyl halide (e.g., chloroform)^4,33. When performed in the presence of saccharide analytes, the released Cl⁻ anion can be picked up by the saccharide, resulting in the detecting of the sugar in negative-ion mode. In this regard, Jiang and Cole investigated different anion species for oligosaccharide analysis using negative mode MS. It was reported that Cl⁻ anion provides the stable adduct-related product ion³⁵. We report, for the first time, that when subjected to collisional activation in MS/MS, the sugar-chloride adduct anion dissociates to give distinct fragment ions for each structural isomer of sucrose. We provide detailed fragmentation pathways that lead to the formation of diagnostic ions for each isomer. Importantly, we report the application of the chloride anion attachment for complex mixture analysis of honey samples, which confirmed the presence of all known isomers of sucrose in four different honey samples. Other oligosaccharides such as raffinose (trisaccharide), β-cyclodextrin (cyclic heptasaccharide), and maltoheptaose (linear heptasaccharide) were also sensitively analyzed.

EXPERIMENTAL METHOD

Non-Contact nESI Apparatus.

Non-contact nESI was used to deliver all sample solution toward the mass spectrometer and consists of a silver (Ag) metal electrode and a pulled glass capillary prepared from a disposable borosilicate glass (I.D; 1.17 mm, O.D; 1.5 mm) using a micropipette puller (Model P-97, Sutter Instrument Co., Novato. CA, USA). In the non-contact spray mode (Figure 1a), the sample solution (5 μL of methanol/chloroform, 3:1 v/v) present in the pulled glass capillary is not in physical contact with the Ag electrode. There is an air gap between the Ag electrode and analyte solution. The application of 1.5 kV direct current (DC) voltage to the Ag electrode induces electric field, which charges the analyte solution electrostatically, causing the release of charged microdroplets from the tip of the pulled glass capillary. As illustrated in Figure 1a, the nESI setup is positioned in front of the mass spectrometer inlet. Thus, the generated charged droplets containing the sugar-chloride adduct is directly transferred to the proximal mass spectrometer for subsequent analysis in the negative-ion mode. Because physical contact with the analyte solution is not required during nESI-MS analysis, very small volumes of sample are needed. All analyses were performed using the non-contact nESI platform unless stated otherwise.

Figure 1:

a) Schematic representation of non-contact nESI setup. Negative-ion mode mass spectra showing b) chloride ions formed from a solution of methanol and chloroform mixture, c) pure methanol, analyzed in the absence of chloroform, and d) non-contact nESI MS mass spectrum showing chloride adduct of leucrose, after background subtraction.

Mass spectrometry.

Mass spectra data were collected using a Thermo Fisher Scientific Velos Pro ion trap mass spectrometer at a full MS range (San Jose, CA, USA). The MS parameters used were: 250 °C inlet capillary temperature, 3 microscans, and 100 ms ion injection time. Spectra were recorded for at least 30 s. The distance between the nESI tip and the MS inlet was kept constant at 5 mm. Collecting and processing of data were achieved using the Xcalibur 2.2 SP1 software (Thermo Fisher Scientific). Isomer identification and characterization were done utilizing tandem MS with collision-induced dissociation (CID) at 30% (manufacturer’s unit) and 1.5 Th (mass/charge units) for isolation window of normalized collision energy.

Chemicals and Reagents.

Sucrose, turanose, maltulose, leucrose, and palatinose were purchased from Sigma-Aldrich (St. Louis, MO). Isomaltose, maltose, gentiobiose, cellobiose, and trehalose were purchased from VWR International (Radnor, PA). Chloroform and methanol were purchased from Acros organics (New Jersey, USA) and Sigma-Aldrich (St. Louis, MO), respectively.

Preparation of honey samples.

Four US grade A honey samples were obtained from Nature Nate’s Natural 100% raw unfiltered honey, McKinney, TX (honey sample 1), raw honey from Union County, OH (honey sample 2), Kirkland’s Signature Organic Raw Honey, Seattle, WA (honey sample 3), and Honeyrun Farm, Raw Ohio Buckwheat honey, Williamsport, OH (honey sample 4). 50 mg/mL of honey samples was prepared by diluting 500 mg of each honey sample in water. 5 mL of each diluted sample was centrifuged using a Microsep^™ Advance 3k MW cutoff Centrifugal Filter (Pall Corp., Ann Arbor, MI) at ~3000 rpm for 1 hour. 250 uL of each of the filtered honey samples in 100% water was mixed with 3:1 methanol/chloroform solution and analyzed.

RESULTS AND DISCUSSION

Process Optimization.

The non-contact nESI approach (Figure 1a) was chosen for this work because it can withstand higher spray voltages without damaging the tip of the glass capillary. The ability to use high spray voltage is necessary for our long-term goal to combine the nESI setup with atmospheric pressure chemical ionization (APCI) for analysis of a variety of structurally different compounds. In the current study, we found that higher voltage is also beneficial for sugar-chloride adduct formation (Figure S1), where increased signal was observed with increasing spray voltage up to 3 kV (maxima) after which the signal dropped. We have previously shown that the spray voltage in non-contact nESI can be further increased to as high as 6 kV without damaging the glass tip when it is combined with APCI. This extra tip stability is due to the well-known cooling effects of corona discharge in the APCI process⁴⁵. In the current work, the maximum signal in non-contact nESI was >2X higher than the traditional nESI operated under optimized spray voltage of 2 kV. Although previous reports indicated plasma discharge is needed for Cl⁻ generation from methanol/chloroform solution⁴, we observed otherwise. We employed the non-contact nESI approach operated at −1.5 kV (in the absence of APCI) to generate Cl⁻ ions in high abundance.

Anion attachment (including halides) is a general mechanism for the formation of negative ions. In the presence of molecules with polar functional groups (e.g., OH in sugars), formation of adducts is highly favorable. The adduct can lead to the formation of [M-H]⁻ where proton is transferred from the neutral (or acidic) molecule to the anion. In this work, we chose to use halide anions where stable adduct is known to form³⁵; Cl⁻ and Br⁻ are common anions because of their diagnostic 3:1 and 1:1 isotopic peak intensities for the [M]⁻ and the [M+2]⁻, respectively, that can be used for easy identification. For example, Figure 1b represent the mass spectrum recorded for a blank methanol/chloroform solution (3:1, v/v), showing the expected diagnostic ion peak of 3:1 isotope ratio at m/z 35 and 37 for ³⁵Cl:³⁷Cl and clearly indicating the presence of chloride anions in solution. As stated above, the −1.5 kV used here does not induce a plasma, and yet high intensity Cl⁻ ions were observed. Further experiments utilizing a non-electrical mode of electrospray via a gas-assisted process (no voltage applied, Figure S2) also showed the presence of Cl⁻ anions (albeit with lower intensity, Figure S3). This experiment confirms that the Cl⁻ anions are generated in solution upon mixing methanol and chloroform, with minimal influence from the spray voltage used in the non-contact nESI spray mode. To confirm that the species at m/z 35 and 37 are due to chloride isotopes, we performed additional experiment where only methanol was sprayed at −1.5 kV using the non-contact nESI setup. The result of this experiment is shown in Figure 1c in which the peaks at m/z 35 and 37 disappeared due to the absence of chloroform.

Analysis of Sucrose Isomers.

Following the characterization of the non-contact nESI setup, the methanol/chloroform solution was used as solvent for the direct analysis and characterization of isomers of sucrose. We began the analysis with sucrose (100 μM) via Cl⁻ adduct formation in the negative-ion mode non-contact nESI-MS. We anticipated that the Cl⁻ ions generated in solution will interact with the hydroxyl group of the sucrose to form an adduct. This expectation was met in which m/z 377 peak [M+Cl]⁻ was registered in high abundance. This peak corresponds to the reaction between sucrose (MW 342 Da) and chloride anion in the negative-ion mode. We extended the analysis to structural isomers of sucrose, namely turanose α(1→3), maltulose α(1→4), leucrose α(1→5), and palatinose α(1→6). Figure 1d represents the full MS recorded when a mixture of leucrose and methanol/chloroform solution was analyzed. This also registered a high intensity peak at m/z 377, which indicates the formation of leucrose (MW 342 Da)-chloride (MW 35 Da) adduct. Full MS of all other isomers produced the most abundant peak at m/z 377 (Figure S4), as well as the isotopic peak at m/z 379 due to the adduction of ³⁷Cl anion as illustrated in Figure 1d. The full MS spectra also registered a minor peak at m/z 341, which represents the deprotonated sugar (MW 341 Da). We found that the [M−H]⁻ peak does not provide higher sensitivity (about 10X less sensitive) for characterization as compared to the m/z 377 chloride-adduct peak. The sugars also formed chloride-bound dimers [2M+Cl]⁻ during electrospray, which registered a signal at m/z 719. This was confirmed by tandem MS analysis (Figure 2f). Of the five sucrose isomers, the data for trehalulose is not presented here because of delays in the synthesis of this isomer.

Figure 2.

Negative-ion mode tandem MS analysis of a) Leucrose, b) Sucrose, c) Palatinose, d) Maltulose, and e) Turanose, all at m/z 377. f) Tandem MS of ion at m/z 719 confirming the formation of chloride-bound dimers of the disaccharides. As shown, some regions of the spectra were amplified 5X.

To differentiate the structural isomers of sucrose, we performed CID MS/MS on the m/z 377 [M+Cl]⁻ species. We observed both cross-ring and glycosidic bond breakages during these MS/MS experiments and in the process, we identified diagnostic fragments for each isomer. For example, chloride adducts of leucrose (m/z 377) fragmented in CID to give a relatively abundant diagnostic ion at m/z 233 (Figure 2a), which was not observed for any of the other sucrose isomers. The chloride adduct of the leucrose first loses HCl (MW 37 Da) to form a deprotonated leucrose (M−H)⁻ at m/z 341. The deprotonated leucrose subsequently undergoes cross-ring breakage to lose C₃H₆O₃ and H₂O to afford a diagnostic ion at m/z 233, as illustrated in Scheme S5.

Tandem MS analysis of palatinose and maltulose also showed cross-ring breakages. That is, the chloride adduct of palatinose α(1→6 linkage) lost neutral HCl to form a deprotonated palatinose (m/z 341). Subsequent fragmentation led to cross-ring breakage to produce a diagnostic ion at m/z 221 due to loss of two molecules of C₂H₄O₂ (Figure 2c). Similar fragmentation patterns has been reported by other α(1→6) linked disaccharides²⁷, leading to subsequent neutral losses with masses 60 (C₂H₄O₂), 90 (C₃H₆O₃), and 120 Da (2C₂H₄O₂). MS/MS of maltulose α(1→4 linkage) produced a diagnostic ion at m/z 263 (Figure 2d), which was not found in any of the other isomers. This product ion resulted from loss of C₂H₄O₂ and H₂O from the deprotonated maltulose. Similar observation has also been reported earlier where α(1→4) linked disaccharides like maltulose, which lost neutral species with masses 60 and 90 Da. Different from leucrose, palatinose, and maltulose, sucrose fragments via glycosidic bond cleavage. This lack of cross-ring breakage has been reported to occur because the anomeric oxygen which enable ring opening via deprotonation is blocked²⁷. Hence, chloride adducts of sucrose (m/z 377) fragmented in CID via glycosidic bond cleavage to yield high abundant ion at m/z 215 with subsequent loss of H₂O to give a diagnostic ion at m/z 197. While m/z 215 was found in the fragmentation pattern of leucrose, only sucrose was found to undergo subsequent water loss to produce a distinct ion at m/z 197, which was not found in the fragmentation pattern of the other isomers. The tandem MS of turanose was found to follow similar fragmentation pattern as leucrose but produce a distinct diagnostic ion at m/z 203 (Figure 2e). The different relative ion intensities recorded during tandem MS of different isomers may be related to the structural stability of the different fragment ions. The detail dissociation pathways for each isomer have been summarized in Schemes S6 – S9.

Thus, the current study represents the first systematic comparison of MS/MS fragmentation patterns of five disaccharides isomers where unique diagnostic fragment ions are found to be associated with each isomer. Therefore, without any prior chromatographic separation, our data demonstrate, and for the first time, that structural isomers of sucrose can be characterized in direct infusion MS experiments using only 5 μL sample. In addition to Cl⁻ adduct formation, we also studied Br⁻ adduction using methanol/bromoform solution. We found that the formation of the bromide adduction occurs competitively with Cl⁻ adduction when using bromoform (Figure S10). This systematic appearance of Cl⁻ adducts when using bromoform might be related to trace impurities derived from the synthesis of bromoform after treatment of chloroform with aluminum tribromide. The competitive Cl⁻ adduct formation can be undesirable since it tends to distribute the analytical signal into multiple peaks. Because of this, most experiments were performed using methanol/chloroform where only the Cl⁻ adducts of the saccharides were detected. Nevertheless, the fragmentation of Br⁻ adducts of the isomers in MS/MS gave higher intensity of diagnostic ions as summarized in Figure S11.

Analysis of Mixtures Prepared from Standard Isomers.

To further explore the capabilities of the chloride adduct formation based on non-contact nESI MS platform for sugars analysis, a mixture of the five structural isomers was analyzed. That is, leucrose, maltulose, palatinose, sucrose, and turanose were mixed into methanol/chloroform solution at 100 μM and the presence of each isomer was confirmed without any difficulty. Figures 3a and b represent the full MS and tandem MS at m/z 377, respectively, during the analysis of the five-component sugar-mix. The tandem MS clearly shows distinct product ions from each sugar where m/z 233, 263, 221,197, and 203 are attributed to leucrose, maltulose, palatinose, sucrose, and turanose, respectively. To ensure that scrambling does not contribute to the observed diagnostic fragment ions, the isomers were mixed in pairs and similarly analyzed. That is, two-component mixtures consisting of leucrose and turanose, sucrose and leucrose, sucrose and turanose, and palatinose and turanose were analyzed separately. In each case, only the expected diagnostic fragments ions for each isomer were observed (Figure S12). No trace of the excluded isomers was detected in the two-component mixtures.

Figure 3.

a) Negative-ion mode non-contact nESI MS mass spectrum showing chloride adduct with standard mixture of leucrose, maltulose, palatinose, sucrose and turanose with background subtraction. b) Tandem MS analysis of m/z 377 derived from the standard mix of the isomers. c) Negative-ion mode mass spectrum showing chloride adduct with sugars in honey Sample 1. d) Tandem MS analysis of m/z 377 derived from complex honey Sample 1. Some regions are amplified 10X and 5X.

Analysis of Complex Honey Samples.

Following these remarkable results, complex mixtures of honey were analyzed using the non-contact nESI MS approach, with minimal sample preparation. Honey is known to be rich in monosaccharides such as glucose and fructose (about 75%). It also contains disaccharides such as sucrose, palatinose, maltulose, turanose (about 10–15%)^49–53, among others. For this experiment, honey samples from four different sources were analyzed. Figures 3c and d show the full MS and tandem MS of honey Sample 1, respectively, analyzed in negative-ion mode. The full MS recorded a peak at m/z 377 indicating the formation of disaccharide (MW 342 Da)-chloride (MW 35 Da) adduct. The full MS of all of the other honey samples registered the peak at m/z 377 as shown in Figure S13a. Tandem MS analysis shows fragmentation pattern that confirms the presence of the disaccharide sucrose (diagnostic ion m/z 197), leucrose (diagnostic ion m/z 233), isomaltose (diagnostic ion m/z 269), and turanose (diagnostic ion m/z 203) in all four honey samples (Figure S13b).

The full MS spectra for the honey samples also showed the presence of other peaks, the most abundant of which occurred at m/z 215. This peak (M+Cl)⁻ matches the monosaccharide constituents of honey, which has been reported to be primarily glucose and fructose (MW 180 Da)^51,52. To confirm this assignment, standards of glucose and fructose were purchased, and solutions were prepared in methanol-chloroform (3:1, v/v) mixture. Tandem MS analyses of the standard monosaccharides were compared with MS/MS of m/z 215 detected from the complex honey samples, which revealed high probability that the glucose component in honey formed a stable adduct with Cl⁻ ions (Figure S14). Based on MS/MS data the presence of fructose cannot be excluded although its confirmation will require additional information, for example chromatographic retention time data. It should be noted that other peaks were detected in some honey samples that were not present in others. For example, intense peaks were observed at m/z 195 and 191 for honey samples 2 and 3. Although these species were not characterized, they might be unique to the specific honey brand.

Analysis of Other Selected Disaccharide Isomers.

The method was extended to the analysis of other closely related disaccharides (Scheme 3) namely, isomaltose α(1→6), maltose α(1→4), gentiobiose β(1→6), and cellobiose β(1→4). The full mass spectra of all these disaccharides (MW 342 Da) recorded high intensity peaks for [M+Cl]⁻ at m/z 377 confirming the formation of adducts (data not shown). To investigate their fragmentation patterns, tandem MS analysis was performed on chloride adducts (Figure S15a) of each selected disaccharide. Tandem MS analysis revealed that the chloride adduct first undergoes deprotonation to form m/z 341, followed by cross-ring breakages to produce diagnostic ions. For example, isomaltose was found to produce a diagnostic ion at m/z 269, resulting from the loss of C₂H₃O and H₂CO from its deprotonated fragment (Figure S14a).

Scheme 3:

Structures of other selected disaccharides isomers: isomaltose, maltose, gentiobiose, and cellobiose

In a similar fashion, maltose fragmented to produce m/z 221 through the loss of C₂H₄O₂, H₂O, and C₂H₂O (Figure S15b). Likewise, gentiobiose and cellobiose were found to fragment to produce m/z 237 and 333, respectively. The product ion at m/z 237 was due to loss of C₂H₄O₂ and CO₂ from the deprotonated fragment from gentiobiose (Figure S15c) whereas product ion at m/z 333 resulted from loss of CO₂ from the adduct (Figure S15d). We also analyzed trehalose α (1→1), which is a non-reducing disaccharide like sucrose, but fragments to produce two diagnostic ions at m/z 297, resulting from the loss of C₃H₈O₃ from m/z 333 (Figure S14e). Sucrose α(1→2) did not produce the m/z 297 and 333 fragment ions, instead yielding clean ions at m/z 197 and 215 (Figure S16).

Importantly, notice that gentiobiose β(1→6) differs from palatinose α(1→6) not only from the orientation of the glycosidic bond, but also their monomeric sugar units are different. Gentiobiose is composed of two glucose units whereas palatinose is made up of glucose and fructose. Thus, our method was able to differentiate them with ease, with gentiobiose showing diagnostic ion at m/z 237 and palatinose affording m/z 221 as the diagnostic ion (Figure S17). Isomaltose α(1→6) with two glucose monomeric units produced diagnostic ion at m/z 269, allowing it to be differentiated from gentiobiose and palatinose (Figure S18). Similarly, maltose and maltulose are both α(1→4) but they differ in that maltose has two glucose units while maltulose has glucose and fructose units. Thus, they can be differentiated by careful observation of MS/MS patterns of the chloride adducts, with maltose and maltulose yielding diagnostic ions at m/z 221 and 263, respectively (Figure S19). On the other hand, cellobiose β(1→4) and maltose α(1→4) did not produced distinct diagnostic ions since they both have two unit of glucose that differ only by the orientation of the glycosidic linkage (Figure S20). Though cellobiose produces diagnostic ion at m/z 333 that allows it to be differentiated from maltose, effective differentiation of maltose from cellobiose will require a front-end separation. We also believe that complete identification and isomer characterization could be boosted by the development of a computer program/software that will utilize the entire fragmentation pattern (holistically, including differences in intensities of similar fragment ions) for complete identification, in addition to the use of unique diagnostic ions. Similarly, maltose and palatinose share the ion at m/z 221, but maltose produce two additional peaks (m/z 185 and 263) can distinguishes it from palatinose. It is important to recognize that in some applications separation does not need to happen online. For example, the rare sugars, which are the focus of the current study, are often synthesize in solution, followed NMR characterized of the purified sample. But NMR typically requires milligram quantities of material. The MS-based method described in this work can use similarly purified samples, but with much reduced quantities (microgram). The simplicity of current method makes it highly significant as it uses halide adducts and CID MS/MS ― readily available on almost all commercial mass spectrometers ― to achieve disaccharide isomer differentiation. Even as the direct infusion work is novel, we believe will further provide the framework work to characterize rare sugars on LC-MS, especially for co-eluted species.

Positive-Ion Mode Analysis.

Positive-ion mode MS has been widely used to analyze sugars via sodium or potassium adduct formation. We analyzed sucrose and its structural isomers via positive-ion mode non-contact nESI MS. We found that the sodium adduct (M+Na)⁺ was the most favorable positive ion. Even here, the sugar with most intense (M+Na)⁺ ion was about 10X less sensitive than the detection of (M+Cl)⁻ species (Figure S21). Importantly, the tandem MS analyses of the (M+Na)⁺ species at m/z 365 for the five sucrose isomers did not yield obvious diagnostic fragments for each isomer (Figure S22). It might be possible to use the overall MS/MS profiles for identification although this task might require software development.

Analysis of Higher-Order Oligosaccharide.

Lastly, we expanded the applicability of the non-contact nESI method from monosaccharides and disaccharides to oligosaccharides. In this case, we selected raffinose, β-cyclodextrin, and maltoheptaose for analysis. Raffinose is a non-reducing trisaccharide sugar consist of galactose linked to sucrose via the glucose moiety in α(1→6) glycosidic linkage. In the negative ion mode MS, raffinose (MW 504 Da) forms an adduct with Cl⁻ ions to produce m/z 539 (Figure S23). The adduct undergoes competitive fragmentations upon CID to give a highly intense but less informative product ion at m/z 503 due to the loss of HCl and an informative minor peak at m/z 377 via the release of a hexose moiety (C₆H₁₀O₅; MW 162 Da). The major fragment at m/z 503 underwent subsequent dissociation to give minor peaks at m/z 341 and 179 via the two sequential losses of a hexose moiety (C₆H₁₀O₅).

β-Cyclodextrin is a cyclic heptasaccharide, which finds many applications in food, biotechnology, pharmacy, textiles, medicine, and cosmetics^56,57. β-Cyclodextrin (MW 1135 Da) is composed of seven glucose subunits joined together by α(1→4) glycosidic bonds. The non-contact nESI analysis of β-cyclodextrin in methanol/chloroform solution produced a peak at m/z 1170 formed by its adduction with chloride. Tandem MS of the precursor ion gave only one product ion at m/z 1134 resulting from the loss of HCl (Figure S24) with no possibility to identify the sequence of the sugar. Thus, multiple stage tandem MS (MS³) was performed on fragment m/z 1134, which subsequently dissociated to ions at m/z 1014 and 852 through the sequential losses of two sets of neutral species C₄H₈O₄ via cross-ring breakage followed by the loss of C₆H₁₀O₅ via glycosidic bond cleavage. This sequence is repeated in the reverse, where species at m/z 852 first fragments via the loss of C₆H₁₀O₅ to give ion at m/z 690, which subsequently fragments to produce species at m/z 570 via the release C₄H₈O₄. Maltoheptaose, which is the open form of β-cyclodextrin was also analyzed via the Cl⁻ adduct formation in the negative-ion mode. Maltoheptaose (MW 1153) forms an adduct peak with Cl⁻ ion at m/z 1188 (Figure S25). Tandem MS analysis of the precursor ion first loses HCl to give a product ion mass at m/z 1152. This undergoes subsequent fragmentation via successive glycosidic bond breakage to produce major ions at m/z 990, 828, 666, and 504.

CONCLUSIONS

We presented a simple non-contact nano-electrospray ionization approach that allow halide (Cl⁻ and Br⁻) adduct formation with saccharides in the absence of plasma discharge. The resultant adduct was observed to dissociate in tandem MS producing unique diagnostic fragment ions for all five structural isomers of sucrose tested. This allowed a mixture of consisting of the sucrose isomers, differing only by the linkage, to be distinguished with ease. Subsequently, we applied the method to analyze complex raw honey samples where we unambiguously confirmed the presence of sucrose, turanose, isomaltose, and leucrose by mass spectrometry alone. The analysis of oligosaccharide by halide adduct formation in the non-contact electrospray process was also found to be favorable. With these results, we believe halide adduct formation with saccharides can provide a facile approach toward rare sugar analysis and isomeric differentiation where minute quantities of sample can be analyzed. This method can also be easily implemented on liquid chromatography-MS to enable both quantitation and differentiation of more complex saccharide mixture.