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Direct analysis in real time mass spectrometry for characterization of large saccharides Huiying Ma, Qing Jiang, Diya Dai, Hongli Li, Wentao Bi, and David D. Y. Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00242 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 23, 2018
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Analytical Chemistry
Direct analysis in real time mass spectrometry for characterization of large saccharides Huiying Ma1ǂ, Qing Jiang1ǂ, Diya Dai1, Hongli Li1*, Wentao Bi1*, David Da Yong Chen1,2* 1
Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China 2 Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada
ǂ
Huiying Ma and Qing Jiang contributed equally to this work.
Corresponding Authors *Phone: +86 25 85891349. Fax: +86 25 85891707. E-mail:
[email protected]. *Phone: +86 25 85891859. Fax: +86 25 85891707. E-mail:
[email protected]. *Phone: +1 604 8220878. Fax: +1 604 822 2847. E-mail:
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Abstract Polysaccharides characterization posts the most difficult challenge to available analytical technologies compared to other types of biomolecules. Plant polysaccharides are reported to have numerous medicinal values, but their effect can be different based on the types of plants, and even regions of productions and conditions of cultivation. However, the molecular bases of the differences of these polysaccharides are largely unknown. In this study, direct analysis in real time mass spectrometry (DART-MS) was used to generate polysaccharides’ fingerprints. Large saccharides can break down into characteristic small fragments in the DART source via pyrolysis, and the products are then detected by high resolution MS. Temperature was shown to be a crucial parameter for the decomposition of large polysaccharide. The general behavior of carbohydrates in DART-MS was also studied through the investigation of a number of monoand oligo-saccharide standards. The chemical formula and putative ionic forms of the fragments were proposed based on accurate mass with less than 10 ppm mass errors. Multivariate data analysis shows the clear differentiation of different plant species. Intensities of marker ions compared among samples also showed obvious differences. The combination of DART-MS analysis and mechanochemical extraction method used in this work demonstrates a simple, fast and high throughput analytical protocol for the efficient evaluation of molecular features in plant polysaccharides.
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Analytical Chemistry
Introduction Plant polysaccharides have numerous therapeutic effects including modulating immune system, regulating biological functions such as blood pressure and even apoptosis, and have shown anti-tumor and anti-oxidation activities.1-3 Saccharides extracted from traditional Chinese herbs are of great variety and importance. The majority of the herbs are listed in the Pharmacopoeia of the People’s Republic of China are due to the medicinal values of their polysaccharides.4-6 Different polysaccharides act differently, and the saccharides from different herb species and even the same species but from different regions of productions and cultivation conditions can vary significantly, which can directly affect their therapeutic value and may even lead to the reverse of functions.7,8 Evaluation, differentiation and characterization of polysaccharide components is important for quality control of saccharide-based medicinal products, and for providing more insights into their biological functions. However, plant polysaccharides are mixtures of polymers with large molecular weights and have highly sophisticated structures from monomer composition, linkage and branching features, stereochemistry to three-dimensional configuration,9-11 that pose great challenges for analytical techniques currently in use. Due to the large molecular weight (tens of kDa to thousands of kDa) and structure complexity, getting any information from intact polysaccharide is difficult. Size exclusion chromatography is commonly used to separate saccharide polymers, even though normally only a few wide peaks can be obtained,12-14 because there are no efficient ways to resolve complex polysaccharide mixtures. Usually, large polysaccharide is first digested into small molecules through chemical, physical or enzymatic methods,15-17 and the products are then analyzed by other analytical techniques. The enzymes required to hydrolyze the polysaccharides are expensive and mostly unavailable and the digestion methods normally takes hours if not longer.17,18 Analytical techniques including thin layer chromatography, liquid chromatography (LC),19,20 gas chromatography (GC)21,22 or LC coupled to mass spectrometry (LC-MS)23,24 are then used for the identification of saccharide hydrolysates. Until now, the majority of studies have focused on the determination of monomer composition and acquisition of fingerprints of polysaccharides on the basis of small digested products.25-27
Monomer composition is
determined by matching the known monosaccharide standards such as glucose and mannose with
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the complete hydrolysis products with LC.26 Sample derivatization is often required because carbohydrates do not have chromophoric groups. To set up the evaluation standards and compare the differences among various plant polysaccharides, liquid chromatographic profiles of digested fragments of Chinese herb polysaccharides were considered as the fingerprints for quality control.28,29 Sugar hydrolysates with similar or isomeric carbohydrates can co-elute in LC due to its limited resolution, and impurities or unknowns cannot be identified using LC alone. Mass spectrometry is a powerful tool that can be used for compound identification in many applications.22,24,30 However, direct infusion of large polysaccharides using traditional ion source such as electrospray ionization (ESI) is difficult due to inefficient ionization and mass range limitation. LC in combination with MS can provide much better sensitivity for the analysis of the digested mixtures since MS measures m/z values for every single compound eluted from LC.22,23 However, these methods involve tedious multi-step sample preparation (saccharide extraction, purification and hydrolysis), and method development and optimization are typically timeconsuming and labor-intensive since polar carbohydrates show close to no retention on reverse phase columns. More sensitive and higher throughput characterization and differentiation techniques are needed for the large diversities of polysaccharides to generate characteristic fingerprints for quality assessment. Ambient ionization mass spectrometry is fast, high throughput and simple in operation, requiring little or no sample pretreatment.31,32 Among which, direct analysis in real time (DART) source allows direct ionization of gas, liquid and solid samples in the open air in seconds.32,33 Neutral molecules are charged either through energy transfer from excited metastable molecules (radical ions) or through proton transfer from ionized gaseous water clusters (protonated ions).3436
The instrumental design and ionization details have been summarized in several recent review
papers.34-36 Due to the speed and the ease of operation, DART-MS has shown remarkable advantages in applications such as illicit drug screening, food safety, environmental monitoring and contamination analysis.37-39 DART has been mainly used for small molecules because it is thought to be not capable of ionizing molecules larger than 500 Da. Interestingly, a couple of studies have shown that large glycosaminoglycan and lipopolysaccharide can break down into small fragments instantaneously in DART source.40,41 Nemes et al. reported that heparin and its infamous adulterant, over-sulfated chondroitin sulfate, can be differentiated through the characteristic small products (m/z
