Development Of A Qualitative And Quantitative Method For The Characterization Of Chitosan

ObjectiveChitosan is one of the most abundant resources.It is an alkaline polysaccharide with low toxicity,good degradability and high level of bioactivity,and has been widely used in the field of agriculture,food,medical dressing and drug delivery system?DDS?.Although chitosan showed low toxicity,it is necessary to establish a quantitative method for the supervision of chitosan.This method is of great significance for the content determiantion of chitosan medical dressing and DDS.Moreover,the method can also be used for the study on quality control of chitosan.Mass spectrometry?MS?is a perfect structure analysis technique,which detects the specific mass to charge ratio?m/z?.However,chitosan is a macromolecular copolymer that composed of repeated deacetylated monomer?GlcN?and acetylated monomer?GlcNAc?,therefore,determination of chitosan using MS technique is really a challenging task.Up to now,many published papers have emplied a MS method for the analysis of Chito-oligosaccharide?COS,<2 kDa?.However,no researches involved chitosan analysis by MS method have been reported.As discussed above,the molecule of chitosan only composed of GlcN monomer and GlcNAc monomer.So the degraded ions or mulitipule charged ions of chitosan that detected in the ESI source may be related to the Mw,DD and concentration of chitosan samples.This study aimed to estimate the obtained MS patterns of COS and chitosan,and then tried to develop a reliable MS method for the qualitative and quantitative analysis of chitosan.The sensitive,specific and rapid MS method will provide an optional way for the application study of chitosan and its derivatives.Part 1 Purification and Mw determination of chitosanThe Mw of 9 kinds of commercial chitosan samples was usually determined by a size exclusion chromatogram tandem refractive index detector?SEC-RID?system.The introduced COS impurities in the chitosan samples should be accessed accurtately.In this study,a chitosan 7 sample with a wide range of viscosity?50-800 mPa·s?was purified and the Mw and COS impurities of the purified and commercial chitosan samples were evaluated.The purification method consists of two steps:?1?After dissolved with acidic solution,the chitosan solution was then filterd.This step was aimed to remove the chitosan with higher Mw or lower DD.?2?Reprecipitation of chitosan in alkaline solution.This step was to remove chitosan with lower Mw?e.g.COS?.The determined Mws of the 9 kinds of chitosan were range from 106.1 to 485.1 kDa.After purified,the Mw of chitosan 7 decreased from 485.1 kDa to 367.7 kDa,reduced by 24.20%.No COS impurities were detected in commercial chitosan samples or purified chitosan sample.However,duo to the poor sensitivity of the SEC-RID method,low concentration of COS in chitosan could not be detected.Therefore,a more sensitive method is needed for the evaluation of the COS impurities in commercial chitosan samples.Part 2 DD determination of chitosan samplesPapers on the DD determination were published a lot.The most commonly used method is acid base titration method and ¹H NMR method.In this research,these two methods were adopted for the DD detection of chitosan.The determined results were also compared and discussed.The DDs determined by acid base titration method were in the range of 63.80-86.19%.While higher DDs?range from 69.97 to 89.06%?were obtained by ¹H NMR method.The deviation of these two methods was range range from-8.82 to-0.80%.Most researches drew a conclusion that the ¹H NMR method is a more accurate method.This method was always used for the DD determination of chitosan samples that can be dissolved in acid solution.And the accuracy of determined DD will not be affected by weight measurement of chitosan.Moreover,the pH of chitosan solutions will not be changed before and after the detection,so the detected results can not be influenced by the chitosan precipitation.However,the expensive instrument and complicated parameters are challenges for many researchers.The DD determined by acid base titration method was a little lower than that determined by the ¹H NMR method,but the difference was not statistically significant.Therefore,the acid base titration method could be used for the qualitative analysis of chitosan.This method needs simple instrument and easy to be operated,but weight measurement of chitosan samples and the judgment of titration end point should be considered carefully.Part 3 Determination of COS impurities in chitosan samples using an UPLC-MS/MS methodChito-oligosaccharide?COS?is usually prepared by the degradation of chitosan and shows perfect biological characteristics.Since both chitosan and COS are composed of randomly distributed GlcN monomer and GlcNAc monomer,the structures of these two compounds are very similar.Therefore,in the ESI source,the generated ions of COS impurities may interfere with the ions that origined from chitosan.In this part,a sensitive UPLC-MS/MS method was established to determine the COS impurities in chitosan samples.The elution of COSs?DP 1-7?was evaluated on an XBRIDGE BEH130 C18 column and a BEH Amide column.XBRIDGE BEH 130 C18 is a kind of reversed phase column.The RTs of COS?DP1-7?were 0.48,0.45,0.43,0.41,0.40,0.39 and 0.38 min,repectively.However,the RT of chitosan was 0.30 min.Even though COS and chitosan were not successfully separated on this column,this method could be suitable for the qualitative analysis of chitosan or COS.Moreover,COS transitions were detected in the chitosan samples at0.30 min,which indicated that?1?COS impurities detected in chitosan were below low limit of detection,?2?chitosan were prone to be interrupted in the ESI source.BEH Amide is a HILIC column and always used for the analysis of peptide and polysaccharide.The RTs of COS?DP 1-5?on the column were 2.98,3.32,3.58,3.79and 4.10 min,repectively.COSs?DP 1-5?were completely separated on the column.However,when the DP was above 5,COS can not be eluted.As the DP of chitosan molecule was above 5,chitosan can not be eluted on this kind of column and the in source dissociation of chitosan was not observed as well.Nevertheless,the in source dissociation of COSs was also observed.Part 4 MS patterns analysis of COS and chitosan in the ESI sourceAs demonstrated in part 3,COS and chitosan were easy to be interrupted in the ESI source.In this part,a Synapt G2-Si Q-TOF system was adopted to determine the MS spectra?100-1200 m/z?of COS and chitosan and the MS patterns were then characterized.Both COS and chitosan were prone to be degraded in the ESI source and the degradation features of these two compounds were similar.Even though the Mw of 9kinds of chitosan were different,after degraded in the ESI source,there were no differences among the MS spectra of different chitosan samples.The ions were mainly composed of GlcN?161.07 Da?and GlNAc?203.08?units,and dehydrated ions were also observed in the MS spectra.The intensities of ions decreased with the increase of mass to charge ratio?m/z?,when the m/z was above 1200,limited abundance of species were observed which indicated that the degradation of chitosan in the ESI was completely.And ions that had stronger intensities were composed of GlcN units.Moreover,capillary voltage was demonstrated to be a key parameter for catalyzing the dissociation.The ion intensities increased with the increase of capillary voltage and then decreased when the capillary voltage was above 2.4 kV.Part 5 Research on correlation coefficient?R?between the MS Patterns and DDs of chitosanIn this part the ions that composed of GlcN units were detected.As discussed above,glycosidic linkages of chitosan were prone to be cleaved in the ESI source.Therefore,same product ions may occur when these precusor ions were interrupted in the collision cell.In order to avoid further dissociation,we developed an UPLC-pseuedo-MS/MS method?no collision energy was introduced?to detect these specific ions.The detected tranditions were 323.17?323.17?2 GlcN?,484.22?484.22?3 GlcN?,645.28?645.28?4 GlcN?,806.33?806.33?5 GlcN?and 967.20?967.20?6 GlcN?m/z,repectively.We also evaluated the correlation coefficients between the ion intensities and DDs of chitosan samples.The relative abundance of these ions was also analyzed.The obtained relative abundance of 323.17,484.22,645.28,806.33 and 967.20 m/z in 9 kinds of chitosan samples were close to 100.00:63.12:43.85:27.04:11.82.The coefficient of correlations?R?between the determined DD and the intensities of GlcN units were calculated,and the R of 323.17,484.22,645.28,806.33 and 967.20 m/z were 0.9578,0.9612,0.9795,0.9891,0.9765 for ¹H NMR method and 0.9878,0.9722,0.9807,0.9786,0.9648 for acid base titration method,respectively.As verified in the research,good linearities were observed between the intensities of degraded GlcN units and the determined DDs.Therefore,this UPLC-pseuedo-MS/MS method should be suitable for the DD determination of chitosan.Moreover,the concentration of determined chitosan samples was 500 ng/mL and each run only took 1 min.So the established method proved to be more sensitive and rapid in comparison with the conventional methods.However,chitosan standards with known DD and accurate weight measurement of chitosan samples were necessary for this method.Part 6 Research on quantitative analysis of chitosanAs proved in part 5,the degradation ratio of chitosan molecule in the ESI source was similar.So these degraded ions could be related to the concentration of chitosan.In this part,484.22?484.22 m/z,with a higher intensity and little interference,was used for the quantitation of chitosan.The intensities of the ion were determined by an UPLC-pseudo-MS/MS method.Then,the chitosan concentration of chitosan composite nanosheet and a commercial chitosan styptic powder was determined using this method.Moreover,a series of spiked chitosan plasma sample were also prepared to access the established method.Good linearity was obtained between the intensities of the specific ion and chitosan at different concentrations?0.1-10?g/mL,dissolved in solvent?,and two chitosan hemostatic materials were determined by this MS method.Before the determiantion,chitosan of the hemostatic materials was extracted with 1%acetic acid.The determined percentage of chitosan in the chitosan composite nanosheet was 19%.However,the determined concentration of chitosan in the styptic powder was 20%higher than the theoretical value.During the research on determination of chitosan in chitosan composite nanosheet and chitosan styptic powder,we found that the determined results were significantly influenced by the extraction of chitosan in its composite products and the different DD between chitosan standards and samples.Since the chitosan is a copolymer with high Mw and polarity,its extraction and detection in the biological matrix is a challenging task.In this research,a protein precipitation method was used to extract the chitosan in the spiked plasma samples.But the extraction recovery was poor and need further investigation.ConclusionIn this research,Mw and DD of 9 kinds of chitosan samples were determined by traditional methods.Then an UPLC-MS/MS method was established to access the COS impurities in chitosan samples and no COS impurities were observed.The MS patterns of COS and chitosan in the ESI source were discussed for the first time in this study.We demonstrated that these two compounds were prone to interrupted and dehydrated in the ESI source.The ions in the mass spectra of chitosan can be calculated according to the equation:m/z=m·D+n·A+0/1·H₂0+H⁺?m,n?N,D=161.07 Da,A=203.08Da?.After the in source dissociation,most observed species were below 1200 m/z indicating that the in source dissociation was completely.The degraded ions of chitosan were then detected by a developed UPLC-pseudo-MS/MS method.The intensities of in source dissociation induced ions,which were composed by monomer GlcN,were proved to be related to the DD of chitosan.So the calibration curve,wihich was established using chitosan standards with known DD,could be used for the DD determination of unknown chitosan samples.This study provided an optional novel method for the DD determination of chitosan.Moreover,good linearity was also observed between the concentration of chitosan and the intensities of its specific ions.Therefore,this UPLC-pseudo-MS/MS should be suitable for the qualitative and quantitative analysis of chitosan and its composite products.