ResearchGate See discussions, stats, and author profiles fr this publication at 0» vesearchgatenet/pubiicavon/asnerasa2 Chitosan analysis using acid hydrolysis and HPLC/UV Article > Carbohydrate Polymers January 2011 crraTIons EADS 7 2,413 authors, including: Xun Yan Amway Corp, ADA, Ml BPUBLICATIONS 170 CITATIONS SEE PROFILE ‘Some of the authors ofthis publication are also working on these related projects: a carbohydrates View project AAllcontent following this page was uploaded by ¥\> Yo" on 10 January 2018. The userhas requested enhancement he downloaded fle.Casey Poe 872012) 1774-178 ELSEVIER Contents its available at SeiVorse ScienceDi Carbohydrate Polymers journal homepage: www.elsevier.com/lecate/earbpol Wes | 4 Chitosan analysis using acid hydrolysis and HPLC/UV Xun Yan", Heidi M. Evenocheck Anaya Secs Revere nd Deepen Amey Crpraton, 7575 lon tA M4355; United Ses Ianto inthis stdy, 2 method for the characterization and quantification of chitosan was designed wing acd ech 201 Idroiss,gucoramine derivatization and igh peforiancelquldchomatgraphy ased on Kinetic Bi septeer 21 ‘SSE eno ober 031 oa study of acd ydtelynis, we have demonstrate that chitosan can be quantitatively hydraied nto ‘hucosamine in Gh witheither 106 hydrochorie aid (HCY at 105°C. or 12 MHCI at 90°C Following N43 fluorenyimethoxycarbonyoxy)sucinimide (mos-0Su) desivatiaaton, the glucosamine content canbe Separate fom the rest of he hiyétolysates and quanfeg using evese-phase HPLC with UV detection. Twos iran Hyats Dieta supplement ‘his method was validated fr lines. precision (repeatability ad reproduibity and accuracy sing both chitosan and a dietary supplement formulation containing chitosan, 9.2011 Hsewer ad. Alright reserved 1. Introduction Chitosan is 3 copolymer of glucosamine (2-amino-2-deoxy-0- alucose) and N-acetyl glucosamine, which can be derived ffon chitin (B{1-4)-2-acetamido-2-deoxy-n-ghican).a natural amino polysaccharide (Toan, Ng, Ave, Trang. & Stevens, 2005). Chitosan hhas been shown to be non-toxic, biodegradable, biocompati- bile, and highly Soluble in aqueous acidic solutions and it has ound widespread application in food, dietary supplement, agri- cultural. pharmaceutical, and biotechnological industres(Li, Dunn. GGrandmatson, & Goosen, 1996: Ravi Kumaf, Muzzarell, Muzzarel. Sashiwa, & Domb, 2004: Yi eta, 2005). To characterize and quantily chitosan, vatious direct analytical _methods can be used, These include capillary electrophoresis, size ‘exclusion chromatography, and colorimetric detection. Capillary zone electrophoresis can separate chitosan and its acti detiva- lives to provide quantitative information (Fu, Huang, Zhai, Li, & Liu, 2007). Average molecular weights of chitosan can be deter- ‘mined using size exclusion chromatography (Nguyen, Wink, Buschmann, 2008; Terbojevich, Cosan, Focher, & Marsano, 1993) ‘These methods are useful in characterizing chitosan products but they are not specific enough and too labor intensive for routine analysis of chitosan in complex matrices, Uniizing the reactivity of the primary arses present in chi- tosan, ninhydrin, o-phthalaldehyde (OPA), and anionic dyes are used for colorimetric determination of chitosan purity and 144-6173 see ant matter © 2011 evi dA igh reserved content (Larionova, Zubaerova, Guranda, Pechyonkin. & Balabushevich, 2009; Leane, Nankervis, Smith, & illum, 2004; “Muzzareli, 1998). However, due to the wide presence of amine sroups, these colorimetric methods lack satisfactory selectivity for analyzing chitosan content in complex formulations due 10 Inerference with other ingredients n indirect characterization methods, chitosan is hydrolyzed {nt glucosamine whose concentration is determined by colorime- tty, ion exchange, or HPLC methods (Jang et a, 2005; Yu Ip, Veda Robert, & Hennessey, 1992: Zh, Ca, Yang, & Su, 2005). Hydeochlo- sullurie, phospboric, or trifluoroacetic acids have been used for acid hydrolysis of chitosan (Novikov, 2004; Nue'ga, Petrova, Kever, & Makarova, 2002: Zaman}, Jelhanipout, Edebo, Niklasson, {&Taherzadeh, 2008). Chitinase or chitosanase, derived from bacte- "a, have been used for enzymatic hydrolysis ina, Zueva, Lopatin, '& Vatlamov, 2008; Ramirer-Coutino, Marin-Cervantes, Huerta, Reval, & Shiri, 2006). The analytical methods for quantiving glucosamine are well documented, Glacosamine levels ean be determined by colortnet- ‘methods using the reactive primary amine or the reducing aldehyde groups (Cheng. Labavitch, & VanderGheynst, 2011 Rondle & Morgan. 1955) It can be derivatized to aldononitile acetate and analyzed with gas chromatography (Whiton, Lau, Morgan, ilbart. & Fox, 1985). Capillary electrophoresis and ionic curomatography, equipped with electrochemical detectors, have also been used for separating and determining glucosamine (Lee 1996; O'Shea, Lunt, & LaCoutse, 1993}, HPLC, coupled with pre-column desivatizaton, is another ‘method for glucosamine analysis. AOAC International has pub- lished an official method for the analysis of glucosamine in rawA Yon. Evenetek/ Cable Pers 472012) 176-1778 ms AVS ZX ae “XP | Acitanctest A ake e-GleN-Free PoGieN Five Fig 1 Sextet of ehtaran acd yale and glcaramine Fnac OSe materials and finished products (OMA, 2008; Zhou, Wascltc, & ‘Mohammed, 2005).Inzhis method, glucosamine is derivatized with Fmoc-Su (N-8-fluotenyimethoxyearbonyloxy) suecinimide),and the derivatives are separated and quantified by HPLC with UV detection In this study, we demonsteated an indirect chitosan analytical method in which we hydrolyzed chitosan under acidic condi- ‘ions into glucosamine, derivatized the liberated glucosamine with Fmoc-OSu, and quantified the levelof glucosamine derivative inthe resultant mixture with HPLC (Fig 1). The method was validated for routine laboratory analysis, 2, Experimental 2.1, Materials All the reagents were used as purchased without further purification. Glucosamine hydrochloride (C,H,,NOsHC), N49- ‘yorenylmethoxycarbonyloxy) sucinimige (Fmoc-0Su). Acetoni- trile(ACN) and sodium borate were purchased from Sigma-Aldrich (St.Louis, MO). Chitosan (95% purity) was obtained from Wilke Resourees (Lenexa, KS}, Triuoroacetic acid (TFA), acetic acid, and hydrochloric acid (HCL, trace metal grade) were purchased from Fisher Chemical (Fairlawn, N). Different concentrations of hydrochloric acid were prepared by diluting concentrated HCI with deionized (D1) water. Chitosan was blended with excipients (silicon dioxide, mal- odextrin, stearic aid plant extracts, and siliiied microcrystalline cellulose) to a target content of 32% by weight and the dietary formulation was used to analyze method precision and accuracy, 22. Chromatographic conditions and analysis HPLC analysis was conducted using an Agilent 1200 sys- ‘em equipped with degasser, quaternary pump, autosampler, and photodiode array detector (PDA). Agilent Zorbax SB-C18, 5 am. 150mm 4.6mm column was used for the chromatography. To analyze glucosamine Fmoc derivative, the HPLC conditions were as described in the AOAC method (OMA, 2008: Zhou etal, 2005) In brief, the elution gradient begins at 70x of 0.05% TFA in water (A) and 30% ACN (B). After Gmin. B is elevated (0 100% ACN in Smin. The elution rate was held at 8 m/min and the injection volume was 10m for both samples and standards. The chro tatogram Was recorded at 265m UV, integrated, and analyzed with Agilent Chemstation, a chromatography data system, 2.3. Sample hydrolysis and derivatization Samples containing either 50mg chitosan or glucosamine were Weighed into thick-walled glass digestion tubes, 2m. of 1% acetic acid solution was added, and the mixture vortexed nti it formed 8 consistent gel. Chitosan was hydrolyzed following the addition of 1OmL concentrated HCI (6,10, of 12M) and heat (90 or 105°C) ‘Altera designated period of hydrolysis (for kinetic studies, the sam- ples were taken at 30min intervals. the digestion mixture was Cooled to room temperature and subsampled (1 mi) intoa mixture of sodium borate (38) and DI water (20m), The pli was adjusted to 7.00 with 10M HCl and the solution volume adjusted to SO ml. using 022M borate buffer, pH 70. {Glucosamine in the hydrolysate was derivatized by mixing 1 mL ofthe dilute neutralized solution with I ml.of 10 mg/ml. Fmoc-OS in acetonitrile and the reaction was allowed to progress to com- pletion at ambient temperature for a least 4h without agitation ‘Alter derivatization, the sample was diluted with 3mL HPLC mobile phase (0.05% TFAJACN, 1:1 (vv) for analysis CCitosan content was calculated as follows: sme(ome) cea [161.2 +DAt%) mat 2 A2 4 (1. =DA(Z)) x Ma] x 100% here Cis the chitosan content (2); mg isthe glucosamine content as determined by HPLC: 215.7 isthe glucosamine (HC!) molecular Weight; mis the sample weight for analysis; 112 is the mole Weight of glucosamine repeating unit in chitosan: DAis the degree of aceiylationin chitosan: 42 the mole weight of one acetyl group and My is the molecular weight of counter acid in chitosan 24, Method validation For validation ofthe method, we analyzed the response linearity ofthe glucosamine standard, and method precision (repeatability and reproducibility) and accuracy. Five different weights of glu cosamine standard (30-80mg) were dissolved in 12 acetic acid Solution, The standard solutions were processed and derivatized as described forthe derivatization of sample hydrolysates. The deriva- tized standards were analyzed for linearity of detection. To test ‘method repeatability, fve replicate samples were prepared and analyzed following the procedure outlined in the previous sections For method reproducibility. asecond setof replicate samples(n~5) were prepared and analyzed by a second analyst using 3 different instrument The results were then compared to the results ofthe first analyst. Method accuracy was determined using ablankmatrix spiked with chitosan a 50,100, of 150% ofthe formulation target content (33% chitosan) and analyzed. 4, Results and discussion 2:1, Glucosamine separation and quantitative determination Chitosan anda formulation containing chitosan were hydrolyzed, derivatized, and analyzed with HPLC. The chro- rmatogtams for the hydrolysates were the same as those for slicosamine standards. As shown in the chromatogram (Fig. 2) licosamine derivatives were detected in two peaks at 6 and 7.5:in, This is due to the presence of two possible conformations of free glucosamine (a~ and B-glucosamine) in solution (Zhow6 A Yon HM Everechek/Carbldrate Poles #7 (2012) 1741778 ouae [lcasamie Fine aes eucsaie ee deca ‘etal, 2005). The sum ofthe two peak areas was used to calculate {otal glucosamine content. 22, Chitosan hydrolysis ‘The mechanism of chitosan hydrolysis was studied by Shabrukova, Shestekova, Zainetcinova, and Gamayurova (2002) and Einbu, Grasdalen, and Varum (2007). In strong acd, chitosan could be fully hydrolyzed to acetic acid and glucosamine. When the hydrolysisis incomplete, acetyl glucosamine is observed. When ‘weak acid is used at low temperature. oligosaccharides are domi- natin the hydrolysate products 52.1, Bec of acid concentration and temperarute on _lucosamine yield (Chitosan was firs dissolved in acetic acid to ai ite dispersion inthe concentrated HCI solutions. The dispersed chitosan was then hydrolyzed at two different remperatures. The glucosamine con- tent in the hydrolysate solution was analyzed and plotted against hhydrolyss time, Fg. 3 shows the glucosamine yield during the hydrolysis process with respect to hydrolysis time at various aid concentrations and temperatures, ‘As shown in Fig. 3, using8 MHL after8h of hydrolysis, chitosan 4s not folly hydrolyzed into glucosamine, and less than 40% glu- ‘osamine yield i detected at 90°C. The glucosamine yield reaches ‘98% using a digestion condition of 10M HCl and 105°C in Sh. We also observed similar recovery using 12M HCl and 80°C in Gh. Based on these results, hydrolysis conditions of 10M HCl, 105°C. and six hour duration were selected as the oprimum conditions for method validation. The hydrolysis conditions of 12M HCL and 105 ‘Cresultin complete glucosamine conversion intwohours. but ‘he endpoint is difficult to control because glucosamine decom- poses rapidly under these conditions (Fig. 3) 3.22. Activation energy of chitosan hydrolysis The initial stage ofthe hydrolysis (up to Sh) was fed with a first order reaction model, x= 1—e¥, where xis glucosamine veld. kethe reaction constant and f the reaction time. The correlation coefficient is above 098, The reaction constant is correlated with temperature (9) using Arthenius equation, k= Ae", where Ais constant, the gas constant and Eis the activation energy. The activation energy is determined to be 78KI/mol and isindepencent bf acid concentration, 423, Effects of acd concentration and temperature on _lucosamine decomposition Since glucosamine is known tobe deaminated by acid hydrolysis (Shabrukova ec al, 2002), sis necessary to minimize the decom- position to achieve an accurate resull for chitosan quantitation. To Aetermine the degree of decomposition, glucosamine samples were \Welahed into digestion tubes and treated withthe selected hydrol- ysis condition (105°C, JOMHCH}, Atthe end ofeach hour interval. sample was taken and analyzed for glucosamine. The results were recorded and are shown in Fig. 4. The degradation of glucosamine fits a first-order decomposition model. After Gh of hydrolysis, we observed approximately 2 20% loss of glucosamine. However. the decomposition kinetics during the chitosan hydrolysis pro= cessare complicated by the processes of chitosan depolymerization Sand deacetylation, and glucosamine generation, The glucosamine {ecomposition during chitosan hydrolysis does not appear to have 4 significant impact on chilosan recovery. The glucosamine con- tent of the chitosan plain material is 3.6% (w/w, average results fom two analysts, Table 1) which is experimentally close to the ‘nominal chitosan content of the plain material (95%). The recov tery of glucosamine from the formulation was greater than 99%. ‘demonstrating that the decomposition of glucosamine during chi- osan hydrolysis atthe specified conditions has minimel impact on assay accuracy o (clucosamine Conversion (%) Time (Hours) Teno Hews) 2: its fa ocncton n epee rein yin a yt 8} Pen nmin omen long een ya with Stare (a cep tah yer 90 ©A Yon. HM Evenchek/ Carla Pers 47 (2012) 176-1778 um conversion | OH (Gucosami H ie. cnn acon ed non cena 108 3056) Re=n9009 (Glucosamine Concentration (mgmt) Fe 5. Loe cacelalonbetween UY dren a lucerne derhanes oe nal guosamie cones 34, Method validation Five concentrations of glucosamine standards were analyzed and the results plotted as peak area counts against glucosamine ‘concentration (Fig 5). The correlation between the two parameters ts linear with a correlation coefficient R?>0.999, The asay repeatability and intermediate precision were calu- lated by determining the relative standard deviation ofthe results, asrecorded in Table |. The relative standard deviations (RSD) of five faces (2) 3D, peng Randa eon RSD, pean elite Randa replicate analyses (repeatability) forthe chitosan and formulations are0.93% and 1.67%, respectively, for analyst, and 25% and 5.49%, Fespectively, for analyst 2. The overall RSD (Feproducibility) were 452% for the analysis of chitosan and 3.84% forthe analysis ofthe formulation. The assay reproducibility for measuring glucosamine content was within Sv of the nominal content for both chitosan and the formulations The recovery of chitosan from the matstx samples was calcu= lated as percentage of the glucosamine pike. Chitosan recovery for the three formulations (50, 100, and 150% of target chitosan) was between 98 and 103%, demonstrating the accuracy of the method 4. Conclusions Based on a kinetic study of acid hydrolysis, we demonstrated that chitosan can be quantitatively hydrolyzed into glucosamine tn Gh with either 10M HCl at 105°C, or 12M HC at 60°C. Chi- tosan content is calculated based on the ghicosamine content in the hydrolysate Using derivatization and HPLC the current method ore selective than colorimetric methods, The simplicity of the ‘method makes it suitable fr routine quantitative analysis of chi- tosan in both plan material and dietary supplement formelations icalSciences Department (Dr-MarkProefke and Dr. Jlfery North) for providing facility. support and approval for conducting this research and release of information References Coen ¥ abe 3 Vang | 5291) Hehe a fio A uaenH_& Yao KML BOT) Kas of is of hubs Sgt cee bye ao, ane ier sas ose se Reg al i a0, Ams mr crt Soni mele co ca oan val a lati aan yma ys Jang, J iia H.C. The M. noe, C. Fajita M. & Yoshida T.(2005). 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