USP Hypromellose Assay Procedure

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1 July 13, 2015 USP Hyprmellse Assay Prcedure Purpse This dcument is written t prvide guidance fr analysts cnducting the USP Hyprmellse assay test methd. The prcedure utlined in this dcument is based n the prcedure prvided in the current USP and EP mngraphs and includes additinal guidance and ntes as a reference t the analyst. It is based n the mngraph test prvided in USP 37/EP 8.3. Safety Each analyst shuld be acquainted with ptential hazards f the reagents, prducts, and slvents befre beginning labratry wrk. SOURCES OF INFORMATION INCLUDE: MATERIAL SAFETY DATA SHEETS, LITERATURE, AND OTHER RELATED DATA. Safety infrmatin n nn-dw prducts shuld be requested frm the supplier. Dispsal f reagents, reactants, and slvents must cmply with lcal, state, and federal laws and regulatins. Hydridic acid and its reactin byprducts are highly txic. Perfrm all steps f the assay preparatin and the standard preparatin in a prperly-functining hd. Specific safety practices t be fllwed are t be identified and fllwed by the analyst perfrming this test. Review the Material Safety Data Sheet (MSDS) fr this material prir t wrking with this chemical. Persnal prtective equipment t be wrn during the prcedure includes chemical gggles and acid-resistant glves. If the glves becme cntaminated, dispse f them immediately. All wrk with hydridic acid must be dne in a fume hd and all samples shuld be stred in the hd. During the reactin, the glass vials are under pressure at high temperature. Exercise cautin in handling the vials and cnduct the reactin and agitatin behind a safety shield and in a fume hd. Apparatus Fr the reactin vial, use a 5-mL pressure-tight serum vial, 50-mm in height, 20-mm in utside diameter, and 13-mm in inside diameter at the muth. The vial is equipped with a pressure-tight septum having a plytetraflurethylene (PTFE)-faced butyl rubber and an air-tight seal using an aluminum crimp r any sealing system that prvides sufficient air-tightness. Use a heater having a heating mdule that has a square-shape aluminum blck with hles 20-mm in diameter and 32-mm in depth, int which the reactin vial fits. The heating mdule is als equipped with a magnetic stirrer capable f mixing the cntents f the vial, r use a reciprcal shaker that perfrms a reciprcating mtin f apprximately 100 times per minute. Ntes: The design f the reactin apparatus is critical t ensuring accurate and repeatable results. At Dw, the reactin step is carried ut using an electrically-heated, thermstat-cntrlled, insulated, aluminum blck with a lid (made f aluminum r plastic) designed t fully cver the sealed vials during the mixing step. This heating blck is munted n a linear shaker. The blck has multiple (e.g. 8) wells

2 machined t clse tlerance s that the vials fit in while minimizing the space between the walls f the vial and the well ensuring all vials are heated cnsistently. When using a stirred ReactiTherm set up, the slutin is nt necessarily reaching all surfaces f the vial by the stirring mtin, s Hyprmellse slid may get stuck n the upper walls abve the slutin level. Dw recmmends shaking the vials twice thrugh the reactin time (nce at 20 min. and nce at 40 min.). The Hyprmellse sample will disslve nce the slutin is ht, s the shaking will ensure that all f the slid gets int slutin and reacts. The depth f the hles in the aluminum blck must be deep enugh s that the entire vial is within the hle, t minimize any amunt that extends beynd the heated zne f the blck. The heating f the blck shuld be checked peridically with a certified thermmeter t ensure that the temperature reaches 130 C. Check the blck in the same cnfiguratin as it will be used during the sample derivatizatin step, The temperature within each hle f the heating blck shuld be checked peridically t ensure that each hle reaches 130 ± 2 C. This temperature can be checked by filling a reactin vial with a lw viscsity silicn il t the same height as the reactin slutin, capping it, and inserting a thermcuple wire thrugh the septum and int the il. Place the il-filled vial int a hle and mnitr the temperature. After remving the vial frm ne hle, let the il cl, then insert the vial int the next heating blck hle t be tested. Reagents & Slutins Hydridic Acid Use a reagent having a typical cncentratin f Hydridic Acid (HI) abut 57%. Ntes: Use f hydridic acid cntaining <55% cncentratin has been identified as a rt cause fr lwer than expected results. Use f 57% hydridic acid is recmmended (this is the highest purity available n the market). Use f nn-stabilized hydridic acid ver 6 mnths ld has been identified as a rt cause fr lwer than expected results. Dw uses HI stabilized with hypphsphrus acid within 3 mnths f pening the bttle. Strage cnditins prvided by the supplier are recmmended. Internal Standard Slutin Transfer abut 3 g f n-ctane, accurately weighed, t a 100-mL vlumetric flask cntaining 10 ml f -xylene, dilute with -xylene t vlume, and mix. Standard (Calibratin) Slutin Standard slutin: Int a suitable serum vial, weigh between 60 and 100 mg f adipic acid, and add 2.0 ml f Hydridic acid and 2.0 ml f Internal standard slutin. Clse the vial securely with a suitable septum stpper. Weigh the vial and cntents, add between 15 µl and 22 µl f isprpyl idide thrugh the septum with a syringe, weigh again, and calculate the weight f isprpyl idide added, by difference. Add 45 µl f methyl idide similarly, weigh again, and calculate the weight f methyl idide added, by difference. Shake the reactin vial well, and allw the layers t separate. Use the upper layer as the Standard slutin. Ntes: It is suggested that the exact purity f the methyl idide and isprpyl idide be btained frm a supplier Certificate f Analysis r via analytical testing and used when calculating the amunt f idides in the standard preparatin. Failure t crrect values fr the purity f the slutins will cause inaccurate assay results t be btained. Dw uses methyl idide and prpyl idide within 6 mnths f pening the bttles. Care must be taken t ensure the purity f the adipic acid. If it is cntaminated with trace levels f Hyprmellse, the results fr hydrxyprpxy (-OC 3 H 6 OH, HP) and methxy (-OCH 3, MeO) will be higher than expected.

3 The quantity f adipic acid affects the results fr hydrxyprpxy (-OC 3 H 6 OH, HP) and methxy (- OCH 3, MeO). T minimize variability f results Dw uses 85 ± 5mg f adipic acid. Strage cnditins prvided by the supplier are recmmended. Assay Preparatin Sample slutin: Transfer g f dried Hyprmellse t a 5-mL thick-walled reactin vial equipped with a pressure-tight septum-type clsure, add between 60 and 100 mg f adipic acid and pipet 2.0 ml f Internal standard slutin int the vial. Cautiusly pipet 2.0 ml f Hydridic acid int the mixture, immediately cap the vial tightly, and weigh. Using the magnetic stirrer equipped in the heating mdule, r using a reciprcal shaker, mix the cntents f the vial cntinuusly, heating and maintaining the temperature f the cntents at 130 ± 2 fr 60 min. If a reciprcal shaker r magnetic stirrer cannt be used, shake the vial well by hand at 5-min intervals during the initial 30 min f the heating time. Allw the vial t cl, and weigh. If the weight lss is 0.50% f the cntents r there is evidence f a leak, discard the mixture, and prepare anther Sample slutin. Ntes: Sample Drying Ineffective drying can be the cause f unexpected lw results fr bth hydrxyprpxy and methxy. Dry the sample in an ven equilibrated at 105 C fr at least 1 hur. The dried sample shuld be cled in a clsed weighing bttle in a desiccatr fr nly the minimum time necessary prir t weighing. It is suggested that minutes shuld be the maximum time in the desiccatr t minimize the chance fr re-absrptin f misture which wuld lead t unexpected lw results fr bth HP and MeO. Past investigatins have shwn that strage in a desiccatr vernight can result in misture increases as high as 1.5% - leading t significantly lwer than expected final assay results. Accurate Weighing A 5-place balance is suggested (weighing t the nearest g). Care must be taken t ensure stable accurate weight readings are btained. Factrs t cnsider and eliminate/minimize include air currents, static charge, and humidity (misture pick-up). Care needs t be taken t cmplete the assay preparatin steps in such a way that misture is nt added t the cntents f the vial. Quick and careful weighing with capping f the vial in between additins is suggested. Adipic Acid Integrity False high results fr HP r MeO can be btained if the adipic acid is cntaminated with the Hyprmellse pwder. Care shuld be taken t maintain the integrity (purity) f the adipic acid during strage. The adipic acid shuld be weighed exactly t be in the range f 85 ± 5mg. Derivatizatin Reactin This is a very critical step in the assay testing and has multiple surces f ptential rt causes fr unexpected results. At Dw, the reactin step is carried ut using an electrically-heated, thermstat-cntrlled, insulated, aluminum blck with a lid (made f aluminum r plastic) designed t fully cver the vials during the mixing step. This heating blck is munted n a linear shaker with a prgrammable strke rate. The blck has multiple wells (e.g. 8) machined t clse tlerance t ensure cnsistent heating in all wells. D nt mix the cntents f the vial by hand prir t placing in the heater. The reagents and sample in the vial are nt sluble at rm temperature and will adhere t the sides f the vial. This will lead t ineffective slubilizatin and derivatizatin f the Hyprmellse granules resulting in lwer than expected results. It is strngly suggested that a reciprcal shaker r magnetic stirrer be used during the reactin. The USP methd allws fr shaking the vials by hand at 5-minute intervals during the initial 30 minutes f heating time. This intrduces a pssible safety risk (thermal r chemical expsure due t ver-pressurizatin f the vials during the reactin) and can als lead t lwer than expected results (due t incmplete derivatizatin resulting frm sample slutin temperatures belw 130 C). Mixing can be accmplished with a linear shaker r with a magnetic stir bar, but mixing/stirring f the sample slutin must be as cntinuus and vigrus as pssible t ensure cmplete

4 derivatizatin. With a linear shaker, it is suggested that at least 100 strkes per minute (clser t 200 strkes per minute is suggested) be used t ensure cmplete derivatizatin. It is critical that the cntents in the vial are cmpletely within the wells f the heating unit during the heating step. A cver ver the wells hlding the vials is als recmmended t better ensure unifrm heating f the entire cntents and headspace f the sealed vials. If multiple vials are heated at the same time, it is imprtant t ensure that the heating prfile fr the heating element is cnsistent frm vial t vial. This has been identified as a rt cause fr incnsistent and lwer than expected results during investigatins with custmer and cntract labs. Temperature f the sample slutin is critical. As stated in the mngraph, the cntents f the vial are t mix cntinuusly while heating at 130 ± 2 C fr 60 minutes. If the slutin is t ht, n- prpyl idide can frm and HP numbers will be lwer than expected. If the slutin is nt ht enugh, if it takes significant time t get t the target temperature (130 C), r if the temperature varies during the 60 minute heating time, the derivatizatin reactin may nt prceed t cmpletin and bth the MeO and HP results may be lwer than expected. If unexpectedly lw results are btained, the heating blck shuld be checked fr the amunt f time it takes fr each hle t reach 130 C. A waiting perid t allw the vial cntents t reach 130 C culd be added prir t the 60 minute reactin time. The mngraph allws a maximum weight lss f 0.50% f the cntents f the reactin vial. Dw uses a maximum allwable weight lss f 10 mg. If crimp caps are used, test the seal by hand after crimping t make sure that the cap cannt be rtated. If the crimp ring appears defrmed/wrinkled/dented after crimping, the seal shuld be cnsidered suspect, and the crimping tl shuld be readjusted. If the septum is t thick fr the metal crimp ring, the metal will nt extend far enugh arund the glass lip, and the seal is mre likely t fail. Transfer f Xylene Extract: Take up the xylene phase with the pipet in a single step, eject the extract int the GC vial, and cap the vial immediately. Avid sucking up the extract in the reactin vial and ejecting it int the reactin vial again t suck it up a secnd time. Taking up the xylene phase and re-ejecting int the reactin vial results in the lss f methyl idide and isprpyl idide ut f the xylene phase. T eliminate the manual transfer f the xylene phase Dw uses autsamplers frm PAL. After cling the reactin vial t rm temperature it is transferred t the PAL autsampler. The PAL autsampler extracts an aliqut f the xylene phase and injects it directly int the GC, thus eliminating the need f a manual transfer. Chrmatgraphic System The gas chrmatgraph is equipped with a thermal cnductivity detectr r hydrgen flame-inizatin detectr and a 3- t 4-mm 1.8- t 3-m glass clumn packed with 20% liquid phase G1 n 100- t 120-mesh supprt S1D that is nt silanized. Helium is used as the carrier gas fr use with the thermal cnductivity detectr; helium r nitrgen can be used fr the hydrgen flame-inizatin detectr. The temperature f the clumn is maintained at 100 ºC. Chrmatgraph the Standard preparatin, and adjust the flw rate s that the retentin time f the internal standard is abut 10 minutes. Use a clumn giving well reslved peaks f methyl idide, isprpyl idide, and the internal standard in this rder. Ntes: Representative chrmatgrams f a calibratin standard and sample are prvided alng with example calculatins in Figures 1 and 2, respectively. These plts were generated using a thermal cnductivity detectr. The flw rate f the Dw system was ptimized t ensure efficient analyses withut sacrificing peak tailing r peak reslutin. The retentin time f the internal standard (n-ctane) is arund 4.5 minutes. Cnditining f the packed clumn is recmmended prir t initial use t ensure stability during sample analysis. At Dw, this is accmplished by the fllwing thermal and chemical cnditining prcedures: Thermal Cnditining

5 After the clumn is first installed, heat t 200 C at rate f 2 C per minute and hld fr apprximately 16 hurs (r vernight). It is critical that the clumn nt be expsed t any air during this thermal cnditining. Return the ven and clumn t the nrmal analysis temperature f 100 C. Chemical Cnditining Make a mixture f the methyl idide and isprpyl idide by adding 300 L f each t a standard vial. If the clumn has been repacked, make three separate 5-L injectins f the 1:1 idide standard. If the clumn has been replaced, make five 5-L injectins f the 1:1 idide standard. Prepare a Standard (calibratin) Slutin and inject 2-L a minimum f 20 times until the respnse factrs are stabilized within 5% relative standard deviatin. Run a blank t generate a baseline. If the baseline is flat, inject a reference standard and cmpare results t previus trend charts t supprt integrity f the system. It is recmmended that the GC packed clumn be replaced annually unless system suitability r ther trending results indicate clumn degradatin and mre frequent replacement is required. If samples are analyzed infrequently, it is recmmended that injectin f a previusly-prepared sample slutin be cnducted t cnditin the clumn. The results frm this analysis shuld nt be used fr quantitatin purpses. It is recmmended that the helium carrier gas be passed thrugh xygen and misture traps t better ensure clumn integrity and lngevity. Flw rates used at Dw are as fllws: Carrier gas, Helium: 40 ml/min (adjusted as needed) Reference gas, Helium: 100 ml/min The mngraph des nt specify a temperature fr the injectr r detectr. Dw uses 200 C fr the injectr and 250 C fr the detectr. The mngraph calls fr the temperature f the clumn t be maintained at 100 C. Dw fllws the elutin with a temperature ramp t remve any residue frm the clumn. The recmmended temperature prgram: Oven temperature: 100 C hld fr 15 minutes, ramp t 200 C and hld fr 15 minutes If testing is cnducted rutinely, it is gd practice t prepare, analyze and cntrl chart substitutin results frm a reference Hyprmellse material t demnstrate cnsistency f the entire system (sample preparatin, derivatizatin, and analysis). At Dw, specific batches f Hyprmellse have been set aside fr use as reference samples fr this purpse. At Dw the reference Hyprmellse material is analyzed with each sample sequence. Obtained results fr Methxyl and Hydrxyprpxyl assay f the reference Hyprmellse material shuld be trend charted and lngterm reprducibility and trending be mnitred. Fr custmers wh d nt have access t a packed clumn GC system, equivalent results can be btained using a capillary clumn such as Phenmenex ZB-5, 30 m x 0.53 mm x 5.0 micrn film thickness in a split mde with a split rati f 5:1. System Suitability Peak Tailing Factr: Inject a 1-µL aliqut f the tp layer f a Sample Slutin int the gas chrmatgraph, and separate accrding t the chrmatgraphic cnditins. Repeat the analysis fur additinal times with the same Sample Slutin (a ttal f five chrmatgrams shuld be btained). Determine the peak tailing factr, T I, fr each peak f interest (methyl idide, isprpyl idide, and the internal standard) using the fllwing frmula: T I = PW I / 2f, in which PW I is the peak width at 5% peak height (minutes); and f is the width (in minutes) between the peak maximum and the frnt edge f the peak at 5% f the peak height. The system is suitable fr use if the average peak tailing factr is between 0.9 and 1.5 and the relative standard deviatin f the five factrs is less than 3.0%.

6 Peak Reslutin: The relative retentin times are abut 1.0, 2.2, 3.6 and 8.0 minutes fr methyl idide, isprpyl idide, tluene, and -xylene, respectively. The reslutin, R, between peaks is determined using the fllwing frmula: R = 2(t 2 -t 1 )/(W 1 +W 2 ), in which t is the retentin time (minutes) fr the peak f interest; and W is the width (in minutes) f the base f the peak f interest. The system is suitable fr use if the reslutin fr all targeted pairs is nt less than 2.0. Due t the many manual steps f this prcedure it is strngly recmmended that system suitability be mnitred n a peridic basis using a sample cntaining bth methxyl and hydrxyprpxyl substitutin r the calibratin standard slutin. Fr each sample sequence either prepare a test slutin f the Hyprmellse reference material r use the calibratin standard slutin t demnstrate cnsistency f the entire system. The slutin is prepared nce and injected five times at the beginning f the sequence. Frm all repeat injectins calculate fr methyl idide respectively isprpyl idide the standard deviatin f the adjusted peak area rati (Ratii) using the fllwing frmula: Rati i = (Area i,sample /Area IS,sample ) (W IS,sample /W sample ) in which Area i,sample is peak area fr the peak f interest btained during analysis f the sample, i is peak f interest (methyl idide r isprpyl idide), W IS,sample is the weight (g) f internal standard (n-ctane) in the sample slutin, Area IS,sample is the peak area fr the internal standard (n-ctane) btained during analysis f the sample, and W sample ) is the weight (mg) f sample in the sample slutin. The system is suitable fr use if the relative standard deviatin f the five adjusted peak area ratis des nt exceed 2.0%. Calibratin Separately inject abut 1 t 2 µl f the upper layer f the Standard preparatin and the Assay preparatin int the gas chrmatgraph, and recrd the chrmatgrams. Calculate the fllwing: Q Ta : Q Tb : Q Sa : Q Sb : the rati f the peak areas f methyl idide t n-ctane in the Assay preparatin; the rati f the peak areas f isprpyl idide t n-ctane in the Assay preparatin; the rati f the peak areas f methyl idide t n-ctane in the Standard preparatin; and the rati f the peak areas f isprpyl idide t n-ctane in the Standard preparatin. Example calculatins fr Q Ta and Q Tb are prvided in Figure 1 fr reference. Example calculatins fr Q Sa and Q Sb are prvided in Figure 2 fr reference. Calculate the percentage f methxy ( OCH 3 ) in the Hyprmellse taken by the frmula: (Q Ta /Q Sa )(W Sa /W U ) in which W Sa is the weight, in mg, f methyl idide in the Standard preparatin; and W U is the weight, in mg, f Hyprmellse, calculated n the dried basis, taken fr the Assay preparatin. An example calculatin is prvided in Figure 2 fr reference. Similarly, calculate the percentage f hydrxyprpxy ( OC 3 H 6 OH) in the Hyprmellse taken by the frmula: 44.17(Q Tb /Q Sb )(W Sb /W U )

7 in which W Sb is the weight, in mg, f isprpyl idide in the Standard preparatin; and W U is the weight, in mg, f Hyprmellse, calculated n the dried basis, taken fr the Assay preparatin. An example calculatin is prvided in Figure 2 fr reference. Please cntact me with any questins regarding this test prcedure. Sincerely, Barbara Serr, PhD Business Analytical Leader Dw Wlff Cellulsics The Dw Chemical Cmpany 2301 Brazsprt Blvd, B-1819 Freeprt, TX USA Phne: (979) brserr@dw.cm

8 Methyl- Isprpyl Octane Vlt Vlt Figure 1. Representative Calibratin Standard Chrmatgram (USP 2910) W Sa = Adjusted weight f methyl idide in the calibratin standard: mg x 99.9% purity = mg W Sb = Adjusted weight f isprpyl idide in the calibratin standard: 27.5 mg x 99.6% purity = 27.4 mg Q Sa = Area(MeI)/Area(n-ctane) in Std Prep = ( / ) = Q Sb = Area(PrI)/Area(n-ctane) in Std Prep = ( / ) = Peak Identificatin Retentin Time (min) Area Cunts 1 Methyl Idide Prpyl Idide n-octane (ISTD) Rear TCD Cal Slutin Cal Slutin 1 USP HPMC.met Name Retentin Time Area Minutes

9 Prpyl n-octane Methxyl Vlt Vlt Figure 2. Representative Sample Slutin Chrmatgram The weight f dried Hyprmellse sample in the assay vial: 65.5 mg Q Ta = Area(MeI)/Area(N-ctane) in Assay Prep = ( / ) = Q Tb = Area(PrI)/Area(N-ctane) in Assay Prep = (907685/ ) = % Methxyl Assay = (Q Ta/ Q Sa ) (W Sa /W U ) = (0.9983/1.3059) (117.0/65.5) = 29.8% % Prpxyl Assay = (Q Tb /Q Sb ) (W Sb /W U ) = (0.1809/0.4381) (27.4/65.5) = 7.6% Peak Identificatin Retentin Time (min) Area Cunts 1 Methyl Idide Prpyl Idide n-octane (ISTD) Rear TCD Name Minutes

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