Mass Balance Method for Purity Assessment of Organic Reference Materials: for Thermolabile Materials with LC-UV Method

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1 Purity Assessment of Thermolbile Compounds with LC-UV Method Bull. Koren Chem. Soc. 2014, Vol. 35, No Mss Blnce Method for Purity Assessment of Orgnic Reference Mterils: for Thermolbile Mterils with LC-UV Method Joonhee Lee nd Byungjoo Kim * Division of Metrology for Qulity Life, Kore Reserch Institute of Stndrds nd Science, Dejon , Kore * E-mil: byungjoo@kriss.re.kr Received My 2, 2014, Accepted July 28, 2014 A mss blnce method for purity ssessment of thermolbile orgnic reference mterils ws estblished by combining severl techniques, including liquid chromtogrphy with UV/VIS detector (LC-UV), Krl-Fischer (K-F) Coulometry, nd therml grvimetric nlysis (TGA). This method ws pplied to three fluoroquinolones like enrofloxcin, norfloxcin nd ciprofloxcin. LC-UV ws used to nlyze structurlly relted orgnic impurities bsed on UV/VIS bsorbnce spectr obtined in combintion with LC seprtion. For ll three orgnic reference mterils, the UV/VIS spectr of the seprted impurities were similr to tht of the mjor component of the corresponding mterils. This indictes tht the impurities re structurlly relted to the respective reference mteril shring common chromophores. Impurities could be quntified by compring their bsorbnces t the wvelength of mximum bsorbnce (λ mx ). The wter contents of the reference mterils were mesured by K-F Coulometry by n oven-drying method. The totl inorgnic impurities contents were ssyed from sh residues in TGA nlysis with using ir s regent gs. The finl purities estimted from results of those nlyticl techniques were ssigned s (99.91 ± 0.06), (97.09 ± 0.17) nd (91.85 ± 0.17)% (kg/kg) for enrofloxcin, norfloxcin nd ciprofloxcin, respectively. The ssigned finl purities would be pplied to the reference mterils which will be used s clibrtors for the certifiction of those compounds in mtrix CRMs s strting points for the trcebility of their certified vlues to SI units. Key Words : Purity ssy, Thermolbile orgnic substnce, LC-UV, K-F coulometry, TGA Introduction Chemicl mesurement society mkes severl endevors to secure the relibility of mesurement results by minimizing bis nd incresing ccurcy in mesurement. In order to chieve the comptibility nd relibility of mesurement results, it hs been emphsized tht the ppliction of wellchrcterized clibrtor with its trcebility to the Interntionl System (SI) of Units 1,2 plys criticl roles. As n effort to link trcebility, Ntionl Metrology Institutes (NMIs) estblish nd mintin the procedures for the purity ssessment of orgnic reference mteril s their core competences. 3-5 Differentil scnning clorimetry (DSC) or quntittive nucler mgnetic resonnce (qnmr) is possible techniques to directly determine the mss frction of the principl components in orgnic substnces. 6,7 However, these techniques re still used s confirmtory methods due to their inherent limittion such s insolubility of impurity in the melt nd requirement of well-chrcterized stndrds nd further vlidtion. The mss-blnce pproch is generlly ccepted to ssy the purity of orgnic reference mterils through mesuring ll detectble impurities obtined by severl nlyticl techniques. 8,9 Previous work performed by our group 10 introduced cse study on the purity ssessment of Endosulfn-II bsed on the mss-blnce pproch. Due to the voltility of Endosulfn-II, GC-FID hd been pplied to nlyze the structurlly relted orgnic impurities. However, vrious orgnic reference mterils re sensitive to high temperture, mking them in-comptible with GC nlysis nd requiring tht LC techniques be pplied to seprte min component nd orgnic impurities. Unlike GC-FID which cn show consistent liner response to ll orgnic compounds, UV detector hs limittion s universl detector. However, the utiliztion of photodiode rry (PDA) detector cn detect nd chrcterize the mjor compound nd impurities bsed on the informtion from their UV bsorption spectr. In most of the pure substnces either chemiclly synthesized or purified from nturl sources, orgnic impurities re inherently structurlly relted with the mjor components. As result, those impurities in reference mteril show similr UV/VIS bsorption spectr with the min component, usully becuse they shre common chromophores resulting in their bsorbnce t the wvelength of mximum bsorbnce (λ mx ) hving similr ppernces. Therefore, the chromtogrphicl purity of the reference mteril cn be obtined by compring the pek res of the min component nd ll of the detected impurities t λ mx. If there re impurities which hve different UV bsorbnce spectrum from the min compound, their contents in the mteril should be crefully evluted bsed on the reltive re rtio of impurities t ech chrcteristic wvelength, otherwise externl clibrtion my be required for significnt impurities. This rticle describes n impurity nlysis pproch using LC-UV for het sensitive orgnic reference mterils including enrofloxcin, norfloxcin nd ciprofloxcin, nd the

2 3276 Bull. Koren Chem. Soc. 2014, Vol. 35, No. 11 Joonhee Lee nd Byungjoo Kim estblishment of purity ssessment bsed on mss blnce method including Krl-Fischer coulometry nd TGA nlysis. This type of purity ssessment will be pplied to primry reference mterils for the certifiction of certified reference mterils (CRM) of defined orgnic species with linking trcebility to SI units. Mterils nd Methods Regents nd Chemicls. Pure substnces, enrofloxcin (Lot No ), norfloxcin (Lot No ), nd ciprofloxcin (Lot No ), were purchsed from Dr. Ehrenstorfer GmbH (Augsburg, Germny) with the purpose to use s primry reference mterils to certify the relted CRMs. Orgnic solvents for LC mobile phses were purchsed from Burdick & Jckson (Muskegon, MI, USA). Other chemicls such s formic cid or mmonium cette were purchsed from Sigm-Aldrich (St. Louis, USA). LC/UV Anlysis. The purity of the reference mterils were nlyzed by Wters 2695 HPLC (Milford, USA) with Wters 2998 PDA (Milford, USA) s detector. Impurity nlysis ws conducted with XDB-Phenyl (I.D.: 3.0 mm, length: 150 mm, prticle size: 3.5 μm) column nd mobile phse consisted of cetonitrile nd wter contining 10 μm EDTA. 11 A grdient mobile phse progrm ws developed to seprte impurity peks from min components for ech reference mteril. Mss frctions of the min component nd detected impurities in the reference mteril were determined s following: A min P LC-UV = A min + A impurity,i where, P LC-UV nd P impurity, i re the mss frctions of the min component nd impurity i, respectively, A min is the pek re of the min component, nd A impurity, i, the pek re of impurity i on the LC chromtogrm (t λ mx ) of the reference mteril. Note tht equtions (1) nd (2) do not include the contents of impurities undetected by the LC-UV nlysis such s inorgnic impurities nd wter. Krl-Fischer Coulometry. The wter contents of the orgnic reference mterils were mesured by C30 Krl- Fischer (K-F) coulometric titrtor connected with n ovendrying smple chnger (Mettler-Toledo, Switzerlnd). To minimize the influence from mbient tmosphere, the K-F coulometry system nd blnce were plced nd operted inside of glove-box filled with nitrogen. Detils of preprtion nd opertion for mesurement were described in our previous pper. 10 The drying oven temperture ws set up for ech orgnic compound ccording to its melting point, for instnce, 150 o C for ll three reference mterils. Six blnk pre-dried empty vils nd single smple vil i A impurity,i P impurity,i = A min + A impurity,i i (1) (2) were mesured in the present study. The six blnk runs were done for the mesurement of the system blnk (sum of wter introduced from crrier gs nd residul wter in the vil). Wter content in the smple ws clculted s followings P wter = (ICEQ/ Time Drift Blnk)/m C (3) where, P wter is the mss frction (kg/kg) of wter in the reference mteril, ICEQ is totl consumed electric chrge, which is converted to totl detected mss of wter (in µg). Time is totl K-F coulometric mesurement time in minute (10 minutes in this cse), Drift (µg/minute) is systemtic wter content mesured by K-F coulometric titrtor before the nlysis, Blnk is the system blnk mesured from the six empty vils, m is smple weight (in g) used for K-F coulometric nlysis, nd C is constnt, , to convert µg/g unit to kg/kg. Thermo Grvimetric Anlysis (TGA). Ash nlysis with thermo grvimetric nlyzer (TGA/DSC1, Mettler Toledo, Switzerlnd) ws pplied to mesure the totl mount of non-voltile inorgnic impurities. Detils of experimentl set-up were s described previously. 10 In this study, ir ws introduced into the TGA oven chmber to burn out orgnic compounds. An externl micro-blnce (MX5, Mettler Toledo, Switzerlnd) ws used for weighing the lumin pn before nd fter loding smple nd fter smple run by the TGA. In this wy, the weight of smple (W smple ) ws loded for mesurement nd the weight of totl residul inorgnic impurities (W non-voltile residues ) tht remined on the pn were precisely determined by weighing-by-difference with the stndrd uncertinty of 0.7 µg. The content of totl nonvoltile impurities (P non-voltile residues ) cn be estimted by following eqution. P non-voltile inorgnic residue = W non-voltile inorgnic residues W smple Results nd Discussion Anlysis of Structurlly Relted Orgnic Impurities by LC-UV. LC-UV nlysis ws crried out to determine the structurlly relted orgnic impurities in reference mterils of three fluoroquinolones including enrofloxcin, norfloxcin nd ciprofloxcin. Anlyses of these fluoroquinolones were performed with XDB-Phenyl column nd mobile phses which consist of cetonitrile nd wter with 10 µm EDTA. Prior to impurity nlysis, vrious concentrtions of the reference mteril solutions were prepred in methnol, nd used to check the dynmic rnge (rnge of liner response) of the detector. Considering intensities of the min compound nd detectble impurity peks, LC/UV nlysis ws conducted with n pproprite concentrtion of smple solution within the dynmic rnge (in the cse of enrofloxcin, 100 mg/kg in methnol). A typicl chromtogrm with UV bsorption spectr (obtined by the PDA) for enrofloxcin nd the seprted peks is shown in Figure 1. The chromtogrm ws drwn bsed on the bsorbnce t 278 nm. In (4)

3 Purity Assessment of Thermolbile Compounds with LC-UV Method Bull. Koren Chem. Soc. 2014, Vol. 35, No Figure 1. LC/UV chromtogrm (278 nm) of the enrofloxcin reference mteril solution in methnol (100 mg/kg). Inserted UV spectr for the min component (enrofloxcin) nd detectble orgnic impurities were obtined from the sme LC run with photodiode rry detector. Figure 2. Overlid chromtogrms from LC run of the enrofloxcin reference mteril, mobile phses nd methnol solvent blnk (), nd of vrious concentrtions ( mg/kg) of enrofloxcin solutions (b). ddition to the min pek (pek 4) representing enrofloxcin, three dditionl peks were obtined t 278 nm. These three peks were not found in either mobile phse or solvent blnk (methnol) under identicl LC conditions (Figure 2()), nd

4 3278 Bull. Koren Chem. Soc. 2014, Vol. 35, No. 11 Joonhee Lee nd Byungjoo Kim Tble 1. Purities for enrofloxcin, norfloxcin nd ciprofloxcin determined bsed on individul nlyticl methods for ech reference mteril Reference Mterils Lot No. Purity ssy by LC-UV, P LC-UV Non-voltile Residues by TGA, P non-voltile residue Wter content by KFC, P wter Purity (P mteril) b Enrofloxcin ± ± ± ± Norfloxcin ± ± ± ± Ciprofloxcin ± ± ± ± 0.17 In P LC-UV, P non-voltile residue, P wter, the number following ± is the stndrd uncertinty. b In P mteril, the number following ± is the expnded uncertinty with level of confidence of 95% for the mesurement vlue. tend to increse the pek re with concentrtion of the min compounds (Figure 2(b)). These two criteri were used to confirm tht peks 1 to 3 were impurities from the reference mteril nd not from the LC system. In compring their UV spectr, we noticed tht peks 1 to 3 hd very similr spectrl ptterns with the min compound. Though theses peks show wek bsorption spectr due to low concentrtions, they lso hve similr spectrl ptterns with enrofloxcin (Figure 1). Therefore, these three peks were considered s orgnic impurities tht were structurlly relted to the min compound, nd 278 nm ws suitble wve length to quntify those impurities in the selected enrofloxcin reference mteril. From the pek res obtined from the chromtogrm t 278 nm, the mss frction of enrofloxcin (P LC-UV,enrofloxcin ) ws estimted, ccording to Eq. (1), s 99.91% (kg/kg). Mss frction for the three orgnic impurities in enrofloxcin ws 0.09% (kg/kg). The stndrd uncertinty ( u PLC-UV,enrofloxcin ) during the purity ssy by LC-UV ws evluted with the stndrd devition ( s PLC-UV,enrogloxcin ) from multiple mesurements (n = 5) (Tble 1). All the impurities observed by LC- UV ws less thn 0.1%, nd therefore smll differences in their molr bsorptivities from the corresponding min components re ssumed to cuse little influences in the observed purities. Following similr procedures, the chromtogrphic purities of reference mterils of norfloxcin nd ciprofloxcin were nlyzed nd results were summrized in Tble 1. These findings indicte tht noticeble orgnic impurities in the mteril re lso structurlly relted with the min components nd tht their reltive contents in the mteril cn be estimted by compring their pek res on LC chromtogrm t its λ mx, 278 nm. Similr ptterns were obtined for both norfloxcin nd ciprofloxcin reference mterils. Two nd five impurity peks were detected in norfloxcin nd ciprofloxcin reference mterils, respectively. Mesurements of Wter by Krl-Fischer (K-F) Coulometry. Mesurement of the mss frction of wter (P wter ) in the reference mteril ws performed by Krl-Fischer (K- F) coulometric titrtion method. 10 The mgnitude of the system blnk nd its stndrd devition ( s KFC system blnk ) from multiple mesurements hve significnt influence upon the ccurte determintion of wter contents in orgnic mterils when only limited mount of smple cn be tken for nlysis. This is especilly true for pure orgnic substnces tht re usully very precious nd supplied in smll mounts per unit. In this cse, only few mg of the smple is vilble for wter nlysis, therefore reducing the mgnitude of the system blnk is criticl to improve mesurement qulity. In n effort to minimize these min fctors, Krl- Fischer (K-F) coulometric titrtor ws plced inside glovebox filled with nitrogen gs pssed through moisture trp. The closed glove-box system helped to protect the moisture from environment nd improved the relibility of results from K-F coulometry. Under the dried nitrogen tmosphere, the vlue of the system blnk ws down to round 160 μg compred to 390 μg in mbient ir, nd the stndrd devition of multiple mesurements for system blnk ( s KFC system blnk ) ws mintined t round 10 μg. Mss frctions of wter (P wter ) for enrofloxcin, norfloxcin nd ciprofloxcin were mesured s (0.00 ± 0.04)%, (2.80 ± 0.17)%, nd (8.03 ± 0.10)%, respectively. Mss Frction of Non-voltile Inorgnic Residues by TGA. Inorgnic impurities cn be introduced during synthesis or/nd purifiction of the mteril. TGA mesurement under inert gs cn mesure residues of inorgnic impurities nd non-voltile high-moleculr weight orgnic impurities. However, most of the compounds tht hve low voltility or/ nd thermo-lbile property produce residues of blck crbon soot in TGA nlysis under inert gs. To overcome this phenomenon, it is necessry to conduct the nlyses using infused ir s the regent gs to burn out ll orgnic compounds in the smple. Therefore, the mount of residues from the TGA nlysis in this study represents totl nonvoltile inorgnic impurities. Due to limited smple size, single TGA nlysis ws conducted for ech reference mteril s discussed previously. 10 The mss frctions of non-voltile impurities (P non-voltile residue ) were ssigned s (0.02 ± 0.02)% (kg/kg) for norfloxcin, (0.00 ± 0.05)% (kg/ kg) for enrofloxcin, nd (0.00 ± 0.06)% (kg/kg) for ciprofloxcin. Here the uncertinty represents the stndrd uncertinty (u non-voltile residue ) which ws estimted from the weighing uncertinty (u weighing-by-difference ) of 0.7 μg in 2 mg of smple (W smple ). Determintion of the Finl Purity nd Evlution of its Uncertinty. All of the mesurement results from individul nlyticl techniques re summrized in Tble 1. The finl purities of the enrofloxcin, norfloxcin nd ciprofloxcin were clculted to be (99.91 ± 0.06), (97.09 ± 0.17) nd (91.85 ± 0.17)% (kg/kg), respectively, which were determined using the following Eq. (6).

5 Purity Assessment of Thermolbile Compounds with LC-UV Method Bull. Koren Chem. Soc. 2014, Vol. 35, No P = (1 P wter P non-voltile residue ) P LC-UV (5) Here, the expnded uncertinties (level of confidence of 95%) were obtined by combining stndrd uncertinties of individul mesurement results. The ssigned vlues were produced by comprehensive exmintion nd evlution of the purities by mss-blnce method by pplying severl vilble nlyticl techniques to detect nd quntify most of the possible mjor impurities. Therefore, the ssigned purity by this best-prctice pproch is more relible nd scientificlly resonble under the current technicl limits nd economic burdens. Conclusion This study estblished the mss-blnce method for purity ssessment on thermolbile reference mterils. The purities of three fluoroquinolones including enrofloxcin, norfloxcin, nd ciprofloxcin were determined bsed on the results of LC-UV, Krl-Fischer coulometry nd TGA nlysis. The ssigned results will be utilized to the reference mterils which re subjected s clibrtors for certifiction of mtrix CRMs, intended to mintin the trcebility to SI units. Acknowledgments. This work ws supported by the Kore Reserch Institute of Stndrds nd Science under the project Estblishing mesurement stndrds for orgnic nlysis, grnt References 1. De Bivère, P.; Tylor, P. D. P. Metrologi 1997, 34, De Bivère, P. Int. J. Environ. Anl. Chem. 1993, 52, BIPM, Primry clibrtors nd comprisons for orgnic nlysis, 4. Gong, H.; Hung, T.; Yng, Y.; Wng, H. Tlnt 2012, 101, Yip, Y.-C.; Wong, S.-K.; Choi, S.-M. Trend. In Anl. Chem. 2011, 30, Americn Society for Testing nd Mterils (ASTM) Interntionl, Stndrd Test Method for Purity by Differentil Scnning Clorimetry, E , ASTM Interntionl, West Conshohocken, PA, USA, Mlz, F.; Jncke, H. J. Phrm. Biomed. Anl. 2005, 38, Westwood, S.; Choteu, T.; Direus, A.; Jesephs, R. D.; Wielgosz, R. I. Anl. Chem. 2013, 85, Ishikw, K.; Hnri, N.; Shimizu, Y.; Ihr, T.; Nomur, A.; Numt, M.; Yrit, T.; Kto, K.; Chib, K. Accred. Qul. Assur. 2011, 16, Kim, S.-H.; Lee, J.; Ahn, S.; Song, Y.-S.; Kim, D.-K.; Kim, B. Bull. Koren Chem. Soc. 2013, 34, Lee, S.; Kim, B.; Kim, J. J. Chromtor. A 2013, 1277, 35.