HPLC Method Development and Validation for Simultaneous Quantification of Vitamins C and K 3

Transcription

1 Annls of Clinicl Reserch nd Trils Open Access Full Text Article Reserch Article HPLC Method Development nd Vlidtion for Simultneous in Hrd Geltin Cpsules for their Potentil Benefit in Postopertive Totl Joint Arthroplsty Yousif Rojeb 1 *, Mohmd Hssoun 1, Ellen Hzelet 1 nd Deirdre Myers 2 1 Deprtment of Phrmceuticl nd Biomedicl Sciences 2 Deprtment of Phrmcy Prctice, College of Phrmcy, Ohio Northern University, Ad, Ohio, USA This rticle ws published in the following Scient Open Access Journl: Annls of Clinicl Reserch nd Trils Received August 21, 2017; Accepted September 18, 2017; Published October 6, 2017 Abstrct Efficient, selective, rugged, simple nd sensitive isocrtic RP-HPLC method for simultneous quntifiction of vitmins C nd ws developed nd vlidted. This method consisted of UV-detection t 254 nm nd seprtion by reversed-phse (RP)-C 18 column. Mobile phse ws composed of 50% methnol, 49% wter nd 1% glcil cetic cid, t flow rte of 1 ml/min. Injection volume (100 µl) consisted of vried concentrtions of both vitmins ( µg/ml) mixed with vitmin E (internl stndrd) in 1:1:2 volume rtio. Cler bseline resolution ws chieved for ll three compounds with retention times of 1.9, 3.3 nd 4.3 min for vitmins C, nd E, respectively. The method exhibited excellent linerity over the entire concentrtion rnge for both vitmins with R 2 of Intrdy (n = 6) ccurcy rnged from % nd % for vitmins C nd, respectively, while those for the inter-dy ssys (n = 12) rnged from % nd %. This HPLC method ws successfully implemented in quntifiction of vitmins C nd in compounded cpsules contining the two ingredients to ensure content uniformity. For vitmin C, content ws found to be 101 ± 4% (men ± stndrd devition) of the lbel clim of 500 mg. As for vitmin, content ws found to be 87 ± 6% of the lbel clim of 5 mg. These cpsules were subsequently dministered orlly in clinicl tril imed t evluting ny beneficil effect(s) of this vitmin mixture on postopertive totl joint rthroplsty. Keywords: UV-HPLC, Isocrtic, Vitmin C, Vitmin, Simultneous quntifiction *Corresponding uthor: Yousif Rojeb, Deprtment of Phrmceuticl nd Biomedicl Sciences, USA, Tel: , Fx: , E-mil: y-rojeb@onu.edu Introduction Arthritis comprises more thn 100 different rheumtic diseses, the most common of which is osteorthritis. Common symptoms include pin, stiffness, nd swelling in or round the joints. It is estimted tht 50 million U.S. dults report doctor-dignosed rthritis, which, consequently, is Americ s leding cuse of disbility [1]. Totl Joint Arthroplsty (TJA), lso known s totl joint replcement, is highly successful nd definitive reconstructive tretment for sufferers of severe rthritis of the mjor joints. About one million Americns ech yer elect to undergo TJA to enjoy the benefits of joint pin relief, restortion of joint function nd n overll increse in the qulity of life. Despite TJA s proven clinicl success, smll but significnt number of ptients fll victims to procedure filure. These filures re frequently ttributed to detrimentl biologicl/inflmmtory response triggered by the genertion of wer micro-prticles tht dislodge from the prosthetic implnt surfces [2-4]. Prticle-induced inflmmtory septic loosening of totl joint prosthetic components remins the gretest thret to the longevity of the rtificil device [4-6]. This septic loosening occurs when the bone bed djcent to the implnt, nd to which the implnt is ttched, is resorbed leving fibrous tissue-filled gp between the bone nd prosthetic device. With the loosening of the device, there is return of pin nd loss of function. It hs lredy been demonstrted tht combintion of vitmins C (scorbic cid) nd K (in the form of ; mendione) in rtio of 100:1 exhibited nti-tumor ctivity both in vitro nd in vivo [7]. This is due to the effective ntioxidnt nd ntiinflmmtory properties of this combintion. Therefore, it is suggested tht if we could extrpolte those nti-inflmmtory effects by dministering this vitmin combintion to people suffering from joint pin nd inflmmtion due to TJA, we could see potentilly significnt therpeutic benefits in these situtions. For this purpose,

2 Pge 2 of 7 stndrd operting procedure (SOP) ws developed nd implemented by our Phrmcy Skills Lb for the compounding of hrd geltin cpsules contining this proprietry rtio of vitmins C nd. Those cpsules were to be dministered in clinicl tril initited by Summ Helth System nd Crystl Clinic Orthopedic Center in Akron, OH imed t evluting the potentil for the forementioned clinicl benefits of this vitmin combintion. Once compounded, n essentil qulity control test to be performed on these cpsules is content uniformity nlysis. This qulity stndrd ensures tht the finlly formed cpsules contin the specified mount of ech of the two ctive ingredients. It lso ensures consistency of the preprtion/compounding procedure nd it quntifies ny possible btch-to-btch content vrition(s). While High Performnce Liquid Chromtogrphy (HPLC) nlyticl methods, in both queous nd plsm smples, lredy exist for quntifiction of vitmins C [8-11] nd [12-14] whether individully or in combintion with other components, no method currently exists for simultneous quntifiction of these two vitmins in single, simple nd efficient HPLC run. This is due, primrily, to the significnt difference in their solubilities nd consequently retention times on the HPLC column. While 1 grm of Vitmin C (scorbic cid) dissolves in bout 3 ml wter, vitmins (mendione) nd E (α-tocopheryl cette; used s the internl stndrd) re prcticlly insoluble in wter [15]. The three vitmins chemicl structures re depicted in Figure 1. For this reson, wter-soluble form of vitmin (mendione sodium bisulfite) ws utilized in this study. While this form of vitmin did ddress the solubility issue of this otherwise lipidsoluble vitmin, still no method currently exists in literture for simultneous quntifiction of the two vitmins (C nd ) in single, HPLC run. Therefore, the objective of this reserch ws two-fold; first, to develop nd vlidte n isocrtic RP-HPLC method for simultneous quntifiction of vitmins C nd nd second, to pply this method in content uniformity nlyses of the different btches tht were compounded to contin this vitmin mixture which ws subsequently dministered orlly in clinicl tril imed t evluting ny beneficil effect(s) of this vitmin mixture on postopertive TJA. Experimentl Chemicls nd regents Vitmin C (in the form of sodium scorbte, USP, pure mteril) ws purchsed from Letco Medicl (Dector, AL) while Vitmin Figure 1: Chemicl structures of () vitmin C (scorbic cid), (b) vitmin (mendione) nd (c) vitmin E (α-tocopheryl cette; internl stndrd). (in the form of mendione sodium bisulfite, 95%) ws supplied by the Aptone Development Center (Akron, OH). Vitmin E (in the form of α-tocopheryl cette 500 IU/gm; internl stndrd; I.S.) ws obtined from PCCA (Houston, TX). HPLC-grde wter nd methnol were purchsed from Fisher Scientific (Fir Lwn, NJ) nd were used in preprtion of the mobile phse, clibrtion curve solutions, qulity control (QC) smples nd in preprtion of vitmin C- nd -contining nlyticl solutions upon cpsule content uniformity nlyses. Regents (glcil cetic cid used for mobile phse preprtion nd dimethylsulfoxide (DMSO) used for preprtion of the I.S. stock solution) were mnufctured by EMD Chemicls nd were purchsed from Fisher Scientific, USA. HPLC conditions This RP-HPLC method consisted of UV detection (Wters Model# 2487, Milford, MA) t 254 nm nd seprtion by XTerr RP-C 18 column (4.6 x 150 mm; 5 µm). An in-line degsser/filter (polypropylene, 0.2 µm from Whtmn, Florhm Prk, NJ) ws connected between the mobile phse reservoir nd the pump (Wters Binry System Model# 1525). Mobile phse ws composed of methnol, wter nd glcil cetic cid (50:49:1), t flow rte of 1 ml/min t room temperture of 20 C. Injection volume of 100 µl (vi Wters Model# 717 uto-smpler) consisted of vried concentrtions of both vitmins ( µg/ ml) mixed with the I.S. in 1:1:2 volume rtio. Totl run time of the ssy ws 6 min. This HPLC system ws operted by Breeze TM Softwre (Wters Corp., Milford, MA, USA). Cpsules compounding procedure Compounding of the vitmin C- nd -contining cpsules ws performed using semi-utomtic ProFill 3006 benchtop cpsule filling mchine (Custom Cpsules, Pvt. Ltd., Indi). Briefly, pre-determined weights for vitmins C nd were mesured for every btch of 300 cpsules ( totl of 31 btches were prepred). Then, vitmin ws triturted in mortr nd pestle nd combined with pre-ground vitmin C using geometric dilution; phrmceuticl process implemented to thoroughly mix two ingredients tht exist in given formultion in different proportions where first, the smller mount, vitmin, is thoroughly mixed with n equl mount of vitmin C. Then, this mount is mixed with n equl mount of the remining vitmin C nd the process is continully repeted until no pure vitmin C is left over. The powder mixture ws then poured into V-type multi-purpose mixer (Gllipot Inc., St. Pul, MN) nd rotted for 20 minutes. After tht, empty hrd geltin cpsules (size 1 white cpsules, Gllipot Inc., St. Pul, MN) were loded onto the filler unit of the cpsule filling mchine fter which cpsule cps were removed with the id of cps try. Cpsule bodies were evenly filled with the vitmin mixture by hnd spreding the powdered mteril bck nd forth cross the filler unit with occsionl tmping to pck the mixture into the cpsule bodies. Lstly, the cps try ws plced bck on top of the filler unit nd cpsule locking ws chieved by pressing the two trys together. Once formed, cpsules were poured onto clen, dry surfce, visully inspected nd were subject to weight vrition tests where 6 btches of 10 cpsules ech s well s 10 individul cpsules were rndomly selected from ech btch nd weighed on n electronic blnce (weight vrition llowed ws ± 5% of trget vlue). If more thn individul cpsule or ny

3 Pge 3 of 7 one btch of 10 cpsules fell out of this rnge, ll 300 cpsules in tht btch were weighed, individully, nd those out of rnge were rejected. All finished cpsules were then kept refrigerted t 2-5 C in properly lbeled mber vils from which 3 cpsules were rndomly selected from ech btch for subsequent content uniformity nlysis within few months (preliminry stbility dt hs indicted no significnt degrdtion of either vitmin t this storge temperture for up to 10 months). Preprtion of clibrtion curves nd content uniformity nlyses Stock solutions of vitmins C, nd I.S. were prepred by dissolving 100 mg of the vitmin in 1 ml wter, mobile phse nd DMSO, respectively (due to their different solubilities [15]), ided by vortexing until complete dissolution. For vitmins C nd, proper seril dilution scheme, with the mobile phse, ws followed to prepre the working solutions nd obtin the finl concentrtions for the 5-point clibrtion curves of 0.5, 1, 5, 10 nd 50 µg/ml for ech vitmin. As for the I.S., seril dilution with the mobile phse ws followed to chieve finl concentrtion of 62.5 µg/ml (this vlue ws chosen becuse UV bsorbnce for the I.S. t this concentrtion under the HPLC conditions pplied wvelength of 254 nm resulted in pek height comprble to those of the mid-rnge concentrtions of vitmins C nd within the clibrtion curve which ws deemed pproprite). Since this method ws designed for simultneous quntifiction of vitmins C nd in single run, clibrtion curve smples consisted of pired concentrtions consisting of the lowest concentrtion of 0.5 µg/ml of ech vitmin for the first point. The next point on the clibrtion curve consisted of finl concentrtion of 1 µg/ml of ech vitmin nd so on. These finl concentrtions were obtined fter ccounting for the dilution fctor involved upon mixing the two vitmins nd the I.S. in 1:1:2 volume rtio. Below is summry of the finl concentrtions of clibrtion stndrds used in quntifiction of vitmins C nd (Tble 1). For quntifiction of vitmins C nd in cpsules from the different btches, 100 mg of the cpsule content ws weighed nd dissolved in 1 ml wter nd properly diluted with the mobile phse to within the rnge of the clibrtion curve, mixed with the I.S. nd injected ( dilution fctor of 1/2000 ws pproprite for this purpose nd ws corrected for upon clculting the content % for ech vitmin in the cpsule). All clibrtion, btch nd QC smples were prepred in medicl-grde polypropylene microcentrifuge tubes (Axygen Scientific Inc., Union City, CA) t room temperture of ~20 C. Method vlidtion The method ws vlidted for selectivity, linerity, Limits of Clibrtion dt point Concentrtion (µg/ml) Vitmin C Vitmin I.S. # # # # # Tble 1: Concentrtions of the three components, vitmins C, nd I.S., in clibrtion stndrds. Detection (LOD) nd quntifiction (LOQ), ccurcy, precision nd ruggedness. For purposes of evluting those vlidtion prmeters, pek height rtios of the nlyte (vitmin C or ) normlized by tht of the I.S. were used. Liner regression nlysis ws followed where those rtios were plotted ginst the corresponding nlyte concentrtion nd stright-line eqution of the generl formul: Y = mx ± B ws generted for ech clibrtion curve where Y: nlyte/i.s. pek rtio; m: slope of the line; X: nlyte concentrtion nd B: y-intercept. Intr-dy (n = 6) nd inter-dy (n = 18) clibrtion curves were constructed over period of 5 months nd it ws from those preprtions tht the linerity, represented by correltion coefficient (R 2 ), ws estblished over the clibrtion curve rnge (finl concentrtions of 0.5, 1, 5, 10 nd 50 µg/ml for ech vitmin). In this method, selectivity ws defined ccording to Ddgr D nd Burnett PE [16], s the bility of the nlyticl method to ccurtely differentite the nlyte from other components in the mixture. This ws chieved by evluting the chromtogrms for ech one of the 3 components (vitmins C, nd the I.S.) individully nd compring those to chromtogrms contining ll 3 components together. LOD nd LOQ were clculted ccording to the signl-to-noise rtio [17] where LOD = 2 (H/h) nd LOQ = 10 (H/h), where H is the pek height corresponding to the nlyte; h is the bsolute vlue of the lrgest noise fluctution from the bseline of the chromtogrm of blnk solution (in our cse the mobile phse). The Upper Limit of Quntifiction (ULQ) ws determined by injecting incresingly higher concentrtions of ech vitmin until signl sturtion ws observed (i.e., no more proportionl increse in the pek height upon incresing the nlyte s concentrtion). Accurcy of n nlyticl procedure expresses the closeness of greement between the vlue which is ccepted s either conventionl true vlue or n ccepted reference vlue nd the vlues found (mesured). Precision, on the other hnd, of n nlyticl procedure expresses the closeness of greement (degree of sctter) between series of mesurements obtined from multiple smpling of the sme homogenous smple under the prescribed conditions [18]. For ccurcy nd precision evlutions, independent runs (from freshly prepred stock solutions for ech vitmin) consisting of three concentrtions ech: 2 (low), 8 (medium) nd 40 (high) µg/ml within the rnge of our clibrtion curve ( µg/ml) were prepred nd injected upon mixing with the I.S. These injections were considered QC stndrds nd were used to ccess the intr- (n = 6) nd inter-dy (n = 12) precision nd ccurcy. Finlly, ruggedness of n nlyticl procedure is the degree of reproducibility of results obtined by nlysis of the sme smple under vriety of norml test conditions, i.e., different nlysts, lbortories, instruments, regents, ssy tempertures, smll vritions in mobile phse, etc. [17]. Ruggedness of our method ws evluted where the 18 clibrtion curves were prepred by 2 nlysts in our lb (12 prepred by one nd 6 by the other), nd dt generted ws comprtively evluted. Results Selectivity Cler bse-line resolution over the entire rnge of concentrtions encountered for vitmins C, nd the I.S.

4 Pge 4 of 7 with no pek interference ws chieved s evidenced by the chromtogrms (Figures 2 nd 3-c). The retention times for vitmins C, nd the I.S. verged (men ± stndrd devition) 1.94 ± 0.003, 3.26 ± 0.05, nd 4.30 ± 0.01 min, respectively, bsed Figure 2: Authentic chromtogrm corresponding to vitmins C (retention time 1.9 min), (retention time 3.5 min) nd E (I.S.; retention time 4.3 min) with cler bseline resolution. The peks represent 3 concentrtions (0.5, 1, nd 5 µg/ml) of vitmins C nd nd tht of the I.S. t 62.5 µg/ml AU Figure 3: Overly of the HPLC chromtogrms from ll 31 btches nlyzed demonstrting high consistency in vitmin C content. Peks for vitmin nd the internl stndrd look much smller thn those in figure 1 becuse of the uto scle djustment to the reltive sizes of the peks within the chromtogrm AU Figure 3b: Up-close depiction of vitmin C peks in figure 3.

5 Pge 5 of AU Figure 3c: Up-close depiction of vitmin nd the I.S. peks in figure 3 demonstrting, gin, the high consistency in vitmin content mong the different btches nlyzed. Component Intr-dy (n = 30) Inter-dy (n = 90) R t (min) b C.V. (%) R t (min) C.V. (%) Vitmin C 1.94 ± ± Vitmin 3.26 ± ± I.S ± ± R t : Retention time; represented s men ± stndrd devition b C.V. (%): Coefficient of vrition percent = (men / stndrd devition) x 100% Tble 2: Intr- nd inter-dy vribility in retention times for ll 3 components (vitmins C, nd E (I.S.)) within the HPLC chromtogrm. Anlyte Intr-dy ssys Inter-dy ssys Vitmin C y = (0.772 ± 0.038) x - (0.60 ± 0.112) y = (0.833 ± 0.065) x - (0.462 ± 0.196) Vitmin y = (0.409 ± 0.012) x + (0.221 ± 0.016) y = (0.401 ± 0.013) x + (0.235 ± ) y-vrible in these expressions represents vitmin C or pek height over tht of the I.S., while the x-vrible represents vitmin s concentrtion in µg/ml. Tble 3: Liner regression equtions for the two nlytes, vitmins C nd. on the intr-dy vribility (n = 30 ech). As for the inter-dy vribility, corresponding vlues were 1.94 ± 0.01, 3.45 ± 0.08, nd 4.33 ± 0.03 min, respectively, for the 3 vitmins (n = 90 ech) (Tble 2). A chromtogrphic run time of 5 min ws sufficient for complete elution of ll three compounds. Linerity, clibrtion, rnge, nd limits of detection nd quntifiction LOD nd LOQ were found to be nd µg/ml, nd nd µg/ml for vitmins C nd, respectively. ULQ ws determined to be 500 µg/ml for vitmin C nd 1500 µg/ml for vitmin. The method exhibited excellent linerity over the entire concentrtion rnge for both vitmins within the clibrtion curve rnge ( µg/ml) with R 2 vlues of nd for vitmin C nd nd for vitmin from intr- (n = 6) nd inter-dy (n = 18) ssys, respectively. Averge liner regression equtions for vitmins C nd re provided in Tble 3. Accurcy nd precision For the intr-dy ssys (n = 6), ccurcy rnged from % nd % for vitmins C nd, respectively, while those for the inter-dy ssys (n = 12) rnged from % nd % for the two vitmins, respectively (Tble 4). The precision of the method, s represented by C.V. (coefficient of vrition; lso known s reltive stndrd devition; defined s (stndrd devition / men) x 100%) ws clculted to be between % nd % for the intr-dy ssys for vitmins C nd vitmin, respectively. Corresponding vlues for the inter-dy ssys were % nd % for the two vitmins, respectively (Tble 4). Appliction This method ws pplied for simultneous quntifiction of vitmins C nd to ensure content uniformity in cpsules compounded to contin the two ctive ingredients in the predetermined weights. Thirty-one btches were nlyzed, in triplictes, for vitmins C nd content. A pre-determined rnge of % of the lbel clim ws considered cceptble s per USP [19]. Btch preprtions were crried out over period of over 1 yer. For vitmin C, content ws found to be 101 ± 4% (men ± stndrd devition) of the lbel clim of 500 mg (rnge: %). As for vitmin, content ws found to be 87 ± 6% (men ± stndrd devition) of the lbel clim of 5 mg (rnge: 64 b - 103%) (Figure 4). This vlue for vitmin C content (%) ws exceptionlly high since the next highest ws only 109%; closer to the collective rnge for ll btches nlyzed.

6 Pge 6 of 7 Intr-dy Theoreticl Conc. (µg/ml) Mesured Conc. (µg/ml) Accurcy (%) b C.V. (%) c n Vitmin C ± b This vlue for vitmin content (%) ws exceptionlly low nd ws regrded s n outlier since the next lowest ws only 76%; closer to the collective rnge for ll btches nlyzed. The vitmin btch with only 64% of lbel clim ws deemed uncceptble for inclusion in the clinicl tril. Discussion The method described in this work ws custom developed for simultneous quntifiction of vitmins C nd in single HPLC run. In our cse, hrd geltin cpsules were purposely compounded to contin the two gents in proprietry rtio. These cpsules were subsequently dministered orlly to evlute ny potentil therpeutic benefit(s) of this vitmin mixture on postopertive TJA. A key feture of this method ws its simplicity, nd hence, pplicbility whether in cdemic or industril lbs for similr sort of nlyses. Efficiency ws nother feture of this method; while smple run time ws set t 6 min, complete seprtion nd bse-line resolution for ll three compounds (Vitmins C, nd E) ws chieved within 5 min indictive of high cost effectiveness rtio. Even when other groups reported methods for simultneous quntifiction of hydrophilic vitmins such s vitmin C in presence of lipophilic ones such s vitmins A (in the form of β-crotene) nd E, much longer run times of ± ± Vitmin ± ± ± Inter-dy Theoreticl Conc. (µg/ml) Mesured Conc. (µg/ml) Accurcy (%) C.V. (%) n Vitmin C ± ± ± Vitmin ± ± ± Defined s (mesured conc./ theoreticl conc.) x 100% b C.V. (%): Coefficient of vrition percent; representtive of method precision, clculted s (men / stndrd devition) x 100% c Number of replictes Content (%) Vitmin C Vitmin Figure 4: Averge content (%) for vitmins C nd upon nlysis of compounded cpsules. Pre-determined cceptble rnge for content (100 ± 15%) is represented by dshed lines (error brs denote stndrd devition). Tble 4: Intr- nd inter-dy ccurcy nd precision for vitmins C nd. 15 nd 25 min were necessry for complete elution [20,21]. Owing to the short retention time of our method, we were ble to nlyze lrge numbers of btches on ny given dy in reltively short mount of time. While cceptble rnge for ccurcy should be within ± 15% of the expected true vlue [22], our method demonstrted high ccurcy indictive of method relibility, t lest within the concentrtion rnges selected, the selection of which ws bsed on the expected vitmins concentrtion encountered in the compounded cpsules. Likewise, smll vritions in the intrnd inter-dy ssys for QC smples estblished the method s repetbility nd high intermedite precision [18]. High sensitivity, represented by 2-digit nno-grm per ml rnge for the LOQ, ws nother chrcteristic of this method lthough the lowest quntifible concentrtion mesured nd nlyzed in cpsules ws more thn 10 times the LOQ for either vitmin. High sensitivity is ttributed, t lest in prt, to the selection of detection UV wvelength of 254 nm, which ws in close proximity to mximum UV bsorption for vitmin C of 245 nm [23] nd tht for vitmin of 252 nm [24], under cidic ph. Though its robustness hs not been thoroughly evluted, this method demonstrted high degree of ruggedness [17] owing to the fct the clibrtion curves/qc smples for both vitmins were prepred by two nlysts in our lb nd dt were pooled together. It is worth mentioning tht ll clibrtion curve points/ QC smples were ccounted for during the method development/ vlidtion (no points were dropped). In this method, vitmin E ws used s the internl stndrd. However, vitmin E is common ingredient in multi-vitmin contining products so with slight modifiction, such s setting UV bsorbnce wvelength t 300 nm which hs been shown to llow for mximum bsorbnce for vitmin E [21], this method could be effectively utilized for quntifiction of those three vitmins in single, simple run. This method ws successfully pplied in simultneous quntifiction of vitmins C nd to ensure content uniformity in cpsules compounded to contin mixture of the two ctive ingredients nd for the vst mjority of btches, cpsules content fell within the pre-determined ccepted rnge of 100 ± 15% of the lbel clim. It is this method s overll relibility tht gve us confidence in our cpsule compounding

7 Pge 7 of 7 procedure nd in dministering those cpsules, except for those tht filed the content uniformity testing, in the clinicl tril for which they were prepred. Conclusion An efficient, selective, rugged, simple nd sensitive isocrtic RP-HPLC method for simultneous quntifiction of vitmins C nd ws developed nd vlidted. This HPLC method ws successfully implemented in quntifiction of vitmins C nd in compounded cpsules contining the two ingredients to ensure content uniformity. These cpsules were compounded for orl dministrtion in clinicl tril imed t evluting ny beneficil effect(s) of this vitmin mixture on postopertive TJA. Acknowledgement We would like to cknowledge the contributions of the multiple PhrmD students t The College of Phrmcy t Ohio Northern University who ssisted in the compounding of the vitmin C- nd -contining hrd geltin cpsules nd for performing the necessry weight vrition tests. References 1. Arthritis: Meeting the Chllenge of Living Well. CDC (Centers for Disese Control nd Prevention), Atlnt, GA Hynes DR, Rogers SD, Hy S, Percy MJ, Howie DW. The differences in toxicity nd relese of bone-resorbing meditors induced by titnium nd coblt-chromium-lloy wer prticles. J Bone Joint Surg Am. 1993;75(6): Shnbhg AS, Jcobs JJ, Blck J, Glnte JO, Glnt TT. Humn monocyte response to prticulte biomterils generted in vivo nd in vitro. J Orthop Res. 1995;13(5): Kovcik MW, Grdisr IA, Toksh JC, et l. An introduction of vrious spectroscopic methods to identify in vivo metl wer in totl knee rthroplsty. J Biomed Mter Res A. 2008;84(4): Athnsou NA, Quinn J, Bulstrode CJ. Resorption of bone by inflmmtory cells derived from the joint cpsule of hip rthroplsties. J Bone Joint Surg Br. 1992;74(1): Chib J, Schwendemn LJ, Booth RE Jr, Crossett LS, Rubsh HE. A biochemicl, histologic, nd immunohistologic nlysis of membrnes obtined from filed cemented nd cementless totl knee rthroplsty. Clin Orthop Relt Res. 1994;299: Tper HS, Jmison JM, Gilloteux J, Summers JL, Clderon PB. Inhibition of the development of metstses by dietry vitmin C: combintion. Life Sci. 2004;75(8): Rose RC, Nhrwold DL. Quntittive nlysis of scorbic cid nd dehydroscorbic cid by high-performnce liquid chromtogrphy. Anl Biochem. 1981;114(1): Behrens WA, Mdère R. A highly sensitive high-performnce liquid chromtogrphy method for the estimtion of scorbic nd dehydroscorbic cid in tissues, biologicl fluids, nd foods. Anl Biochem. 1987;165(1): Liu LS, Lee BL, New AL, Ong CN. Determintion of plsm scorbic cid by high-performnce liquid chromtogrphy with ultrviolet nd electrochemicl detection. J Chromtogr. 1993;612(1): Ptil SS, Srivstv AK. Development nd vlidtion of rpid ion-pir RPLC method for simultneous determintion of certin B-complex vitmins long with vitmin C. J AOAC Int. 2012;95(1): Po ES, Ho JW, Gong BY. Simultneous chromtogrphic nlysis of eight ft-soluble vitmins in plsm. J Biochem Biophys Methods. 1997;34(2): Chtzimichlkis PF, Smnidou VF, Ppdoynnis IN. Development of vlidted liquid chromtogrphy method for the simultneous determintion of eight ft-soluble vitmins in biologicl fluids fter solid-phse extrction. J Chromtogr B Anlyt Technol Biomed Life Sci. 2004; 805(2): Xue X, You J, He P. Simultneous determintion of five ft-soluble vitmins in feed by high-performnce liquid chromtogrphy following solid-phse extrction. J Chromtogr Sci. 2008; 46(4): O Neil MJ, Smith A, Heckelmn PE, et l. (13 th ed). The Merck Index, Merck Reserch lbortories, Whitehouse Sttion, NJ. pp. 141, 1042& Ddgr D, Burnett PE. Issues in evlution of bionlyticl method selectivity nd drug stbility. J Phrm Biomed Anl. 1995;14(1-2):23: A Guide to Vlidtion in HPLC for Stndrd Bse Anlyticl Chemistry Lbortory Mesurements, Hungry. 2012; p: EMEA Europen Medicinl Agency. Vlidtion of Anlyticl Procedures: Text nd Methodology, London. 1995; pp: The United Sttes Phrmcopei 26-Ntionl Formulry 21 (2003) Rockville, MD: U.S. Phrmcopeil Convention. 20. Zho B, Thm SY, Lu J, Li MH, Lee LK, Moochhl SM. Simultneous determintion of vitmins C, E nd bet-crotene in humn plsm by high-performnce liquid chromtogrphy with photodiode-rry detection. J Phrm Phrm Sci. 2004;7(2): Pulo MG, Mrques HM, Moris JA, Almeid AJ. An isocrtic LC method for the simultneous determintion of vitmins A, C, E nd bet-crotene. J Phrm Biomed Anl. 1999;21(2): Shh VP, Midh KK, Findly JW, et l. Bionlyticl method vlidtion revisit with decde of progress. Phrm Res. 2000;17(12): Eitenmiller RR, Ye L, Lnden Jr WO. (2 nd ed). Vitmin Anlysis for the Helth nd Food Sciences, CRC Press, Boc Rton, FL. 2008;pp Hui YH. Hndbook of Food Science, Technology, nd Engineering, CRC Press, Boc Rton, FL, Ch. 11, 2005;19:pp. 26. Copyright: 2017 Yousif Rojeb, et l. This is n open-ccess rticle distributed under the terms of the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl uthor nd source re credited.