HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF GENTAMICIN SULFATE REFERENCE STANDARDS AND INJECTION USP

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1 , pp Aville online t HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF GENTAMICIN SULFATE REFERENCE STANDARDS AND INJECTION USP CHUONG M.C.*, CHIN J., HAN J.W., KIM E., ALHOMAYIN W., AL DOSARY F., RIZG W., MOUKHACHEN O. AND WILLIAMS D.A. College of Phrmcy, MCPHS University, Boston, Msschusetts, USA. *Corresponding Author: Emil- monic.chuong@mcphs.edu Received: July 31, 2013; Accepted: August 12, 2013 Astrct- The USP-NF 2013 descries 4 minoglycoside chromtogrphy peks with n elution order of C1, C1, C2 nd C2. The method clcultes gentmicin sulfte from single stndrd concentrtion. The USP method for Gentmicin Sulfte Injection is microil ssy, not HPLC method. Therefore, the purposes were to evlute the performnce of different C18 columns for seprting the minoglycosides in the USP reference stndrd, gentmicin sulfte powder met USP testing specifictions, nd commercil injection product, nd to construct multi-point stndrd curve for clculting ech of minoglycosides rther thn from single concentrtion. Three LC columns from the sme mnufcturer were selected: (1) AquÒ C18, 5 micron, (2) LunÒ C18, 5 micron, nd (3) Nuc leosilò C18, 3 micron (ll in 4.6 x 150 mm). When smples prepred from gentmicin sulfte powder met the USP test specifictions were injected, only one pek t 8.8 min ws displyed in the 35-min runtime chromtogrms for Aqu C18 column, while three peks were displyed for the Nuc leosil C18 column, nd four peks for the Lun column. Stndrd linerity ws otined from 0.2 to 2 mg/ml. Using the Lun column, the minoglycosides eluded from the smples prepred from the USP Reference Stndrd nd from commercil injection USP were noted s three. Keywords- o-phthlldehyde solution, Aqu C18, Lun C18, Nuc leosil C18, pre-column derivtiztion, sodium 1-heptnesulfonte Cittion: Chuong M.C., et l. (2013) High Performnce Liquid Chromtogrphy of Gentmicin Sulfte Reference Stndrds nd Injection USP., ISSN: & E-ISSN: , Volume 4, Issue 1, pp Copyright: Copyright 2013 Chuong M.C., et l. This is n open-ccess rticle distriuted under the terms of the Cretive Commons Attriution License, which permits unrestricted use, distriution nd reproduction in ny medium, provided the originl uthor nd source re credited. Introduction Gentmicin, isolted in 1958 nd reported in 1963 y Weinstein, et l., is ctive ginst mny common Grm negtive pthogens [1]. This drug is otined commercilly from Micromonospor purpure, nd four minoglycosides hve een identified in the USP-NF 2013 (cusp) s C1, C1, C2 nd C2 [2] with three structures were listed in the compendium [Fig-1]. Crem nd ointment dosge forms re used in the tretment of skin infections nd urns complicted y Pseudomonemi while n injectle solution including 10- nd 40- mg/ml gentmicin sulfte is used for serious systemic nd genitourinry trct infection [3]. The USP-NF 2013 descries single-point content 0.65 mg/ml determintion y dissolving Gentmicin Sulfte USP in wter prior to the ddition of other ingredients for HPLC. The cusp method descries the elution order s gentmicin C1, gentmicin C1, gentmicin C2, nd gentmicin C2 with 5-mm 10-cm column tht contins 5-µm pcking L1 [2]. L1 is defined s octdecyl silne chemiclly onded to porous or nonporous silic or cermic microprticles, 1.5 to 10 µm in dimeter, or monolithic silic rod [2]. The purposes of this project were () to compre the performnce of different LC columns for conducting gentmicin sulfte ssy, () to explore whether multi-point stndrd curve ws fesile to estlish so tht smples of unknown concentrtions my e determined, nd (c) to exmine whether the moile phse might e simplified or modified under the modern LC column technology. Fig. 1- Structures of gentmicin sulfte [2,5] Mterils nd Methods Mterils USP Gentmicin Sulfte Reference Stndrd (RS) (Lot M1J001) ws purchsed from USP, Rockville, MD. Gentmicin Sulfte powder met the USP test specifictions (Amresco LLC, Lots 2562C059 nd 2712C327, Solon, OH), mmonium hydroxide, oric cid, chloroform, glcil cetic cid, isopropyl lcohol, methnol, o- phthlldehyde, potssium hydroxide, sodium 1-pentsulfonte, sodium 1-hexnesulfonte, sodium 1-heptnesulfonte, thioglycolic cid were purchsed from VWR (Bridgeport, NJ). Gentmicin Sul- Bioinfo Pulictions 25

2 High Performnce Liquid Chromtogrphy of Gentmicin Sulfte Reference Stndrds nd Injection USP fte for Injection USP (APP, Lots 03012DK, ) ws otined from Crdinl Helth. High Performnce Liquid Chromtogrphy Method Smple Preprtion Solutions of Gentmicin Sulfte in wter t 0.2, 0.4, 2, 4, 8 nd 20 mg/ml were prepred using the USP Gentmicin Sulfte RS nd the gentmicin sulfte powder met the USP test specifictions, respectively. Ten ml of ech solution ws trnsferred to 25 ml volumetric flsk. Five ml of isopropyl lcohol nd 4 ml of o- phthlldehyde solution were then dded nd mixed [2]. The o- phthlldehyde solution ws prepred y dissolving 1.0 g of o- phthlldehyde in 5 ml methnol, nd dding 95 ml of 0.4 M oric cid which hd een djusted with potssium hydroxide (8 N) to ph After two ml of thioglycolic cid ws dded, the resulting solution ws djusted gin with potssium hydroxide (8 N) to ph 10.4 for pre-column derivtiztion. The flsk ws then filled with isopropyl lcohol to the mrk of 25 ml. Flsks were heted in 60 C wter th (Precision Scientific, Model 183, Chicgo, IL) for 15 minutes, nd then cooled to room temperture [2]. LC Conditions Moile phse ws prepred y mixing methnol, wter, nd glcil cetic cid (70:25:5). Sodium 1-heptnesulfonte ws next dissolved. Its composition in the moile phse ws 0.5% w/v. Filtrtion with deertion ws followed. Three LC columns were selected for the study. They were () Aqu, 5 micrometers [6], () Lun, 5 micrometers [7], nd (c) Nuc leosil, 3 micrometers (ll were mm, Phenomenex, CA). The Agilent Series 1100 (Hewlett Pckrd) LC system contined vcuum degsser, inry pump, uto smpler, column thermosttic comprtment, nd vrile wvelength detector. The detection wvelength ws set t 330 nm with flow rte t 1.1 ml/min nd run time 37 min/cycle. Moile phse ws methnol/wter/glcil cetic cid/sodium 1-heptnesulfonte in rtio of 700/250/50/5 (v/v/v/w) [2]. For testing Aim (c), sodium 1- heptnesulfonte ws either removed from the moile phse or replced with sodium 1-pentnesulfonte or sodium 1- hexnesulfonte. Results Using the ssy method descried in the cusp [2] nd the stndrd concentrtion of 2 mg/ml prepred from the gentmicin sulfte powder met the USP test specifictions, y the end of 35 min, there were only one remrkle pek t 8.8 min ws displyed in the chromtogrm of Aqu C18 column [Fig-2], while there were three peks t the retention time of 18.3, 24.0 nd 27.7 min were eluted from Nuc leosil column (in three repetitions, [Fig-2]), nd the sme numer of peks t 10.5, 13.5 nd 15.2 min were eluted from the commercil Injection USP [Fig-2c]. The third column, Lun, seprted the USP Gentmicin Sulfte RS into three peks with retention time of 18.3, 24.0 nd 30.0 min [Fig-3] nd lso seprted the commercil Injection USP into three peks t 14.8, 19.2 nd 23.7 min [Fig-3]. But this Lun column ws le to seprte the gentmicin sulfte powder which met the USP test specifictions into four minoglycoside peks with the retention time of 19.0, 25.0, 28.3 nd 31.0 min [Fig-3c]. The linerity of the gentmicin sulfte stndrd curve rnged from 0.2, 0.4 nd 2 mg/ml were presented in ll peks eluted y either the Nuc leosil column [Fig-4] or the Lun column [Fig-4]. In the derivtiztion temperture of 60 C ll stndrd smples were in cler solution form ut expressing different colors. The smple of 0.2 mg/m ws slightly tinted yellow; 0.4 mg/ml tinted yellow; 2 mg/ml drk wine red. The color t 4 mg/ml or higher still to reduce the redness with 4 mg/ml in right wine red, 8 mg/ml slight reddish pech, nd 20 mg/ml in pech color. After ll smples were llowed to cool to room temperture nd stored in 4 C overnight, needle shpe of crystl ws found in the solution of 4 nd 8 mg/ml smples [Fig-5], while the smple of 20 mg/ml or higher formed powder shpe precipitte in cler color solution. The content % of ech component listed in the product informtion of distriutor, cusp-nf nd the column specific results from our study re summrized in [Tle-1]. Fig. 2- The chromtogrms of the first two study LC columns: () Aqu C18, () the Nuc leosil C18 t the concentrtion of 2 mg/ml. The smple in chromtogrms ) nd () ws prepred from the gentmicin sulfte powder met the USP test specifictions, ut eluded through n Aqu C18 nd Nuc leosil column, respectively. Figure (c) ws the chromtogrm of the commercil Gentmicin Sulfte for Injection USP eluded y Nuc leosil C18 column. c Bioinfo Pulictions 26

3 Chuong M.C., Chin J., Hn J.W., Kim E., Alhomyin W., Al Dosry F., Rizg W., Moukhchen O. nd Willims D.A. c Fig. 4- Stndrd Curves of Gentmicin Sulfte nd Precipittes t higher concentrtions. Using the Lun column, the linerity of the gentmicin sulfte stndrd curve rnged from 0.2, 0.4 nd 2 mg/ ml: () the smple prepred from the USP RS; () the smple from the gentmicin sulfte powder met the USP test specifictions. Fig. 3- The chromtogrms of the third columns, Lun C18. Figure () ws the smple prepred from the USP Gentmicin Sulfte RS nd Figure () ws the commercil Injection USP. Both displyed three minoglycoside peks. Figure (c) ws the smple prepred from the gentmicin sulfte powder met the USP test specifictions. All concentrtions were t 2 mg/ml. Fig. 5- Recrystlliztion of Gentmicin Sulfte USP. In the left is powder met the USP test specifictions. In the right is the regulr ciculr crystl. These needles were collected from the three derivtiztion smples of 4 nd 8 mg/ml ech fter eing stored in 4 C overnight, filtered y grvity nd ir dried. Tle 1- Using three LC columns to study the percent of pek re of ech prticulr gentmicin component in the smple prepred from the gentmicin sulfte powder met the USP test specifictions t the concentrtion of 2 mg/ml compred to the product informtion from distriutor nd the cusp-nf [2] Column Type (Smple Size) Pek 1 (%) Pek 2 (%) Pek 3 (%) Pek 4 (%) Sum of Pek 3 nd Pek 4 (%) Aqu C ND ND ND ND Nuc leosil C18 (n=3) 38.63± ± ±7.59 Lun C18 (n=5) 32.87± ± ± ± ±3.52 Distriutor Info < 45 < 30 - < 30 - USP Guidelines Mentioned, ut % not specified Mentioned, ut % not specified from Reference 4; 5-mm 10-cm column contining 5-µm pcking L1 Bioinfo Pulictions 27

4 High Performnce Liquid Chromtogrphy of Gentmicin Sulfte Reference Stndrds nd Injection USP The ph of the forementioned studies moile phse ws In order to exmine whether the moile phse might e simplified under the modern LC column technology, 0.5%w/v sodium 1- heptnesulfonte ws removed from the moile phse. The ph of such modified moile phse ws 3.2. When the sme smple ws injected into the LC system with Lun onded phse C18 (which ws le to seprte gentmicin sulfte stndrd preprtion t 2 mg/ ml) into four peks in the forementioned study) there ws only smll pek ws oserved t 10.5 min (dt not shown). This suggests tht for using Lun onded phse C18, recently developed LC column, sodium 1-heptnesulfonte ws still required in the moile phse to ssy gentmicin sulfte. Among sodium 1- pentnesulfonte, sodium 1-hexnesulfonte, sodium 1- heptnesulfonte, the pek elution of 2 mg/ml smple prepred from USP RS chnged from four into three when the sme smples were injected with the one of the first two regent in the moile phse within the sme dy ut couple hours lter, while sodium 1- heptnesulfonte eluded the USP RS into three minoglycoside peks consistently. Discussion There re four min resons of using derivtiztion in HPLC: to improve detectility, to chnge moleculr structure of polrity of nlyte for etter chromtogrphy, to chnge the mtrix for etter preprtion nd to stilize sensitive nlyte [8]. Post-column derivtiztion is commonly ccomplished using rection detector where the nlyte is derivtized fter the seprtion ut prior to detection. Pre-column derivtiztion requiring less equipment s it my e performed mnully or utomted. Pre-column derivtizing regents most typiclly used for mino cid nlysis re o- phthlldehyde [8], phenyl isothiocynte, fluorescmine, nd dnsyl chloride. Rection cn e either simply mixing t room temperture or requiring heting. There re t lest eight pulictions in the topic of gentmicin HPLC since 1979 [9-17]. Freemn, et l [10]. reported tht utilizing pre-column derivtiztion with n ophthldelhyde/thioglycolic cid regent on Hypersil O.D.S column nd UV detection, four mjor components of gentmicin were resolved (C1, C1, C2 nd C2). The elution sequence of gentmicin components is different from tht of the cusp (1). But ion-pired chromtogrphy ws used to quntify the derivtive. Brends, et l [11] pulished n HPLC method to quntify gentmicin in serum nd identified two chromtogrphic peks for C1 nd C1 + C2. But pek height insted of re under the curve ws reported, since oth peks did not return to the seline t the end of elution. Cnes, et l [12]. quntified gentmicin in iologicl fluids using silic column. Pre-column derivtiztion with o-phthlldehyde ws done to form fluorescent products for detection. They reported stndrd curves in oth plsm nd urine t concentrtion rnging from 0 to 10 mg/l [12], which is much lower thn our finding of 0.2 to 2 mg/ml. Grhm, et l [13] tested degrdtion of gentmicin sulfte in dextrose solution. Pre-column derivtiztion ws not incorported. Insted, 0.11 M queous trifluorocetic cid - methnol (95:5) ws used to dilute the stock of gentmicin sulfte in wter. They reported the elution order s C1, C2, C2 nd C1, with the composition eing 10-12% for C2 nd 1-2% for C2 (the fifth component) [13]. Since the moile phse nd smple preprtion did not follow the cusp-nf method, the results were not comprle. It is lso interesting to find tht gentmicin chromtogrm only contins single pek in ddition the internl stndrd pek from tormycin [14]. Grhek, et l [15]. identified 17 gentmicin impurities y using liquid chromtogrphy tndem mss spectrometry. However, their moile phse ws different from tht of the cusp [2]. The Kowlczuk, et l [16]. determined the gentmicin components s G1, G2 nd G3 y using o-phthlldehyde in comintion with N- cetycysteine (NAC). Like our endevor, Joseph, et l [17]. lso screened columns during method development. Since the detector of their study ws chrged erosol type, ion-pring gents ws required to put into moile phse to provide the required selectivity for gentmicin compound resolution on RP-HPLC column. Three different ion-pring gents were investigted. Eventully trifluorocetic cid ws chosen. The uthors elieved tht gentmicin sulfte is mixture of C1, C1, C2, C2 nd C2 with C1 of 26%, C1 27%, C2 nd C2 s mixture of 47% [17]. The AUC of Pek C1 ws extremely smll, < 1%. Our project ws the first endevor of reporting tht gentmicin sulfte HPLC ssy is reference stndrd specific s well s the performnce specific of ech tested column. We found four peks when Lun onded phse C18 [7] nd the gentmicin sulfte powder met the USP test specifictions ws used. This column is recommended for extremely high ph ppliction, such s ph 10.4 in this project, or when longer retention is desired [7] in order to seprte multiple components. Aqu s polr end cpping produces surfce chemistry tht is well suited for the nlysis of smll peptides [6]. Seprtion mechnism of Nuc leosil column is sed on hydrophoic (vn der Wls) interctions. When o-phthlldehyde solution (ph 10.4) rects with primry mino group of compound in n lkline medium, it will give solule product. This compound hs een exploited for spectrophotometric study y mesuring the sornce t 340 nm [9] nd quntittively estimting of compounds contining primry mino group. At the concentrtion, the rection produced the strongest color. It is importnt to mintin medium lkline [9]. This explins the short liner stndrd curve found in this study (0.2 to 2 mg/ml). The elution order descried in the cusp is C1, C1, C2 nd C2. Bsed on the physicochemicl property of gentmicin nd column chemistry, Kowlczuk, et l [16] reported the correct elution order of C1, C2 nd C1. But their method filed to identify the fourth peks. Joseph, et l [17]. suggested tht the fourth pek, C2, is the epimer of C2. Conclusions The cusp listed gentmicin slt in three different structures [Fig-1], ut descries the HPLC elution sequence s C1, C1, C2 nd C2 with the specifiction of C1 content rnging 25-50%, C %, C2 nd C %. The compendium only provides generl informtion of the column used ( 5-mm 10-cm column tht contins 5- µm pcking L1 [2]) without further specifying which one to use. Among the three different LC columns studied in this project, the AquÒ C18 nd LunÒ C18 re oth leled s 5 micrometers. Both their performnces were totlly different. The estimtion of gentmicin mjor component mde y Nuc leosil column fulfilled the description in the product informtion of distriutor [4]. A Lun column ws cple of identifying four chromtogrphic peks consistent with the description of cusp [2] when the smples were prepred from gentmicin sulfte powder met the USP test specifictions, ut not from the USP Gentmicin Sulfte RS nor from the commercil injections USP. Perhps this ws due to either different mnufcturing process, column performnce or other cuses. This project lso dvnced the single stndrd concentrtion preprtion in the cusp to stndrd curve from 0.2 to 2 mg/ml. Bioinfo Pulictions 28

5 Chuong M.C., Chin J., Hn J.W., Kim E., Alhomyin W., Al Dosry F., Rizg W., Moukhchen O. nd Willims D.A. References [1] Weinstein M.J., Luedemnn G.M., Oden E.M., Wgmn G.H., Rosselet J.P., Mrquez J.A., Coniglio C.T., Chrney W., Herzog H.L., Blck J. (1963) J. Med. Chem., 6, [2] Gentmicin Monogrph (2013) USP36-NF31, USP Convention Inc., Rockville, MD, [3] Block J.H. nd Bele J.M. (2004) Wilson nd Gisvold s Textook of Orgnic Medicinl nd Phrmceuticl Chemistry, Lippincott Willims nd Wilkins, Bltimore, MD, 340. [4] Sigm Aldrich (2012) Distriutor Informtion of Gentmicin Sulfte. [5] O Neil J.M., Smith A. nd Heckelmn P.E. (2001) Merck Index Whitehouse Sttion, NJ, [6] Agilent Technologies (2012) Distriutor Informtion of Aqu Column. [7] Agilent Technologies (2012) Distriutor Informtion of Lun Column. [8] Snyder L.R., Kirklnd J.J. nd Gljch J.L. (1997) Prcticl HPLC Method Development, John Wiley & Sons, NY, [9] Vid F.H.M., Aminuddin M. nd Mehmood K. (2004) Pkistn J. Phrm. Sci., 17, [10] Freemn M., Hwkins P.A., Lorn J.S. nd Sted J.A. (1979) J. Liquid Chromtogr., 2, [11] Brends D.M., Vn Dersndt J.S.F. nd Hulshoff A. (1980) J. Chromtogr., 182, [12] Cnes A., Cjl Y., Hro I., Grci Anton J.M., Reig F. nd Aroix M. (1991) J. Liquid Chromtogr., 14, [13] Grhm A.E., Speicher E. nd Willimson B. (1997) J. Phrm. Biomed. Anl., 15, [14] Lecroz C., Cmpnero M.A., Gmzo C. nd Blnco-Prieto M.J. (2006) J. Antimicroil Chemotherpy, 58, [15] Grhek R. nd Zupncic-Krlj L. (2009) J. Phrm. Biomed. Anl., 50, [16] Kowlczuk D., Pietrs R., Pw B. nd Czerkies A. (2010) Polish J. Environ. Studies, 19, [17] Joseph A. nd Rustum A. (2010) J. Phrm. Biomed. Anl., 51, Bioinfo Pulictions 29