Analytical Chemistry Insights

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1 Anlyticl Chemistry Insights O r i g i n l R e s e r c h Open Access Full open ccess to this nd thousnds of other ppers t Quntittive Mss Spectrometric Anlysis of Ropivcine nd Bupivcine in Authentic, Phrmceuticl nd Spiked Humn Plsm without Chromtogrphic Seprtion Nhl N. Slm 1 nd Shudong Wng 2 1 Ntionl Orgniztion for Drug Control nd Reserch (NODCAR), 6-Abu Hzem Street, Pyrmids Ave, PO Box 29, Giz, Egypt. Emil: slm_nhl4@hotmil.com. 2 School of Phrmcy nd Centre for Biomoleculr Sciences, The University of Nottinghm, University Prk, NG7 2RD, U.K. Abstrct: The present study employs time of flight mss spectrometry for quntittive nlysis of the locl nesthetic drugs ropivcine nd bupivcine in uthentic, phrmceuticl nd spiked humn plsm s well s in the presence of their impurities 2,6-dimethylniline nd lkline degrdtion product. The method is bsed on time of flight electron spry ioniztion mss spectrometry technique without preliminry chromtogrphic seprtion nd mkes use of bupivcine s internl stndrd for ropivcine, which is used s internl stndrd for bupivcine. A liner reltionship between drug concentrtions nd the pek intensity rtio of ions of the nlyzed substnces is estblished. The method is liner from 23.8 to 23. ng ml -1 for both drugs. The correltion coefficient ws.996 in uthentic nd spiked humn plsm. The verge percentge recoveries in the rnges of 95.39% 2.75% ws obtined. The method is ccurte (% RE 5%) nd reproducible with intr- nd inter-ssy precision (RSD% 8.%). The quntifiction limit is 23.8 ng ml -1 for both drugs. The method is not only highly sensitive nd selective, but lso simple nd effective for determintion or identifiction of both drugs in uthentic nd biologicl fluids. The method cn be pplied in purity testing, qulity control nd stbility monitoring for the studied drugs. Keywords: ropivcine, bupivcine, 2,6-dimethylniline, time of flight mss spectrometry, phrmceuticl formultions, spiked plsm Anlyticl Chemistry Insights 9: This rticle is vilble from the uthors, licensee Liberts Acdemic Ltd. This is n open ccess rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution nd reproduction provided the originl work is properly cited. Anlyticl Chemistry Insights 9:4 11

2 Slm nd Wng Introduction Mss spectrometry (MS) is one of the most powerful nlyticl techniques, prticulrly for phrmceuticl nlysis, where good selectivity nd high sensitivity re often needed. In the phrmceuticl industry mesurements of drugs nd their metbolites in plsm re essentil for drug discovery nd development. The more ccurte nd rpid these mesurements, the more quickly drug cn progress towrds regultory pprovl. Time-of-flight mss spectrometer (TOF-MS) delivers high sensitivity, resolution, nd exct mss mesurements. A vriety of ion source nd softwre options mkes MS verstile choice for rnge of nlyticl chllenges. 1 5 Bupivcine (Bup), member of the pipecoloxylidide group (Fig. 1), is the most commonly used locl nesthetic. Commercil Bup is the opticlly inctive rcemic (RS) mixture of R- nd S-Bup. Severl recent studies hve demonstrted tht systemic exposure to excessive quntities of Bup result in crdio toxicity due to its high ffinity for, nd dwell time t, voltgegted sodium chnnels. A promising lterntive to Bup is ropivcine. Ropivcine (Rop) is structurl derivtive of Bup tht differs only by the replcement of the butyl group on the piperidine nitrogen tom of Bup with propyl group (Fig. 1). The minor structurl modifiction leds to reduced hydrophobicity nd the decresed bility to diffuse into the hert nd brin. As result, Rop hs lower systemic toxicity thn Bup. In ddition, Rop is mnufctured s pure S-enntiomer, further lowering the crdiotoxic potentil. Both drugs ct by blocking the conduction of impulses in trget nerve structures, primrily locted within the subrchinoid spce. 6 8 Severl methods using GC with 9, or without MS, 11 high performnce liquid chromtogrphy (HPLC) with UV, mss spectrometery (MS) or mperometric detection, 18 nd cpillry electrophoresis 19 hve been developed to nlyze the drugs nd their impurities in phrmceuticl formultions nd/or biologicl fluids. The im of this study is to develop rpid, ccurte nd simple method for determintion of both drugs in uthentic, phrmceuticl, spiked humn plsm s well s in presence of their impurities without chromtogrphic seprtion. The described method ws not investigted previously. In this work we describe how simple TOF ES-MS nlyticl method cn be used to determine Bup nd Rop in uthentic nd phrmceuticl formultions s well s in spiked humn plsm. The technique hs mny dvntges; no need for method development, short nlyticl time (1.5 min), nd miniml mount of solvent being required, coupled with high sensitivity, selectivity nd exct mss mesurements. Experimentl Mterils nd regents Ropivcine ws kindly supplied by AstrZenec Co., UK, certified to contin 99.%, CAS No Nropin TM vil contining 7.5 mg ml -1 ropivcine hydrochloride (AstrZenec Co., UK) ws purchsed from locl mrket. Bupivcine ws kindly supplied by Al-Debeiky Phrm Co., Egypt, its purity ws found to be 99.% ccording to BP 8. Bucin vil contining 5. mg ml -1 bupivcine hydrochloride (DeltSelect, GmbH, Germny) ws purchsed from the mrket. Humn plsm ws kindly donted from volunteers. The following regents nd Figure 1. Chemicl structures of ropivcine, bupivcine nd their impurity 2,6-dimethylniline. 12 Anlyticl Chemistry Insights 9:4

3 Quntittive mss spectrometric nlysis of ropivcine solvents were purchsed nd used without further purifiction: 2,6-dimethylniline (99.% Aldich UK, CAS No ), methnol, chloroform (HPLC grde, Fisher Scientific, UK), cetonitrile (LC-MS grde, Reidel-dehen, UK), ultr pure wter (ELGA, UK), formic cid (Sigm-Aldrich, UK), sodium hydroxide (BDH, UK) nd hydrochloric cid (Certified Fisher Scientific, UK). TLC-seprtion Pre-coted TLC pltes ( cm, luminium pltes coted with.25 mm silic gel F254) were purchsed from Merck UK. UV-Rdition (Cole-Prmer Instrument, Frnce) detective wvelength ws 254 nm. TOF-ES MS mesurements The TOF-ES-MS mesurements were performed using WATERS 2795 (Wters, UK) equipped with n utosmpler injector ( µl) nd Mss Lynx v 4.1. The system ws operting in the following regime: electrospry voltge, 3 kv; cpillry temperture, 1 C; smple solution flow rte,.1 ml/min. All nlysis ws performed in the positive ion detection mode. All smples were dissolved in % solution of cetonitrile in wter contining.1% formic cid. Preprtion of lkline degrdtion products The degrdtion products were lbortory prepred by heting mg of bupivcine hydrochloride in 1 M NOH ( ml) for 4 hours on hot plte t temperture C. The solution ws neutrlized with 1M HCl then the solution ws evported nd diluted to ml with methnol. Complete degrdtion ws monitored by TLC. The TLC seprtion ws crried out by using chloroform-methnol (9:1 v/v) s the mobile phse. The R f vlue is.71% ±.2% for both drugs. The mjor lkline degrdtion product, nmely 2,6-dimethylniline, which hs n R f vlue of.81% ±.1% s identified by comprison with the reference stndrd. Stndrd solutions nd clibrtion curves Stock solutions of ropivcine nd bupivcine were prepred in methnol t concentrtion of 1 mg ml -1, nd stored t 4 C. These were further diluted with % queous cetonitrile contining.1% formic cid to give the pproprite working solutions. Working solutions of ech drug were prepred to yield finl concentrtions of 23.8, 59.5, 119., 238., 595., 119. nd 23. ng ml -1 by further dilution with the sme solvent. Bupivcine (23. ng ml -1 ) ws used s IS for ropivcine. Ropivcine (23. ng ml -1 ), ws used s IS for bupivcine. Preprtion of spiked humn plsm Aliquots equivlent to ng ml -1 of ech drug nd 71. ng ml -1 of the internl stndrd in 1 ml plsm were sonicted for 5 minutes. Acetonitrile (2 ml) ws dded nd then centrifuged t rpm for minutes. One milliliter of the superntnt ws evported. Lbortory prepred solutions Mixtures of ropivcine nd bupivcine were prepred by mixing different concentrtions of ech drug with its impurity (2,6-dimethylniline) nd lkline degrdtion product, where the rtio.1%.% of the mixtures were obtined. Procedure Ten µl ech of the bove solutions ws injected in the TOF-ES-MS under conditions mentioned bove. The chrcteristic ions used for identifiction nd determintion of Rop, Bup nd 2,6-DMA were = 275, = 289 nd = 122 [M + H] +, respectively. Anlysis of phrmceuticl preprtions Milliliters equivlent to mg from the corresponding drug vil were trnsferred quntittively into ml volumetric flsks nd mde up to the volume with methnol. The procedure ws completed s mentioned bove. Clcultions The clibrtion curves were clculted by unweighted lest-squres liner regression nlysis of the concentrtions of the nlyte versus the pek intensity rtio of ions of nlyzed substnce of ropivcine ( 275) to tht of the IS ( 289). As for bupivcine ( 289) to tht of IS ( 275) ws used. Concentrtions of unknown smples were determined by pplying the liner regression eqution Anlyticl Chemistry Insights 9:4 13

4 Slm nd Wng of the stndrd curve to the unknown smple s pek intensity rtio. Method Vlidtion Limit of quntifiction The limit of quntifiction of the two drugs ws defined s the lowest concentrtion of the clibrtion curve. Precision nd ccurcy Precision nd ccurcy were ssessed by ssying freshly prepred solutions of the two drugs in triplicte t three concentrtion levels; 59.5, 119. nd 23. ng ml -1. Precision is reported s reltive stndrd devition (RSD%) of the estimted concentrtions nd ccurcy (% Reltive error) expressed s [mesured-nominl/nominl ]. Selectivity nd specificity Specificity is the bility of the method to mesure the nlyte response in the presence of impurity nd or degrdtion products. For specificity determintion, synthetic mixtures of different percentges of 2,6-dimethylniline nd degrdtion products of ech drug were dded to ech pure drug smple. The recovery percent ws clculted. Results nd Discussion The work includes (1) mss spectrometric identifiction nd determintions of ropivcine nd bupivcine; (2) genertion of the stndrd clibrtion curves; (3) identifiction nd determintion of drug substnces in spiked humn plsm; (4) determintion of both drugs in presence of lkline degrdtion nd/or impurity(2,6-dma); nd (5) quntittive nlysis of the individul ropivcine, bupivcine in their phrmceuticl preprtions. The mss spectr of ropivcine nd bupivcine nd their internl stndrds re shown in Figure 2. Under the conditions of TOF ES-MS in positive mode, the spectr displys intense peks of [M + H] + with ions of the highest mss to chrge, e.g. = for ropivcine nd for bupivcine, respectively. Linerity rnge ws found to hold good over (A) (B) (C) % Reltive Abundnce % Reltive Abundnce % Reltive Abundnce [M+H] Bupivcine [M+H] Bupivcine Figure 2. The typicl Mss spectr of (A) ropivcine, 23. ng ml -1 (B) bupivcine, 23. ng ml -1 (C) ropivcine (119. ng ml -1, nlyte) nd bupivcine (23. ng ml -1, internl stndrd) in % queous solution of cetonitrile contining.1% formic cid. 14 Anlyticl Chemistry Insights 9:4

5 Quntittive mss spectrometric nlysis of ropivcine concentrtion rnge of ng ml -1 for both drugs. The results of regression dt re presented in Tble 1. Anlysis of studied drugs in spiked humn plsm re shown in Figure 3. The linerity ws in the rnge of ng ml -1 for Rop while it is within ng ml -1 for Bup in 1 ml plsm smple, which is the nticipted concentrtion rnge in clinicl investigtion of drug phrmcokinetics. The mximum plsm level of Rop nd Bup fter different rout of dministrtions were found to be more thn ng ml which could be ssessed by the proposed method. The high sensitivity of the proposed method llowed the determintion of both drugs in spiked humn plsm. Liner regression nlysis of the dt gives the equtions, A =.27 C +.82, r =.996, for Rop nd A =.5441 C.137, r =.999, for Bup. Where A is the pek intensity rtio for = 275/289 for Rop nd 289/275 for Bup, C is the concentrtion in ng ml -1 nd r is correltion coefficient. The dt stted in Tble 1, indicte tht the method is efficient for determintion of the studied drugs in biologicl fluids s there is no significnt differences between the results for determintions of both drugs in uthentic nd spiked plsm smples. Vlidtion Dt Linerity nd limit of quntifiction Clibrtion curves for ropivcine nd bupivcine exhibited good linerity over the concentrtion rnge studied ( ng ml -1 ) for both drugs in uthentic nd spiked humn plsm s stted in Tble 1. From the results, it is cler tht there is no interference from the plsm mtrix demonstrting the efficiency for determintion of the drugs in biologicl fluids by TOF ES-MS. The limit of quntifiction (LOQ) ws chosen s the lowest clibrtion stndrd concentrtion (23.8 ng ml -1 ) for the studied drugs in uthentic nd spiked humn plsm. Precision nd ccurcy Tble 2 summrizes men vlues, precision, nd ccurcy of intr- nd inter-ssy nlysis. Precision nd ccurcy were within the rnges cceptble for nlyticl nd bio-nlyticl purposes. Intr-dy precision rnged from. to 3.61% for ropivcine while 2.16 to 3.33% for bupivcine in drug substnces. While in spiked humn plsm rnged from 1.7 to 7.98% for ropivcine nd from.95 to 5.13 for bupivcine (Tble 3). Inter-dy precision did not exceed 8.% over the three level concentrtions for three dys in drug substnces nd spiked humn plsm. The ccurcy Tble 1. Linerity, recovery nd LOQ of TOF ES-MS ssy for ropivcine nd bupivcine in uthentic nd spiked humn plsm. Prmeters Ropivcine Bupivcine Authentic Spiked humn plsm Authentic Spiked humn plsm Linerity ng ml Regression eqution Slope (b) SE of slope Intercept () SE of intercept Correltion coefficient (r) SE of estimtion Recovery Men b ± RSD% ± ± ± ± 2.88 LOQ ng ml Regression eqution, A = + bc, where A is the pek intensity rtio for = 275. /289. for Rop, nd A is the pek intensity rtio for = 289./275. for Bup, C is the concentrtion. b n = 6. Anlyticl Chemistry Insights 9:4 15

6 Slm nd Wng (A) (B) (C) (D) % Reltive Abundnce % Reltive Abundnce % Reltive Abundnce % Reltive Abundnce [M+H] Bupivcine Bupivcine [M+H].... Figure 3. The mss spectr of spiked humn plsm (A) humn plsm, control (B) bupivcine, 119. ng ml -1 (C) ropivcine, 119. ng ml -1, (D) mixture of bupivcine, 23.8 ng ml -1 (nlyte) nd ropivcine, 23. ng ml -1 (IS) in % queous solution of cetonitrile contining.1% formic cid. Tble 2. Intr nd inter-dy precision nd ccurcy of TOF ES-MS ssy for ropivcine nd bupivcine in uthentic smples. Drug substnces Ropivcine 59.5 Conc. ng ml -1 Precision RSD% Accurcy RE% Bupivcine 59.5 n = Inter Intr Inter Intr Anlyticl Chemistry Insights 9:4

7 Quntittive mss spectrometric nlysis of ropivcine Tble 3. Intr nd inter-dy precision nd ccurcy of TOF ES-MS ssy for ropivcine nd bupivcine in spiked humn plsm. Drug substnces Ropivcine Bupivcine n = 3. Conc. ng ml -1 Precision RSD% Accurcy RE% Intr Inter Intr Inter (A) (B) (C) (D) % Reltive Abundnce % Reltive Abundnce % Reltive Abundnce [M+H]2.6-Dimethylniline [M+H]2.6-Dimethylniline % Reltive Abundnce [M+H]2.6-Dimethylniline [M+H] Bupivcine [M+H] Bupivcine.. [M+H]2.6-Dimethylniline Figure 4. The mss spectr of (A) 2,6-dimethylniline (B) Alkline degrdtion products (C) mixture of ropivcine, 23. ng ml -1 (internl stndrd), bupivcine 119. ng ml -1 (nlyte) nd their impurity 2,6-dimethylniline (D) mixture of both drugs with lkline degrdtion products, in % queous solution of cetonitrile contining.1% formic cid. Anlyticl Chemistry Insights 9:4 17

8 Slm nd Wng of the technique ws considered stisfctory, since between-dy vrition over the concentrtion rnge studied ws found to be less thn 5%. Selectivity nd specificity Ropivcine nd bupivcine re mides expected to lkline degrdtion through clevge of mide linkge with production of 2,6-dimethylniline. Solutions of lkline degrdtions of ech were tested by TLC ginst pure smple of 2,6-dimethylniline. The sme spots of ech hve the sme R f (.81) of the pure compound. For further confirmtion, TOF-ES-MS ws crried for ech of both compounds. The product obtined from lkline degrdtion hs = corresponds to protonted 2,6-dimethylniline. The mss spectrometric determintions of Rop nd Bup in the presence of their lkline degrdtion products re shown in Figure 4. Synthetic compound 2,6-dimethylniline ws used s control, nd in the spectrum (A) the ion of the mss to chrge () corresponding to 2,6-dimethylniline ws identified. The highest ion pek ws = ( ) which might be resulted in cetonitrile solvent interference in the system. The sme peks ppered in the spectrum (B) nd 2,6-dimethylniline ws the mjor degrdtion product of bupivcine. In ddition, reltive low of moleculr ion pek t = ws observed which my be ssigned s m-xylene ion. The spectr (C) nd (D) disply the intensive ion peks with = [M + H] + for 2,6-dimethylniline clerly indicting 2,6-dimethylniline to be the mjor lkline degrdtion product of Rop nd Bup. The specificity ws lso ssessed by nlyzing synthetic mixtures of ech drug with its lkline degrdtion product in concentrtion rnging from.1 to.%. The results revel the high selectivity nd sensitivity of the method which cn determine the impurity in concentrtion down to.1% present in both drugs (Tble 4). Tble 4. Specificity of TOF ES-MS ssy for ropivcine nd bupivcine in uthentic smples. Degrdtion products nd/or 2,6-DMA% Recovery % ± RSD% Ropivcine Bupivcine ± ± ± ± ± ± 1.67 Men of four different experiments. Tble 5. Results for the determintion of ropivcine nd bupivcine in phrmceuticl formultions by the proposed TOF ES-MS method. Preprtions Nropin vil, 7.5 mg ml -1 ropivcine hydrochloride Bucin vil, 5. mg ml -1 bupivcine hydrochloride Men of five experiments. TOF ES-MS Men recovery % RSD% Anlysis of phrmceuticl preprtions The method ws pplied to determine ropivcine nd bupivcine in Nropin nd Bucine vil respectively. The % RSD ws less thn 3.%, indicting the precision of the method, the results re presented in Tble 5. Conclusion In this mnuscript, we described newly developed TOF-MS bsed method for quntittive determintion of ropivcine nd bupivcine in uthentic, phrmceuticl dosge forms nd the spiked humn plsm without chromtogrphic seprtion. The strtegy of this pproch consists in direct multi-ion detection of nlytes with reference to internl stndrds with close structures to the nlyte. The method cn lso be used to identify the degrdtion products in minute mounts in presence of the corresponding ropivcine or bupivcine. The method could be routinely used for quntittive drug nlysis in phrmceuticl formultions nd biologicl medi s well s for ssessing drug purity nd stbility. Acknowledgments N. Slm would like to thnk the NODCAR, Egypt nd School of Phrmcy, The University of Nottinghm, UK, for the visiting scholrship wrds. Disclosure The uthors report no conflicts of interest. References 1. Krtsso YO, Logunov IV, Sergeev MG, Nikolev EN, Vrfolomeev SD, Chistykov VV. Phrmceuticl Chemistry Journl. 7;41: Jysimhulu K, Hunt SM, Kneshiro ES, Wtnbe Y, Giner JL. Am J Mss Spectrom. 6;18: Hn X, Yng K, Yng J, et l. Mss Spectrom. 6;17: Delong CJ, Bker PR, Smuel M, Cui Z, Thoms MJ. Lipid Res J. 1;42: Koivuslo M, Himi P, Heikinheimo L, Kostinen R, Somerhrju P. Lipid Res J. 1;42: Anlyticl Chemistry Insights 9:4

9 Quntittive mss spectrometric nlysis of ropivcine 6. The United Stte Phrmcopoei. The Ntionl Formulry USP United Stte Phrmcopoeil Convection Inc. 7. p Sweetmn SC, Mrtindle. The Extr Phrmcopoei, 34 ed, Phrmceuticl Press, London 5. p. 1371, 1372, Brunton LL, Lzo JS, Prker KL. Goodmn nd Gilmn s. The Phrmcologicl Bsis of Therpeutics, 11th. Ed. Mc Grow Hill, New York, U.S.A. 6. p Throui A, Wtson DG, Skellern GG, Hudson SA, Petrie P, Fccend K. J Phrm Biomed Anl. 1996;15:251.. Wtnbe T, Nmer A, Yshiki M, Iwski Y, Kojim T. J Chromtogr B. 1998;9: Bniceru M, Croitoru O, Popescu SM. J Phrm Biomed Anl. 4;593: Gross AS, Nicoly A, Eschlier A. J Chromtogr B. 1999;728: Einosuke T, Tkko N, Shinichi I, Ktsuy H. J Chromtogr B. 6;834: Rifi N, Hsin O, Hope T, Skmoto M. Ther Drug Monitor. 1;23: Kwno S, Murkit H, Ymmoto E, Askw N. J Chromtogr B Anl Tech Biomed nd Life Sci. 3;792: Koehler A, Oertel R, Kirch W. J Chromtogr B. 5;88: Abdel-Rehim M, Bielenstein M, Askemrk Y. Anl Chim Act. 3;49: Zbigniew F, Emil B, Agt P, Mlgorzt WG. J Phrm Biomed Anl. 5;37: Krisko RM, Schieferecke MA, Willims TD. Lunte CE. Electrophoresis. 3;24:23.. Florey K. Anlyticl Profiles of Drug Substnces nd Excipients; Acdemic Press: Inc., 1993;19: Put O, Schreiber E, Lcroix F, Meyrieux V, Simon N, Lvrut T, Cmboulives J, Bruguerolle B. Br J Anesth. 4;92: Concepcion M, Richrd Arthur G, Susn M, Steele Angel M, Bder BG. Covino Anesth Anlg. 199;:. 23. Covino BG, Feldmn HS, Arthur GR. Anesth Anlg. 1988;67:53. Publish with Liberts Acdemic nd every scientist working in your field cn red your rticle I would like to sy tht this is the most uthor-friendly editing process I hve experienced in over 1 publictions. Thnk you most sincerely. The communiction between your stff nd me hs been terrific. Whenever progress is mde with the mnuscript, I receive notice. Quite honestly, I ve never hd such complete communiction with journl. LA is different, nd hopefully represents kind of scientific publiction mchinery tht removes the hurdles from free flow of scientific thought. Your pper will be: Avilble to your entire community free of chrge Firly nd quickly peer reviewed Yours! You retin copyright Anlyticl Chemistry Insights 9:4 19