Development of n Assy for in Amlodipine y IC nd Second Assy to Simultneously Determine Amlodipine nd y HPLC Brin De Bor nd Jeffrey Rohrer, Thermo Fisher Scientific, Sunnyvle, CA, USA
Overview Purpose: Amlodipine esylte is clcium chnnel locker tht is used for the tretment of hypertension nd ngin. Similr to other clcium chnnel lockers, mlodipine works y relxing the rteril muscles, which decreses peripherl resistnce nd therefore reduces lood pressure. Active phrmceuticl ingredients (APIs) such s mlodipine re commonly mnufctured into cid ddition slts to improve iovilility. Amlodipine esylte is the slt form of mlodipine, which is produced from the rection of mlodipine ( wek se) nd enzenesulfonic cid. It is criticl to quntify the concentrtion of the API nd counterion in the drug formultion to estlish stoichiometry, completeness of slt formtion, nd mss lnce. Method: We developed n isocrtic ion chromtogrphy (IC) method with suppressed conductivity detection for determining esylte in mlodipine esylte. A second method ws developed for the simultneous determintion of mlodipine nd esylte using trimode (reversed-phse, nion-exchnge, ction-exchnge) high-performnce liquid chromtogrphy (HPLC) column with UV detection. The HPLC method used moile phse contining 70% 100 mm mmonium cette/30% cetonitrile. The smple ws prepred in deionized (DI) wter for the IC method nd moile phse for the HPLC method. Results: The linerity, limit of detection (LOD), limit of quntittion (LOQ), precision, nd ccurcy re presented for oth methods. Correltion coefficients for the stndrd curves rnged from 0.9984 to 0.9995 with pek re RSDs <1. The recovery of esylte spiked into the API rnged from 99% to 104% using the IC method. In ddition, the mount of esylte in mlodipine esylte rnged from 28.1 28.4% with the IC method nd 27.6 28.2% with the HPLC method, which is comprle to the theoreticl mount of 27.9% sed on the moleculr weight of the drug formultion. Conclusions: These results demonstrte the ility to ccurtely determine the counterion in drug formultion y IC with suppressed conductivity detection. In ddition, comprle results cn e otined for the counterion when using HPLC with UV detection. The HPLC method lso provides dditionl dvntges y simultneously determining the API nd counterion, which is cost-effective pproch for mny lortories. The two methods re shown to hve good linerity, sensitivity, precision, nd ccurcy. Introduction Reversed-phse liquid chromtogrphy (RPLC) is the most common pproch used to nlyze the API, ut it often fils to retin the more hydrophilic nlytes tht re commonly used s counterions during the drug mnufcturing process. For the determintion of counterions, IC hs proven to e relile, sensitive, nd selective technique. 1 The current U.S. Phrmcopei (USP) method for determining mlodipine esylte (Figure 1) descries RPLC for seprting the API using 50 mm triethylmine (ph 3)/MeOH/MeCN t rtio of 50:35:15 with UV detection t 237 nm. 2 However, there is currently no USP method for determining the esylte counterion. Mixed-mode chromtogrphy comines RPLC nd ion-exchnge properties to simultneously seprte the drug molecule nd counterion. However, the determintion of phrmceuticl counterions y IC provides other enefits, such s improved sensitivity nd the ility to use electrolyticlly generted eluents (i.e., KOH or MSA), which elimintes the lor nd potentil errors of mnul eluent preprtion. In this study, mlodipine nd esylte (i.e., enzenesulfonic cid) were seprted on Thermo Scientific Acclim Trinity P1 column. This trimode column contins unique construction tht provides sptil seprtion of the nion-exchnge, ctionexchnge, nd reversed-phse modes, which llows ech to e independently controlled. After seprtion, mlodipine ws detected y UV t 237 nm nd esylte ws detected t 262 nm. In ddition, seprte IC method ws developed nd compred to the HPLC method for determining esylte in the drug sustnce. FIGURE 1. Structure of mlodipine esylte. 2 Development of n Assy for in Amlodipine y IC nd Second Assy to Simultneously Determine Amlodipine nd y HPLC
Methods Smple Preprtion Amlodipine esylte (1 mg/ml): Weigh pproximtely 10.0 mg of mlodipine esylte on n nlyticl lnce nd dissolve the solid in 10 ml of DI wter for the IC method or 70% NH 4 OAc/30% MeCN for the HPLC method. Perform seril dilutions to chieve the desired trget concentrtion. Method A: IC A Thermo Scientific Dionex ICS-5000 + HPIC Ion Chromtogrphy system ws used for determintion of esylte. The system included n SP Single Pump, EG Eluent Genertor, DC Detector/Chromtogrphy Comprtment, nd n AS-AP Autosmpler. Method B: HPLC Thermo Scientific Dionex UltiMte 3000 Rpid Seprtion LC (RSLC) System including: SRD-3600 Integrted Solvent nd Degsser Rck HPG-3400RS Binry Rpid Seprtion Pump with Solvent Selector Vlves WPS-3000TRS Rpid Seprtion Wellplte Smpler Thermosttted TCC-3000RS Rpid Seprtion Thermosttted Column Comprtment DAD-3000RS Rpid Seprtion Diode Arry Detector TABLE 1. Chromtogrphic conditions. Columns Moile Phses Method A IC Thermo Scientific Dionex IonPc AG18/AS18 set, 2 mm KOH delivered using Thermo Scientific Dionex EGC 500 KOH Eluent Genertor Crtridge with Thermo Scientific Dionex CR-ATC Continuously Regenerted Ction Trp Column Method B HPLC Acclim, Trinity P1, 3 µm Anlyticl, 3 50 mm A: 100 mm NH 4 OAc, ph 5 B: 100% MeCN Conditions 60 mm KOH 0 8 min 30% B Totl Run Time 15 min 8 min Flow Rte 0.25 ml/min 0.50 ml/min Inj. Volume 5 μl 5 μl Column Temp 30 ºC 30 ºC Detection Results Suppressed conductivity, Thermo Scientific Dionex ASRS 300 Anion Self-Regenerting Suppressor, 2 mm, recycle mode, 38 ma UV t 237 nm (mlodipine) nd 262 nm (esylte) This study demonstrtes two methods: n IC method to determine the esylte counterion; nd seprte HPLC method to simultneously determine mlodipine nd esylte. Method A: Determintion of y IC The Figure 2 chromtogrm demonstrtes the determintion of esylte in mlodipine esylte using Dionex IonPc AS18 column with suppressed conductivity detection. As shown, esylte is seprted in pproximtely 12 min using 60 mm electrolyticlly generted KOH eluent. At this eluent concentrtion, trce nions (if present) elute ner the void nd therefore do not interfere with the determintion of the counterion. To vlidte the method, the linerity, precision, nd ccurcy were evluted. Thermo Scientifi c Poster Note PN70869_E 11/13S 3
Tle 2: Linerity, LOD, nd LOQ for esylte. Rnge Coefficient of Determintion (r 2 ) LOD LOQ c 0.5 20 0.9995 0.02 0.07 Qudrtic Fit LOD = 3x S/N c LOQ = 10x S/N FIGURE 2: Determintion of esylte in mlodipine esylte using Method A. 1.5 Columns: Dionex IonPc AG18/AS18, 1 2 mm Eluent: 60 mm KOH Eluent Source: Dionex EGC 500 KOH with Dionex CR-ATC Flow Rte: 0.25 ml/min Inj. Volume: 5 µl µs Detection: Suppressed conductivity, Dionex ASRS 300, 2 mm, recycle mode Smple: USP Amlodipine RS Peks: 1. 2.8 µg/ml 0.5 0 5 10 15 Minutes Precision The method precision ws determined y performing six replicte injections of 10 µg/ml mlodipine esylte USP stndrd tht ws prepred on three seprte dys. TABLE 2: Precision nd recovery of the esylte counterion in mlodipine esylte (Method A). Dy Mesured % in Amlodipine Pek Are RSD Recovery (%) 1 2.84 28.4 0.64 102 2 2.82 28.2 0.81 101 3 2.81 28.1 0.45 101 n = 6 clculted reltive to the theoreticl 27.9% esylte Accurcy Amlodipine esylte ws spiked t 50%, 100%, nd 150% of the trget esylte concentrtion nd the recovery ws clculted. The recovery rnged from 98.7 104.4%. Rpid Seprtion of y High Pressure IC To increse smple throughput, 4 µm Dionex IonPc AS18 column (2 150 mm) ws riefly evluted for the determintion of esylte in mlodipine esylte. As shown in Figure 3, esylte is seprted in pproximtely 5 min on the 4 µm column compred to pproximtely 12 min using the column nd conditions descried in Method A. 4 Development of n Assy for in Amlodipine y IC nd Second Assy to Simultneously Determine Amlodipine nd y HPLC
FIGURE 3: Seprtion of esylte in mlodipine esylte on 4 µm Dionex IonPc AS18 column. Columns: Dionex IonPc AG18/AS18 4 µm, 2 mm 1.6 1 Eluent: 60 mm KOH Eluent Source: Dionex EGC 500 KOH with Dionex CR-ATC Flow Rte: 0.38 ml/min Inj. Volume: 5 µl µs Detection: Suppressed conductivity, Dionex ASRS 300, 2 mm, recycle mode Smple: 10 µg/ml USP Amlodipine RS Peks: 1. 2.8 µg/ml 0.2 0 2 4 Minutes 6 8 Method B: Determintion of Amlodipine nd y HPLC Figures 4A nd 4B show chromtogrms demonstrting the determintion of mlodipine t 237 nm nd esylte t 262 nm, respectively in mlodipine esylte using n Acclim Trinity P1 trimode column. Although oth compounds cn e quntified simultneously, the primry focus of this method ws to determine the esylte counterion. To vlidte the method, the linerity, precision, nd ccurcy were evluted. TABLE 3: Linerity, LODs nd LOQs for mlodipine nd esylte. Rnge Coefficient of Determintion (r 2 ) LOD c LOQ d Amlodipine 10 150 0.9998 0.14 0.47 10 100 0.9984 2.3 7.7 Prepred s mlodipine esylte ccording to USP protocol; detected t 237 nm Detected t 262 nm c LOD = 3x S/N d LOQ = 10x S/N FIGURE 4: Simultneous determintion of mlodipine nd esylte. Column: Acclim Trinity P1, 3 µm, 3.0 50 mm 120 Eluents: A. 100 mm NH 4 OAc, ph 5.0 A B. 100% MeCN 1 Isocrtic: 30% B from 0 8 min Flow Rte: 0.5 ml/min Inj. Vol.: 5 µl Detection: A. UV, 237 nm; B. UV, 262 nm mau Smple: A. 50 µg/ml USP Amlodipine RS B. 200 µg/ml USP Amlodipine RS Peks: A B 1. Amlodipine 50 µg/ml 2. 60-5 0 2 4 6 8 10 B 1 mau 2-2 0 2 4 6 8 Minutes Thermo Scientifi c Poster Note PN70869_E 11/13S 5
Accurcy Amlodipine esylte ws spiked t 50% nd 100% of the trget esylte concentrtion nd the recoveries were clculted. The recovery t the 50% spiked concentrtion ws 103% nd 98.1% for the 100% spiked concentrtion. Precision The method precision ws determined y performing six replicte injections of 200 µg/ml mlodipine esylte USP stndrd tht ws prepred on three seprte dys. TABLE 4: Precision nd recovery of the esylte counterion in mlodipine esylte using Method B. Dy Mesured % in Amlodipine Pek Are RSD Recovery (%) 1 55.6 27.8 0.90 99.6 2 55.2 27.6 0.75 98.9 3 56.5 28.2 0.51 101 n = 6 clculted reltive to the theoreticl 27.9% esylte Conclusion (i.e., enzenesulfonte) ws relily determined in mlodipine esylte y nion-exchnge chromtogrphy using simple smple prep nd electrolyticlly generted KOH eluent. Amlodipine nd esylte were simultneously determined on trimode HPLC column, which reduces time nd lor ssocited with running second method. The results for determining the esylte counterion were comprle etween the IC nd HPLC methods. References 1. De Bor, B.M.; Rohrer, J.S. Ion Chromtogrphy of Drug Product Counter-Ions nd other Ions in Drug Products. In Applictions of Ion Chromtogrphy for Phrmceuticl nd Biologicl Products; Bhttchryy, L., Rohrer, J.S. Eds.; John Wiley & Sons, Inc.: Hooken, NJ, 2012; pp 259 269. 2. U.S. Phrmcopei Ntionl Formulry 2011: USP 34 NF 29, U.S. Phrmcopeil Convention, Rockville, MD, pp 1872. 6 Development of n Assy for in Amlodipine y IC nd Second Assy to Simultneously Determine Amlodipine nd y HPLC
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