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2S CIENTIFICAThermo Fisher Scientific BrandAB71180-E 06/14S Fast Nevirapine Impurity ProfilingUsing UHPLC-DAD Holger Franz and Susanne FabelThermo Fisher Scientific， Germering， Germany Key Words Active Pharmaceutical Ingredient (API)， Impurity profiling， Ballisticgradient， HPLC to UHPLC transfer tool， ICH requirements Goal To develop a fast ballistic gradient UHPLC method optimized forsimultaneous analysis of an API and impurities in a nevirapine tablet IntroductionNevirapine is a non-nucleoside reverse transcriptaseinhibitor with activity against human immunodeficiencyvirus type 1 (HIV-1)， currently marketed for the treatmentof HIV-1 infected adults. The United States Pharmacopeia(USP) uses a reversed-phase high-performance liquidchromatography (HPLC) separation with UV detectionto determine nevirapine and its impurities. The relatedcolumn is a 4.6×150 mm column packed with L60(spherical， porous silica gel， 5 pm in diameter).2 Due tothe strong retention of impurity C， the USP monographmethod requires about 30 minutes to separate this APIand known impurities. A previous Dionex， now part ofThermo Scientific， application note demonstrated that anHPLC-UV separation can meet or exceed the chromato-graphic requirements of the USP monograph method fornevirapine while requiring about half the analysis timeper sample. Table 1.Reagents and chemicals. Here we report further optimization of this approach，using a state of the art gradient UHPLC-UV method.Applying ballistic gradients with latest-generation UHPLCequipment achieves significantly shorter analysis timewhile maintaining compliance with ICH (InternationalConference on Harmonization of Technical Requirementsfor Registration of Pharmaceuticals for Human Use)requirements. Compound Supplier P/N Nevirapine Anhydrous USP Standard 1460703 Related compound A LGC Standards MM1146.01 Related compound B LGC Standards MM1146.02 Impurity C LGC Standards MM1146.03 Ammonium acetate Fisher Scientific A114-50 Acetonitrile OPTIMALC/MS Fisher Scientific A955-212 Ultra-pure lab water，18.2 MQ·cm at 25°C n.a. n.a. Equipment · Thermo ScientificVanquish UHPLC Systemconsisting of： - Binary Pump H (P/N VH-P10-A) - Split Sampler HT (P/N VH-A10-A) - Column Compartment H (P/N VH-C10-A) -Diode Array Detector HL (P/N VH-D10-A) ·Thermo Scientific"Dionex ChromeleonMChromatography Data System software 6.80 SR14 Experimental Conditions Column： Thermo Scientific" Syncronis" C18， 1.7 pm， 2.1×100 mm Mobile Phase： A) 10 mM NH 0Ac， pH 5.0 with acetic acid/ acetonitrile (85%/15%v/v) Gradient： B) Acetonitrile 0-0.730 min： 30-70% B： 0.730-1.100 min： 70%B； 1.100-1.150 min： 70-30%B； 1.150-2.800 min： 30% Flow Rate： 0.800 mL/min Pressure： 950 bar (max.) lemperature： 50℃ Injection Volume： 1 pL Detection： 240 nm， 100 Hz， 0.05 s response time， 4 nm slit width， 4 nm bandwidth Flow cell： LightPipe， 10 mm Results and Discussion A number of rules should be followed for method transferfrom HPLC to UHPLC， to adapt parameters such as flowrate，injection volume， or gradient profile (if applicable)to the new column characteristics. The Thermo ScientificMethod Transfer Tool is a universal， multi-language toolthat streamlines this processt and was used to transferthe USP method to a high-efficiency Syncronis UHPLCcolumn. The application was accelerated by a factor of1.3 by using the tools boost factor functionality. Theresult was 130% of the initial linear mobile phase velocitythrough the column. With sub-2 um particle columns thiscan easily be done while keeping the chromatographicefficiency almost constant. The USP monograph uses 25 mMNHOAc， pH 5.0/acetonitrile (80%/20% v/v) as mobile phase. In themethod transfer， the buffer concentration was reducedto 10 mM and a gradient was applied with a maximumacetonitrile content of 80%. The buffer concentration wasreduced to maintain compatibility of the buffer with thehigher organic content. The detection wavelength waschanged from 220 nm to 240 nm to eliminate baselinedrift caused by varying absorption over the course of thegradient. Figure 1 shows a calibration curve of the active pharma-ceutical ingredient nevirapine with absorbances up to3000 mAU. Even up to this high absorption， the calibra-tion curve is almost perfectly linear with a correlationcoefficient of R2=0.9998. Figure 2 shows a chromatogram of the 0.66 mg/mLstandard. The zoom into the baseline reveals a numberof impurities， including known compounds A， B， and Cmentioned in the USP method as well as four additionalimpurities. Table 2 identifies the peaks of the chromato-gram， the related signal-to-noise ratio and the relative areaof the individual compound. 0.8 Figure 1. Calibration curve of nevirapine demonstrating excellent linearity up to3000 mAU. Minutes Figure 2. Impurity profiling of Nevirapine (0.66 mg/mL) with UHPLC gradientand zoom into the baseline to show related impurities. Table 2. Peak identification for Figure 2 with related signal-to-noise ratio andarea percentage of the individual compound. PeakNo. Compound Signal-to-Noise Area %] 1 Related compound B 28 0.065 2 Nevirapine 17602 99.790 3 Related compound A 16 0.050 4 Impurity C 53 0.053 5 Unknown 1 8 0.007 6 Unknown 2 17 0.011 7 Unknown 3 10 0.008 8 Unknown 4 11 0.013 The ICH defines the reporting threshold for impuritiesdepending on the maximum daily dose. For nevirapine，a dosage of 400 mg/day translates into a reportingthreshold of 0.05%. According to the common definitionof the Limit of Quantitation (LOQ)6 defined as S/N ratioof at least 10， the nevirapine assay here described allowsquantitation down to 0.008% relative area. The compliance with the ICH guidelines was achieveddespite challenging chromatographic conditions. Weapplied a ballistic 44 s linear gradient， achieving theelution of all relevant impurities within 1.2 min. The totalrun time was 2.8 min， using default detection parametersand 100 Hz data collection rate. This application istherefore a good example that the Vanquish systemperformance easily supports even ambitious analysis goalswithout the need for time-consuming instrumentoptimization. Conclusion This application describes an optimized method for theimpurity profiling of nevirapine using a ballistic gradientmethod. The separation is completed in 2.8 min， com-pared to 80 min of the isocratic USP method. Even underchallenging chromatographic conditions， the VanquishUHPLC system easily enables the simultaneous detectionof the API and related impurities while achieving compli-ance with the ICH guidelines on impurity monitoring. ( References ) ( 1. Pav， J.W.， Rowland， L.S.， Korpalski， D.J： J Pharm Biomed Anal. 1999 Jun； 20(1-2)：91-8. ) ( 2. United States Pharmacopeia， 3 7th Revision. Rockville，MD. US Pharmacopeial Convention 2014， 3973-3974 ) ( 3.Dionex (now part of Thermo Scientific) Application Note 180： Determination of Nevirapine Using HPL C with UV Detection [Online] http：//ww w . t hermoscien- t i fic.com/content/dam/tfs/A T G/CM D / CMD% 20 Documen t s/AN - 1 8 0-D e term i n a tion-Nev i rapine-L PN1 9 34.pdf(a ccessed May 23， 2014) ) ( 4. Franz， H. ； F abel， S.：Thermo Fisher S c ientific Technical Note 75： A Universal Tool for Method Transfer From HPLC to UHPLC，[Online] 2014， ht tp ：//www.ther mo- s ci e ntific.com/conten t /dam/tfs/A T G/CMD/ CM D%20 Documents/Product%20Manuals%20%26% 2 0 S pecifica t ions/Chromatography/Liquid% 2 0Ch r omatog- raphy/Liquid%20C h romatography%20Systemsl Standard%2 0 Sys te ms/TN 75 - RSLC -M e thod-Tr a ns- fer-TN 7 0828-E.pdf ( accessed May 23，2014). ) ( 5. International Conferences o n Harmonization， Guidance on I mpurities in New Drug Products. Q3A(R2)， 2006 ) ( 6. Snyder， L.R.， Kirkland，J.J.， Glajch， J.L.： Practical HPLC m e thod development， 1997， John Wiley 6 Sons，Inc.， Second E dition， p . 645 ) ( ◎ 2014 Th er mo Fisher Scient i fic Inc. A ll r i ghts reserved. ISO is a t rademark o f the Intern a tional Standards Org a nization. All other trademarks are t he p r o pert y of T h erm o Fi sh er Scie n t i f i c a nd its subsidi a ries . This info r mation i s presented as an example of the capabil i tie s of T hermo F isher Scienti f ic product s. It is not intended to e ncourage use of t he s e prod u cts in an y manne r s that m i g ht i n f r in g e t h e i ntellec tu al property rights of o thers. S p e cifications， t e rms an d p ricing are sub j ect to change. Not a l l prod u cts ar e availa b le in all co u ntries. Please c o nsult your l o c al s a les represent at iv e f o r d e t ai ls . ) Thermo Fisher Scientific， Sunnyvale， CA USA is /SO 9001：2008 Certified. 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