PEG 400 by UHPLC with corona ultra

Corona ® ultra Charged Aerosol DetectorUsing UHPLC and Corona ® ultraDetection to Shorten the Analysis Timeof ExcipientsApplication noteNew sub-2 micron column technology in thefield of liquid chromatography has lead to theintroduction of Ultra-High Pressure LiquidChromatography (UHPLC). These changes haveresulted in systems and columns that canoperate at pressures up to 15,000 psi andprovide unprecedented separations. This allowsfor faster peak elution and shorter run times,which reduces the costs associated with analysttime and solvent consumption. Universaldetection of fast peaks with minimal peakbroadening is required to maximize the utility ofthis new technology. ESA Biosciences hasintroduced the next generation of ChargedAerosol Detection (CAD) with its ultra-highperformanceversion of its Corona CAD. TheCorona ® ultra detector incorporates all thefeatures introduced in the Corona CAD detector,along with faster data sampling times, improvedfilter algorithms, nebulizer temperature controland other design features. These keyimprovements allow the Corona ultra to meetthe detector demands required by UHPLCsystems for rapid analysis and sharp peak shape.Polyethylene glycol (PEG) is one of the mostcommercially important polymers in its class.The low molecular weight PEGs are widely usedin many fields, including its use as apharmaceutical excipient. Therefore,characterization of PEG products becomes animportant step in the drug development processand appropriate selection for quality. Thesecompounds have poor chromophoric properties,which often make the final analysis suffer fromboth sensitivity and reproducibility issues. Thework outlined here looks at PEG 400, first on atraditional HPLC platform and then in more130.00120.00110.00100.00mV90.0080.0070.0060.0050.0040.0030.0020.0010.000.000.00 1.00 2.00 3.00 4.00 5.00MinutesFigure 1. PEG 400 (~2 μg on column) Analyzed usingeither HPLC or UHPLC Conditions (inset).detail on a UHPLC platform using the Corona ultra. Theanalytical method was transferred to the UHPLC platformwith no significant loss in resolution, peak shape, or overallcluster profile.Method Parameters - HPLCSystem:Agilent 1200 RRLCColumn:Capcell PAK C18 MGIII4.6 x 250 mm, 5 µmMobile Phase A: DI Water / Acetonitrile (93 : 7)Mobile Phase B: AcetonitrileGradient: HPLC - Table 1Injection Volume: 1 µL of a ~2 mg / mL standard in DI waterDetector: Corona ultraFilter:CoronaRange:100pA

Corona ® ultra Charged Aerosol DetectorApplication noteMethod Parameters - UHPLCSystem:Waters Acquity UPLCColumn:Acquity UPLC BEH C182.1 x 50 mm, 1.7 µmMobile Phase A: DI Water / Acetonitrile (93 : 7)Mobile Phase B: AcetonitrileGradient: HPLC - Table 2Injection Volume: 1 µL of a ~2 mg / mL standardin DI waterDetector: Corona ultraFilter:MediumRange:200pAResults and DiscussionFigure 1 shows a chromatogram from the UHPLCmethod overlaid onto the HPLC chromatogram witha full 20 minute time scale. The resolution for thethird major peak and seventh major peak observedin the cluster for each was calculated, and the comparisonis shown in Table 3. The traditional HPLCapproach did achieve superior resolution for bothpeaks, however the resolution for the UHPLCmethod was within 70% of the HPLC method with afour-fold reduction in analysis time and solventconsumption.TimeFlow Rate% B(min)(mL/min)0 3 115 30 117 3 1Table 1 - HPLC Gradient.TimeFlow Rate% B(min)(mL/min)0 3 0.051 10 12.4 20 13 30 13.4 3 0.5Table 2. UHPLC Gradient.Peak 3 Peak 7HPLC UPLC HPLC UPLCRetention Time (min) 8.67 1.35 12.42 2.24Resolution 7.8 5.6 4.1 3.4Table 3. Comparison of Resolution Between HPLCand UHPLC for Two Selected Analytes.Figure 2. Overlay of Four Individual Analytical Runs on the UHPLC Corona ultra System.

Corona ® ultra Charged Aerosol DetectorApplication noteThe UHPLC method reproducibility is shown Figure2, with overlays of four injections of a 2 µg PEG 400standard on column with the calculated %RSD foreach peak is represented in Table 4. A responsecurve was then prepared from ~2 to 2000 µg/mL. Alimit of quantification for the PEG 400 cluster wasobserved at ~18 ng total mass on column, which isshown in Figure 3. The prepared standards wereanalyzed, and the peak area versus the total masson column is plotted in Figure 4. Assuming that theaverage %-area for each peak from the 2 µg oncolumn injections can be used to adjust for themass on column for each peak over the entirecalibration range, a new adjusted response curvewas calculated and is plotted in Figure 5. This wasdone to illustrate the uniformity of response of theCorona CAD for the different MW fractions of PEG400.Retention TimeResponse(min)(peak area)% RSD1.11 1124 1.68%1.34 2596 1.73%1.56 2282 1.09%1.76 3050 1.32%1.94 1010 0.46%2.09 1730 0.95%2.24 1941 1.48%2.37 649 0.76%2.49 669 1.35%2.60 320 1.29%Table 4. Reproducibility of Ten PEG 400Analytes (n=4).0.200.00-0.20-0.40-0.60mV-0.80-1.00-1.20-1.40-1.600.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00MinutesFigure 3. High Sensitivity Analysis of PEG 400 on the UHPLC Corona ultra System(~18 ng total, on column).

The Corona ® ultraCharged Aerosol DetectorResponse (peak area)3000002500002000001500001000005000001.11 min 1.34 min 1.56 min 1.76 min 1.94 min 2.09 min 2.24 min 2.37 min 2.49 min 2.60 minR 2 = 0.9945R 2 = 0.9925R 2 = 0.9944R 2 = 0.9949R 2 = 0.9965R 2 = 0.9965R 2 = 0.9992R 2 = 0.9991R 2 = 0.9998R 2 = 0.9990 500 1000 1500 2000 2500Total Mass on Column (ng)Figure 4. Response Curves for 18 ng to 2 μg Total Mass (oncolumn).ConclusionSensitive methods for the analysis of PEG400 can be obtained by either HPLC orUHPLC with Charged Aerosol Detection.The transfer of the method from HPLC toUHPLC offered a four-fold decrease inanalysis time and solvent consumption.The ability to detect low nanogramquantities of PEG 400 with limits ofdetection