- SO4 A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside Antibiotics Using Charged Aerosol Detection Zhen Long1, Qi Zhang2, Yan Jin1, Lina Liang1, Bruce Bailey2, Ian Acworth2, Deepali Mohindra3 1Thermo Fisher Scientific, Shanghai, China, 2Thermo Fisher Scientific, Chelmsford, MA, USA and 3Thermo Fisher Scientific, Sunnyvale, CA, USA

Comparison of CAD and ELSD Detection Analysis of Other Aminoglycoside Antibiotics Overview Results Purpose: To develop a sensitive non-derivatization method for impurity assessment of Sample pre-treatment with SPE This method has also been applied to impurity analysis of an additional eleven apramycin sulfate and other aminoglycoside antibiotics. Sample pre-treatment with SPE SPE Column: Dionex OnGuard II A CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin,

Comparison between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient method using hydrophilic interaction liquid chromatography Sample solvent: 80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin impurity assessment with a HILIC method. demonstrated that CAD is much more sensitive than ELSD. As shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin with charged aerosol detection (HILIC-CAD) was developed for direct analysis of Sample: 220.6 mg/mL in 2 mL sample solvent Without sample cleanup, some early eluting impurities were found to be masked under chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC-CAD method. For some other aminoglycoside antibiotics, modification apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to SPE procedure: Condition the SPE cartridge with 6 mL sample solvent, then the huge sulfate peak and could not be detected. A Dionex anion exchange SPE detected with CAD at an injected amount of 49.6 μg apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was pass the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE only 12 impurities were detected with ELSD at this level. When injection amount also analyzed with the SCX-UV method recommended by British pharmacopoeia cartridge with additional 2 mL sample solvent . Combine cartridge while the apramycin and impurities passed through the cartridge and collected decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, (veterinary) 2013, which requires post column derivatization. collected loading eluent and wash solution for analysis. The for further analysis. Sulfate was replaced by bicarbonate after SPE, which has little while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. final concentration of apramycin sample was 49 mg/mL. interference with apramycin analysis, since it is volatile and elutes earlier than the peaks FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and Results: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD A 2 mL volume of sample solvent was treated with the same of interest. As seen in Figure 2, after removing sulfate, more impurities can now be The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. method. The SCX-UV method recommended by British Pharmacopoeia only detected just procedure and used as blank. detected. Recovery of three impurity peaks, labeled as peak 11, 14 and 15 in Figure 3a, to the sigmoidal nature of its response curve, resulting in much lower sensitivity. seven impurities. The HILIC-CAD method is much more sensitive than ELSD. With was calculated to be 107%, 92% and 93%, respectively.

20 μg apramycin sulfate on column, 7 impurities were detected by CAD at S/N > 3, while HILIC-CAD/ELSD method only 3 impurities were detected by ELSD. This method, with or without slight modification,

was also used for impurity measurement of an additional eleven aminoglycoside Comparison of HILIC-CAD method with SCX-UV method Column: ACCHROM (Beijing, China), Click XIon, 4.6 x 150mm, 5µm antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, tobramycin, Temperature: 30 ºC FIGURE 4. Comparison of Detection Sensitivity Between CAD and ELSD. amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. The HILIC-CAD method was compared to the SCX-UV method recommended by British Flow rate: 1 mL/min Pharmacopoeia (veterinary) 2013 version (BP2013) . As shown in Figure 3, the number Mobile Phase A: Acetonitrile of impurity peaks resolved and detected was greatly increased with the HILIC-CAD Mobile Phase B: 500 mM ammonium formate, pH 2.9 approach. About 16 impurity peaks were detected with the HILIC-CAD method at Mobile phase C: Water S/N > 3 (Figure 3A). The SCX-UV method only detected seven impurities, as not all of Introduction Gradient 0 min., 70 %A, 20%B, 10% C them could be derivatized by the SCX-UV approach (Figure 3B). Furthermore, the 30 min., 21% A, 20%B, 59% C improved chromatographic resolution and peak shape allows for a higher sample load Aminoglycosides are a group of structurally similar antibiotics used to treat infections Injection volume: 1 μL with the HILIC-CAD method enabling detection of low level impurities. 1 caused by aerobic gram-negative bacteria . Analytical methods are required for rapid Corona Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, assessment of drug purity and detection of minor degredants. As they lack a strong ELSD: Nebulizer temp. 50 °C , evaporation temp. 70 °C The effect of mobile buffer strength and pH on separation and peak shape was chromophore, these compounds are not amenable to UV detection. investigated. The method was optimized with 100mM ammonium formate at pH 2.9. The sample load was increased to 48.9 μg for the HILIC-CAD method and still maintains good Apramycin is an antibiotic used in veterinary medicine. Reported methods for apramycin SCX -UV method peak shape with half peak width W0.5 = 0.77min. While further increase of sample loading and impurities analysis usually involves pre- or post column derivatization followed by UV amount with the SCX-UV method caused significant peak broadening and results in 2,3 Column: Venusil SCX-F 4.6 x 150 mm, 5 µm detection . Such approaches are tedious and time consuming, and may not be able to Temperature: 30 ºC decreased resolution between apramycin and impurity peaks. detect all impurities. UV Detector: 568 nm Sample: 0.28 mg/mL Aminoglycosides can be measured directly by charged aerosol detection without Conclusions Injection volume: 20 μL FIGURE 3A. Impurity Analysis of Apramycin with the HILIC-CAD Method. derivatization. Corona Veo is a universal mass-sensitive detector, Its response is • The described HILIC-CAD method for apramycin enables more accurate impurity A Sensitive Method forindependent Direct of chemical Analysis structure, and does not require the presence of chromophores.Poster Note 21432 assessment, due to the universal detection of CAD and improved sample loading Chromatographic condition and post-column derivatization procedure are same as in 200 Capable of measuring any nonvolatile and many semi-volatile analytes, charged aerosol British Veterinary Pharmacopoeia 2013 pA capacity. More than 16 impurities were detected. The SCX-UV method of Impurities in Apramycindetection enables and accurate degradationOther studies and improved assessments of product recommended by British Pharmacopoeia only detected seven impurities. purity. The Corona Veo is much more sensitive than other universal detectors like ELSD HILIC-CAD method 150 • Comparison between Corona Veo and ELSD detection showed that CAD is much and RI, offering low nanogram quantitation. This poster presents a sensitive HILIC-CAD Data Analysis 48.9 µg on column ™ ™ ™ more sensitive than ELSD. With 20µg sample on column, 7 impurities were detected method for direct analysis of apramycin and other aminoglycosides. Method performance Thermo Scientific Dionex Chromeleon Chromatography Data System software, 7.2 SO - Aminoglycoside Antibiotics Using Charged at S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. 4 was compared to the British Pharmacopoeia HPLC-UV method and ELSD detection. 100 • Sample pretreatment with anion exchange SPE removes interference of sulfate ion Aerosol Detection FIGURE 2. Effect of SPE Pre-treatment of Apramycin Sulfate Sample. and allows for more accurate determination of impurities. A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside Antibiotics Using Charged Aerosol Detection 14 • This method can also be used for the analysis of many other aminoglycoside 50 15 antibiotics. 1 Zhen Long,1 Qi Zhang,2 2 Yan Jin,1 Lina Liang,1 1 Bruce Bailey,2 Ian Acworth,1 2 and Deepali Mohindra2 3 2 3 11 Zhen Long , Qi Zhang , Yan Jin , Lina LiangMethods, Bruce Bailey , Ian Acworth , Deepali Mohindra 13 1 2 ™ ™ ™ 5 6 7 9 10 Thermo Fisher Scientifi c, Shanghai, China; Thermo Fisher ThermoScientifi Scientific c, Chelmsford, Dionex MA; UltiMate 3000 RSLC system with: 1 2 8 12 16 1 2 3 3 4 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD • Pump: LPG-3400SD min Thermo Fisher3Thermo FisherScientific, Scientifi c, Sunnyvale, Shanghai, CA China, Thermo Fisher Scientific, Chelmsford, MA, USA and Thermo Fisher Scientific, -Sunnyvale,10 CA, USA • Auto Sampler: WPS-3000TSL 0.0 5.0 10.0 15.0 20.0 25.1

• Column Compartment :TCC-3000RS Peak Retention Time (min) S/N - • Diode Array Detector: DAD-3000RS Number SO4 μ • Charged aerasol detector: Corona Veo RS FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. 49 g on column 20 µg on column • CAD ELSD CAD ELSD References Varian ELSD 385-LC 1 10.30 4.9 6.4 - 1. Perzynski, S.; Cannon, M.; Cundliffe, E.; Chahwala, S.B.; Davies, J. Effects of A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside Antibiotics400 Using Charged Aerosol Detection2 11.27 10.4 8.8 - 3 11.94 3.0 - - Apramycin, a novel aminoglycoside antibiotic on bacterial protein synthesis. Eur. SCX-UV method – FIGURE 1. 4 12.61 4.2 - 6.4 - J. Biochem. 1979, 99, 623 628. 1 2 1 1 Structure of Apramycin 2 2 3 Comparison200 of CADμ and ELSD Detection Analysis of Other Aminoglycoside Antibiotics Overview Results 5.8 g on column 5 13.32 24.4 19.4 - Zhen Long , Qi Zhang , Yan Jin , Lina Liang , Bruce Bailey , Ian Acworth , Deepali Mohindra 2. British Pharmacopoeia (Veterinary): 2013. London: Her Majesty's Stationery Purpose: To develop a sensitive non-derivatization method for impurity assessment of Sample pre-treatment with SPE 6 14.10 19.6 19.5 3.9 - This method has also been applied to impurity analysis of an additional eleven 1apramycin sulfate and other aminoglycoside antibiotics. 2 Sample pre-treatment with SPE3 7 14.76 29.0 44.6 6.2 - Office, v.4, p. 38-40, 122-125, 2013. Thermo Fisher Scientific, Shanghai, China,SPE Column:Thermo FisherDionex OnGuard Scientific, II A Chelmsford, MA, USA and Thermo Fisher Scientific, Sunnyvale,CAD0 and ELSD are bothCA, nebulization USA-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, 8 15.78 15.0 14.0 3.2 - 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in

mV Comparison between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient method using hydrophilic interaction liquid chromatography Sample solvent: 80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin impurity assessment with a HILIC method. 9 17.79 18.6 22.9 - 10 18.47 10.1 12.3 - - oral soluble powder by a HPLC method using pre-column derivatization with o- Sample: 220.6 mg/mL in 2 mL sample solvent Without sample cleanup, some early eluting impurities were found to be masked under demonstrated-200 that CAD is much more sensitive than ELSD. As shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin with charged aerosol detection (HILIC-CAD) was developed for direct analysis of phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. SPE procedure: Condition the SPE cartridge with 6 mL sample solvent, then the huge sulfate peak and could not be detected. A Dionex anion exchange SPE chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the 11HILIC -CAD method.18.85 For some other51.7 aminoglycoside137.9 antibiotics,11.8 3.7 modification apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to 12 19.34 4.8 - - pass the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE detected with CAD at an injected amount of 49.6 μg apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was 13 21.55 12.7 19.3 - cartridge with additional 2 mL sample solvent . Combine only-400 12 impurities were detected with ELSD at this level. When injection amount also analyzed with the SCX-UV method recommended by British pharmacopoeia cartridge while the apramycin and impurities passed through the cartridge and collected 14 21.87 79.5 293.9 21.3 5.6 © 2015 Thermo Fisher Scientific Inc. All trademarks are the property of Thermo Fisher Scientific and its decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, (veterinary) 2013, which requires post column derivatization. collected loading eluent and wash solution for analysis. The for further analysis. Sulfate was replaced by bicarbonate after SPE, which has little 15 22.52 77.0 238.2 17.9 4.1 subsidiaries. while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. final concentration of apramycin sample was 49 mg/mL. interference with apramycin analysis, since it is volatile and elutes earlier than the peaks 0 10 20 16 23.52 6.9 - - - This information is not intended to encourage use of these products in any manners that might infringe the A 2 mL volume of sample solvent was treated with the same of interest. As seen in Figure 2, after removing sulfate, more impurities can now be Comparison of CAD and ELSD Detectiontime (min) AnalysisFIGURE of5. ChromatogramsOther Aminoglycoside for impurity Antibiotics analysis of kenamycin, etimicin, ribostamycin and intellectual property rights of others. OverviewResults: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD Results The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. Purpose:method. ToThe develop SCX-UV a sensitive method recommendednon-derivatization by British method Pharmacopoeia for impurity assessment only detected of just Sample pre-treatment with procedureSPE and used as blank. detected. Recovery of three impurity peaks, labeled as peak 11, 14 and 15 in Figure 3a, Sample pre-treatment with SPE to the sigmoidal nature of its response curve, resulting in much lower sensitivity. This method has also been applied to impurity analysis of an additional eleven apramycinseven impurities. sulfate and The other HILIC aminoglycoside-CAD method is antibiotics. much more sensitive than ELSD. With was calculated to be 107%, 92% and 93%, respectively. SPE Column: Dionex OnGuard II A CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, 20 μg apramycin sulfate on column, 7 impurities were detected by CAD at S/N > 3, while SampleHILIC -solvent:CAD/ELSD method 80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin impurity assessment with a HILIC method. Comparison between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods:only 3 impurities A 30min weregradient detected method by usingELSD. hydrophilic This method, interaction with or liquidwithout chromatography slight modification, Sample: 220.6 mg/mL in 2 mL sample solvent Without sample cleanup, some early eluting impurities were found to be masked under demonstrated that CAD is much more sensitive than ELSD. As shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin withwas charged also used aerosol for impurity detection measurement (HILIC-CAD) of was an developedadditional elevenfor direct aminoglycoside analysis of Comparison of HILIC-CAD method with SCX-UV method SPEColumn: procedure: ConditionACCHROM the (Beijing,SPE cartridge China), with Click 6 mL XIon sample, 4.6 solvent, x 150mm, then 5µm the huge sulfate peak and could not be detected. A Dionex anion exchange SPE chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC-CAD method. For some other aminoglycoside antibiotics, modification apramycinantibiotics, sulfate including . Samples neomycin, were gentamicin, pretreated kanamycin,with solid phase streptomycin, extraction tobramycin, (SPE) to Temperature: pass30 ºC the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE FIGUREdetected 4. with Comparison CAD at an of injectedDetection amount Sensitivity of 49.6 Between μg apramycin CAD and sulfate ELSD. on column, while of the gradient may be required for optimized separation and resolution. removeamikacin, sulfate etimicin, ion for netilmicin, more accurate sisomicin, determination ribostamycin of impurities. and paromomycin. The same sample was The HILIC-CAD method was compared to the SCX-UV method recommended by British Flow rate: cartridge1 mL/min with additional 2 mL sample solvent . Combine cartridge while the apramycin and impurities passed through the cartridge and collected only 12 impurities were detected with ELSD at this level. When injection amount also analyzed with the SCX-UV method recommended by British pharmacopoeia Pharmacopoeia (veterinary) 2013 version (BP2013) . As shown in Figure 3, the number Mobile Phase A: collectedAcetonitrile loading eluent and wash solution for analysis. The for further analysis. Sulfate was replaced by bicarbonate after SPE, which has little decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, (veterinary) 2013, which requires post column derivatization. of impurity peaks resolved and detected was greatly increased with the HILIC-CAD Mobile Phase B: 500 mM ammonium formate, pH 2.9 while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. final concentration of apramycin sample was 49 mg/mL. interferenceapproach. with About apramycin 16 impurity analysis, peaks sincewere itdetected is volatile with and the elutes HILIC earlier-CAD thanmethod the atpeaks Mobile phase C: Water FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and Results: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD A 2 mL volume of sample solvent was treated with the same of S/Ninterest. > 3 (Figure As seen 3A). in FigureThe SCX 2, -afterUV method removing only sulfate, detected more seven impurities impurities, can nowas not be all of Gradient 0 min., 70 %A, 20%B, 10% C The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. method.Introduction The SCX-UV method recommended by British Pharmacopoeia only detected just procedure and used as blank. detected.them could Recovery be derivatized of three impurityby the SCX peaks,-UV labeledapproach as (Figure peak 11,3B). 14 Furthermore, and 15 in Figure the 3a, 30 min., 21% A, 20%B, 59% C to the sigmoidal nature of its response curve, resulting in much lower sensitivity. seven impurities. The HILIC-CAD method is much more sensitive than ELSD. With wasimproved calculated chromatographic to be 107%, 92% resolution and 93%, and respectively. peak shape allows for a higher sample load Aminoglycosides are a group of structurally similar antibiotics used to treat infections Injection volume: 1 μL 20 μg apramycin sulfate on column, 7 impurities were detected by CAD at S/N > 3, while with the HILIC-CAD method enabling detection of low level impurities. caused by aerobic gram-negative bacteria1. Analytical methods are required for rapid HILICCorona-CAD/ELSD Veo RS: method55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, only 3 impurities were detected by ELSD. This method, with or without slight modification, assessment of drug purity and detection of minor degredants. As they lack a strong ELSD: Nebulizer temp. 50 °C , evaporation temp. 70 °C was also used for impurity measurement of an additional eleven aminoglycoside ComparisonThe effect of of mobileHILIC- CADbuffer method strength with and SCXpH on-UV separation method and peak shape was chromophore, these compounds are not amenable to UV detection. Column: ACCHROM (Beijing, China), Click XIon, 4.6 x 150mm, 5µm antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, tobramycin, investigated. The method was optimized with 100mM ammonium formate at pH 2.9. The Temperature: 30 ºC μ FIGURE 4. Comparison of Detection Sensitivity Between CAD and ELSD. amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Thesample HILIC load-CAD was method increased was comparedto 48.9 g tofor the the SCX HILIC-UV-CAD method method recommended and still maintains by British good Apramycin is an antibiotic used in veterinary medicine. Reported methods for apramycin Flow rate: 1 mL/min SCX-UV method Pharmacopoeiapeak shape with (veterinary) half peak 2013 width version W0.5 = 0.77min.(BP2013) While . As furthershown increasein Figure of3, samplethe number loading and impurities analysis usually involves pre- or post column derivatization followed by UV Mobile Phase A: Acetonitrile of amountimpurity with peaks the resolved SCX-UV andmethod detected caused was significant greatly increased peak broadening with the andHILIC results-CAD in 2,3 Column: Venusil SCX-F 4.6 x 150 mm, 5 µm detection . Such approaches are tedious and time consuming, and may not be able to MobileTemperature: Phase B: 50030 mM ºC ammonium formate, pH 2.9 approach.decreased About resolution 16 impurity between peaks apramycin were detected and impurity with the peaks. HILIC -CAD method at detect all impurities. MobileUV Detector: phase C: Water568 nm S/N > 3 (Figure 3A). The SCX-UV method only detected seven impurities, as not all of Introduction GradientSample: 0 0.28min., mg/mL 70 %A, 20%B, 10% C them could be derivatized by the SCX-UV approach (Figure 3B). Furthermore, the Aminoglycosides can be measured directly by charged aerosol detection without Conclusions Injection volume: 3020 min., μL 21% A, 20%B, 59% C improved FIGURE chromatographic 3A. Impurity Analysis resolution of andApramycin peak shape with allowsthe HILIC for a- CADhigher Method. sample load Aminoglycosidesderivatization. Corona are a group Veo isof a structurally universal mass similar-sensitive antibiotics detector, used to Its treat response infections is Injection volume: 1 μL with the HILIC-CAD method enabling detection of low level impurities. • The described HILIC-CAD method for apramycin enables more accurate impurity 1 causedindependent by aerobic of chemical gram-negative structure, bacteria and does. Analytical not require methods the presenceare required of chromophores. for rapid Corona Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, assessment, due to the universal detection of CAD and improved sample loading Chromatographic condition and post-column derivatization procedure are same as in 200 assessment of drug purity and detection of minor degredants. As they lack a strong ° ° pA Capable of measuring any nonvolatile and many semi-volatile analytes, charged aerosol ELSD:British Veterinary PharmacopoeiaNebulizer temp. 2013 50 C , evaporation temp. 70 C The effect of mobile buffer strength and pH on separation and peak shape was capacity. More than 16 impurities were detected. The SCX-UV method chromophore, these compounds are not amenable to UV detection. detection enables accurate degradation studies and improved assessments of product investigated. The method was optimized with 100mM ammonium formate at pH 2.9. The recommended by British Pharmacopoeia only detected seven impurities. HILIC-CAD method purity. The Corona Veo is much more sensitive than other universal detectors like ELSD sample load was increased to 48.9 μg for the HILIC-CAD method and still maintains good 150 • Apramycin is an antibiotic used in veterinary medicine. Reported methods for apramycin Comparison between Corona Veo and ELSD detection showed that CAD is much and RI, offering low nanogram quantitation. This poster presents a sensitive HILIC-CAD SCX Data-UV Analysis method peak shape48.9 with µg halfon column peak width W0.5 = 0.77min. While further increase of sample loading ™ ™ ™ more sensitive than ELSD. With 20µg sample on column, 7 impurities were detected andmethod impurities for direct analysis analysis usually of apramycininvolves pre and- or other post columnaminoglycosides. derivatization Method followed performance by UV Column:Thermo Scientific DionexVenusil Chromeleon SCX-F 4.6 xChromatography 150 mm, 5 µm Data System software, 7.2 amount with the SCX-UV method caused significant peak broadening and results in 2,3 at S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. detectionwas compared. Such to approaches the British Pharmacopoeiaare tedious and timeHPLC consuming,-UV method and and may ELSD not detection.be able to Temperature: 30 ºC decreased resolution between apramycin and impurity peaks. 100 detect all impurities. UV Detector: 568 nm • Sample pretreatment with anion exchange SPE removes interference of sulfate ion Sample:FIGURE 2. Effect of SPE0.28 P mg/mLre-treatment of Apramycin Sulfate Sample. Conclusionsand allows for more accurate determination of impurities. Aminoglycosides can be measured directly by charged aerosol detection without Injection volume: 20 μL FIGURE 3A. Impurity Analysis of Apramycin with the HILIC-CAD Method.14 • This method can also be used for the analysis of many other aminoglycoside derivatization. Corona Veo is a universal mass-sensitive detector, Its response is 50 15 • The described HILIC-CAD method for apramycin enables more accurate impurity antibiotics. independent of chemical structure, and does not require the presence of chromophores. 11 assessment, due to the universal detection of CAD and improved sample loading Chromatographic condition and post-column derivatization procedure are same as in 200 13 CapableMethods of measuring any nonvolatile and many semi-volatile analytes, charged aerosol pA 7 ™ ™ ™ British Veterinary Pharmacopoeia 2013 5 6 8 9 10 capacity. More than 16 impurities were detected. The SCX-UV method Thermo Scientific Dionex UltiMate 3000 RSLC system with: 1 2 3 4 12 16 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD detection enables accurate degradation studies and improved assessments of product min recommended by British Pharmacopoeia only detected seven impurities. • Pump: LPG-3400SD -10 purity. The Corona Veo is much more sensitive than other universal detectors like ELSD HILIC-CAD method • Auto Sampler: WPS-3000TSL 150 0.0 5.0 10.0 15.0 20.0 25.1 • Comparison between Corona Veo and ELSD detection showed that CAD is much and RI, offering low nanogram quantitation. This poster presents a sensitive HILIC-CAD Data Analysis 48.9 µg on column • Column Compartment :TCC-3000RS Peak method for direct analysis of apramycin and other aminoglycosides. Method performance Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System software, 7.2 Retention Time (min) S/N more sensitive than ELSD. With 20µg sample on column, 7 impurities were detected • Diode Array Detector: DAD-3000RS Number at S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. was compared to the British Pharmacopoeia HPLC-UV method and ELSD detection. μ • Charged aerasol detector: Corona Veo RS FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. 49 g on column 20 µg on column 100 CAD ELSD CAD ELSD • • ReferencesSample pretreatment with anion exchange SPE removes interference of sulfate ion Varian ELSD 385-LC 1 10.30 4.9 6.4 - FIGURE 2. Effect of SPE Pre-treatment of Apramycin Sulfate Sample. 1.and Perzynski, allows for moreS.; Cannon, accurate M.; determination Cundliffe, E.; ofChahwala, impurities. S.B.; Davies, J. Effects of 400 2 11.27 10.4 8.8 - Apramycin, a novel aminoglycoside antibiotic on bacterial protein synthesis. Eur. 14 3 11.94 3.0 - - • This method can also be used for the analysis of many other aminoglycoside 50 SCX-UV method 15 – FIGURE 1. Structure of Apramycin 4 12.61 4.2 - 6.4 - antibiotics.J. Biochem. 1979, 99, 623 628. 200 5.8 μg on column 11 13 5 13.32 24.4 19.4 - Methods 7 2. British Pharmacopoeia (Veterinary): 2013. London: Her Majesty's Stationery ™ ™ ™ 5 6 8 9 10 6 14.10 19.6 19.5 3.9 - Thermo Scientific Dionex UltiMate 3000 RSLC system with: 1 2 3 4 12 16 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD min 7 14.76 29.0 44.6 6.2 - Office, v.4, p. 38-40, 122-125, 2013. • Pump: LPG-3400SD 0 -10 8 15.78 15.0 14.0 3.2 - • Auto Sampler: WPS-3000TSL 0.0 5.0 10.0 15.0 20.0 25.1 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in mV 9 17.79 18.6 22.9 - • Column Compartment :TCC-3000RS Peak10 18.47 10.1 12.3 - - oral soluble powder by a HPLC method using pre-column derivatization with o- -200 Retention Time (min) S/N • Diode Array Detector: DAD-3000RS Number11 18.85 51.7 137.9 11.8 3.7 phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. μ • Charged aerasol detector: Corona Veo RS FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. 12 19.34 49 g4.8 on column - 20 µg on column - CAD ELSD CAD ELSD References • Varian ELSD 385-LC -400 13 21.55 12.7 19.3 - 1 10.30 4.9 6.4 - 400 14 21.87 79.5 293.9 21.3 5.6 1. © 2015Perzynski, Thermo Fisher S.; Cannon, Scientific Inc. M.; All Cundliffe, trademarks areE.; theChahwala, property of ThermoS.B.; Davies, Fisher Scientific J. Effects and its of 2 15 11.2722.52 10.477.0 8.8238.2 17.9 -4.1 subsidiaries. 3 11.94 3.0 - - Apramycin, a novel aminoglycoside antibiotic on bacterial protein synthesis. Eur. 0 SCX-UV method 10 20 16 23.52 6.9 - - - This information is not intended to encourage– use of these products in any manners that might infringe the FIGURE 1. 4 12.61 4.2 - 6.4 - J. Biochem. 1979, 99, 623 628. Structure of Apramycin 200 μ time (min) intellectual property rights of others. 5.8 g on column 5 13.32 24.4 19.4 - 6 14.10 19.6 19.5 3.9 - 2. British Pharmacopoeia (Veterinary): 2013. London: Her Majesty's Stationery 7 14.76 29.0 44.6 6.2 - Office, v.4, p. 38-40, 122-125, 2013. 0 8 15.78 15.0 14.0 3.2 - 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in

mV 9 17.79 18.6 22.9 - 10 18.47 10.1 12.3 - - oral soluble powder by a HPLC method using pre-column derivatization with o- -200 11 18.85 51.7 137.9 11.8 3.7 phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. 12 19.34 4.8 - - -400 13 21.55 12.7 19.3 - 14 21.87 79.5 293.9 21.3 5.6 © 2015 Thermo Fisher Scientific Inc. All trademarks are the property of Thermo Fisher Scientific and its 15 22.52 77.0 238.2 17.9 4.1 subsidiaries.

0 10 20 16 23.52 6.9 - - - This information is not intended to encourage use of these products in any manners that might infringe the time (min) intellectual property rights of others. - SO4 A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside Antibiotics Using Charged Aerosol Detection Zhen Long1, Qi Zhang2, Yan Jin1, Lina Liang1, Bruce Bailey2, Ian Acworth2, Deepali Mohindra3 1Thermo Fisher Scientific, Shanghai, China, 2Thermo Fisher Scientific, Chelmsford, MA, USA and 3Thermo Fisher Scientific, Sunnyvale, CA, USA

- SO4

OverviewA Sensitive Method for Direct Analysis of Impurities in Apramycin Resultsand Other Aminoglycoside AntibioticsComparison of CADUsing and ELSD Detection Charged Aerosol DetectionAnalysis of Other Aminoglycoside Antibiotics Purpose: To develop a sensitive non-derivatization method for impurity assessment of Sample pre-treatment with SPE This method has also been applied to impurity analysis of an additional eleven apramycin sulfate and other1 aminoglycoside antibiotics.2 1 1 2 2 Sample pre-treatment3 with SPE Zhen Long , Qi Zhang , Yan Jin , Lina LiangSPE Column:, Bruce BaileyDionex OnGuard, Ian II A Acworth , Deepali Mohindra CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, Comparison between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient method using hydrophilic interaction liquid chromatography Sample solvent: 80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin impurity assessment with a HILIC method. 1 2 3 demonstrated that CAD is much more sensitive than ELSD. As shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin with Thermocharged aerosol detection Fisher (HILIC-CAD) Scientific, was developed for direct Shanghai, analysis of China,Sample: Thermo220.6 Fisher mg/mL in 2 mLScientific,sample solvent Chelmsford, WithoutMA, sample USA cleanup, and some earlyThermo eluting impurities wereFisher found to be Scientific, masked under Sunnyvale, CA, USA chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC-CAD method. For some other aminoglycoside antibiotics, modification apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to SPE procedure: Condition the SPE cartridge with 6 mL sample solvent, then the huge sulfate peak and could not be detected. A Dionex anion exchange SPE detected with CAD at an injected amount of 49.6 μg apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was pass the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE only 12 impurities were detected with ELSD at this level. When injection amount also analyzed with the SCX-UV method recommended by British pharmacopoeia cartridge with additional 2 mL sample solvent . Combine cartridge while the apramycin and impurities passed through the cartridge and collected decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, (veterinary) 2013, which requires post column derivatization. collected loading eluent and wash solution for analysis. The for further analysis. Sulfate was replaced by bicarbonate after SPE, which has little final concentration of apramycin sample was 49 mg/mL. interference with apramycin analysis, since it is volatile and elutes earlier than the peaks while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and Results: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD A 2 mL volume of sample solvent was treated with the same of interest. As seen in Figure 2, after removing sulfate, more impurities can now be The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. method. The SCX-UV method recommended by British Pharmacopoeia only detected just procedure and used as blank. detected. Recovery of three impurity peaks, labeled as peak 11, 14 and 15 in Figure 3a, to the sigmoidal nature of its response curve, resulting in much lower sensitivity. seven impurities. The HILIC-CAD method is much more sensitive than ELSD. With was calculated to be 107%, 92% and 93%, respectively. Comparison of CAD and ELSD Detection Analysis of Other Aminoglycoside Antibiotics Overviewμ Results 20Purpose: g apramycinTo develop sulfate a onsensitive column, non- 7 impuritiesderivatization were method detected for by impurity CAD at assessment S/N > 3, while of Sample pre-treatment with SPE HILIC-CAD/ELSD method This method has also been applied to impurity analysis of an additional eleven onlyapramycin 3 impurities sulfate were and detected other aminoglycoside by ELSD. This method, antibiotics. with or without slight modification, Sample pre-treatment with SPE was also used for impurity measurement of an additional eleven aminoglycoside SPE Column: Dionex OnGuard II A Comparison of HILIC-CAD method with SCX-UV method CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, Column: ACCHROM (Beijing, China), Click XIon, 4.6 x 150mm, 5µm antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, tobramycin, Sample solvent: 80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin impurity assessment with a HILIC method. Comparison between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient method using hydrophilic interaction liquid chromatography Temperature: 30 ºC FIGURE 4. Comparison of Detection Sensitivity Between CAD and ELSD. amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Sample: 220.6 mg/mL in 2 mL sample solvent TheWithout HILIC- sampleCAD methodcleanup, was some compared early eluting to the impurities SCX-UV weremethod found recommended to be masked by Britishunder demonstrated that CAD is much more sensitive than ELSD. As shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin with charged aerosol detection (HILIC-CAD) was developed for direct analysis of Flow rate: 1 mL/min SPE procedure: Condition the SPE cartridge with 6 mL sample solvent, then Pharmacopoeiathe huge sulfate (veterinary) peak and could2013 notversion be detected. (BP2013) A .Dionex As shown anion in exchangeFigure 3, theSPE number chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC-CAD method. For some other aminoglycoside antibiotics, modification apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to Mobile Phase A: Acetonitrile pass the sample solution through the cartridge and wash the ofcartridge impurity On peaks Guard resolved II A was and used detected to remove was greatlysulfate. increasedSulfate was with retained the HILIC-CAD on the SPE detected with CAD at an injected amount of 49.6 μg apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was Mobile Phase B: 500 mM ammonium formate, pH 2.9 cartridge with additional 2 mL sample solvent . Combine approach.cartridge while About the 16 apramycinimpurity peaks and impuritieswere detected passed with through the HILIC the- CADcartridge method and atcollected only 12 impurities were detected with ELSD at this level. When injection amount also analyzed with the SCX-UV method recommended by British pharmacopoeia Mobile phase C: Water collected loading eluent and wash solution for analysis. The S/Nfor >further 3 (Figure analysis. 3A). TheSulfate SCXwas-UV replaced method onlyby bicarbonate detected seven after impurities, SPE, which as has not littleall of decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, (veterinary) 2013, which requires post column derivatization. Gradient 0 min., 70 %A, 20%B, 10% C Introduction final concentration of apramycin sample was 49 mg/mL. theminterfere couldnce be with derivatized apramycin by theanalysis, SCX- UVsince approach it is volatile (Figure and 3B). elutes Furthermore, earlier than the the peaks while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. 30 min., 21% A, 20%B, 59% C A 2 mL volume of sample solvent was treated with the same improvedof interest. chromatographic As seen in Figure resolution 2, after and removing peak shape sulfate, allows more for impurities a higher cansample now load be FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and AminoglycosidesResults: 16 impurities are a group of apramycin of structurally were detected similar antibiotics at S/N ≥ 3used with to the treat HILIC infections-CAD Injection volume: 1 μL procedure and used as blank. withdetected. the HILIC Recovery-CAD method of three enabling impurity detection peaks, labeled of low level as peak impurities. 11, 14 and 15 in Figure 3a, The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. causedmethod. by The aerobic SCX- gramUV method-negative recommended bacteria1. Analytical by British methods Pharmacopoeia are required only for detected rapid just Corona Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, was calculated to be 107%, 92% and 93%, respectively. to the sigmoidal nature of its response curve, resulting in much lower sensitivity. assessmentseven impurities. of drug The purity HILIC and- CAD detection method of minoris much degredants. more sensitive As they than lack ELSD. a strong With ELSD: Nebulizer temp. 50 °C , evaporation temp. 70 °C The effect of mobile buffer strength and pH on separation and peak shape was 20 μg apramycin sulfate on column, 7 impurities were detected by CAD at S/N > 3, while chromophore, these compounds are not amenable to UV detection. HILIC-CAD/ELSD method investigated. The method was optimized with 100mM ammonium formate at pH 2.9. The only 3 impurities were detected by ELSD. This method, with or without slight modification, μ sampleComparison load was of increasedHILIC-CAD to method48.9 g forwith the SCX- HILICUV-CAD method method and still maintains good Apramycinwas also usedis an forantibiotic impurity used measurement in veterinary of medicine. an additional Reported eleven methods aminoglycoside for apramycin SCX-UVColumn: method ACCHROM (Beijing, China), Click XIon, 4.6 x 150mm, 5µm peak shape with half peak width W0.5 = 0.77min. While further increase of sample loading andantibiotics, impurities including analysis neomycin, usually involves gentamicin, pre- orkanamycin, post column streptomycin, derivatization tobramycin, followed by UV Column:Temperature: Venusil30 ºC SCX-F 4.6 x 150 mm, 5 µm amountThe HILIC- with CADthe SCX method-UV method was compared caused significantto the SCX peak-UV methodbroadening recommended and results by in British FIGURE 4. Comparison of Detection Sensitivity Between CAD and ELSD. detectionamikacin,2,3 .etimicin, Such approaches netilmicin, aresisomicin, tedious ribostamycin and time consuming, and paromomycin. and may not be able to Temperature:Flow rate: 301 ºCmL/min decreasedPharmacopoeia resolution (veterinary) between 2013 apramycin version and (BP2013) impurity . peaks. As shown in Figure 3, the number detect all impurities. UVMobile Detector: Phase A: 568Acetonitrile nm of impurity peaks resolved and detected was greatly increased with the HILIC-CAD Sample:Mobile Phase B: 0.28500 mg/mL mM ammonium formate, pH 2.9 approach. About 16 impurity peaks were detected with the HILIC-CAD method at Aminoglycosides can be measured directly by charged aerosol detection without Conclusions InjectionMobile phasevolume: C: 20Water μL FIGURES/N > 3 (Figure3A. Impurity 3A). The Analysis SCX-UV of Apramycinmethod only with detected the HILIC seven-CAD impurities, Method. as not all of derivatization. Corona Veo is a universal mass-sensitive detector, Its response is • The described HILIC-CAD method for apramycin enables more accurate impurity Gradient 0 min., 70 %A, 20%B, 10% C them could be derivatized by the SCX-UV approach (Figure 3B). Furthermore, the independentIntroduction of chemical structure, and does not require the presence of chromophores. assessment, due to the universal detection of CAD and improved sample loading Chromatographic condition30 and min., post 21%-column A, 20%B, derivatization 59% C procedure are same as in 200 improved chromatographic resolution and peak shape allows for a higher sample load Capable of measuring any nonvolatile and many semi-volatile analytes, charged aerosol pA capacity. More than 16 impurities were detected. The SCX-UV method Aminoglycosides are a group of structurally similar antibiotics used to treat infections BritishInjection Veterinary volume: Pharmacopoeia1 μL 2013 with the HILIC-CAD method enabling detection of low level impurities. detection enables accurate degradation studies1 and improved assessments of product caused by aerobic gram-negative bacteria . Analytical methods are required for rapid Corona Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, recommended by British Pharmacopoeia only detected seven impurities. purity. The Corona Veo is much more sensitive than other universal detectors like ELSD HILIC-CAD method assessment of drug purity and detection of minor degredants. As they lack a strong ELSD: Nebulizer temp. 50 °C , evaporation temp. 70 °C The effect of mobile buffer strength and pH on separation and peak shape was • and RI, offering low nanogram quantitation. This poster presents a sensitive HILIC-CAD Data Analysis 150 48.9 µg on column Comparison between Corona Veo and ELSD detection showed that CAD is much chromophore, these compounds are not amenable to UV detection. ™ ™ ™ investigated. The method was optimized with 100mM ammonium formate at pH 2.9. The method for direct analysis of apramycin and other aminoglycosides. Method performance Thermo Scientific Dionex Chromeleon Chromatography Data System software, 7.2 more sensitive than ELSD. With 20µg sample on column, 7 impurities were detected sample load was increased to 48.9 μg for the HILIC-CAD method and still maintains good at S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. was compared to the British Pharmacopoeia HPLC-UV method and ELSD detection. Apramycin is an antibiotic used in veterinary medicine. Reported methods for apramycin SCX-UV method peak shape with half peak width W = 0.77min. While further increase of sample loading 100 0.5 • Sample pretreatment with anion exchange SPE removes interference of sulfate ion and impurities analysis usually involves pre- or post column derivatization followed by UV Column: Venusil SCX-F 4.6 x 150 mm, 5 µm amount with the SCX-UV method caused significant peak broadening and results in 2,3 FIGURE 2. Effect of SPE Pre-treatment of Apramycin Sulfate Sample. and allows for more accurate determination of impurities. detection . Such approaches are tedious and time consuming, and may not be able to Temperature: 30 ºC decreased resolution between apramycin and impurity peaks. detect all impurities. UV Detector: 568 nm 14 • This method can also be used for the analysis of many other aminoglycoside 50 15 Sample: 0.28 mg/mL antibiotics. 11 Conclusions Aminoglycosides can be measured directly by charged aerosol detection without μ 13 Methods Injection volume: 20 L FIGURE 3A. Impurity Analysis of Apramycin with7 the HILIC-CAD Method. derivatization. Corona™ Veo™ is a universal™ mass-sensitive detector, Its response is 5 6 8 9 10 • Thermo Scientific Dionex UltiMate 3000 RSLC system with: 1 2 3 4 12 16 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD The described HILIC-CAD method for apramycin enables more accurate impurity independent• Pump: of chemicalLPG structure,-3400SD and does not require the presence of chromophores. min assessment, due to the universal detection of CAD and improved sample loading Chromatographic condition and post-column derivatization procedure are same as in -10200 Capable• Auto of Sampler:measuring anyWPS-3000TSL nonvolatile and many semi-volatile analytes, charged aerosol British Veterinary Pharmacopoeia 2013 0.0 pA 5.0 10.0 15.0 20.0 25.1 capacity. More than 16 impurities were detected. The SCX-UV method detection• Column enables Compartment accurate degradation :TCC studies and improved assessments of product Peak -3000RS Retention Time (min) S/N recommended by British Pharmacopoeia only detected seven impurities. purity.• DiodeThe Corona Array Detector:Veo is much DAD more-3000RS sensitive than other universal detectors like ELSD HILIC-CAD method Number 150 49 μg on column 20 µg on column • Comparison between Corona Veo and ELSD detection showed that CAD is much - and RI,• Charged offering aerasollow nanogram detector: quantitation. Corona Veo This RS poster presents a sensitive HILIC-CAD Data Analysis FIGURE48.9 3B. µg Impurity on column Analysis of Apramycin with the SCX-UV Method. SO4 ™ ™ ™ CAD ELSD CAD ELSD References method• for direct analysis of apramycin and other aminoglycosides. Method performance Thermo Scientific Dionex Chromeleon Chromatography Data System software, 7.2 more sensitive than ELSD. With 20µg sample on column, 7 impurities were detected Varian ELSD 385-LC 1 10.30 4.9 6.4 - 1. atPerzynski, S/N ≥ 3 with S.; Cannon, CAD, while M.; only Cundliffe, 3 peeks E.; at Chahwala, S/N ≥ 3 were S.B.; detected Davies, with J. Effects ELSD. of was compared to the British Pharmacopoeia HPLC-UV method and ELSD detection. 400 2 11.27 10.4 8.8 - SO - 100 4 3 11.94 3.0 - - • SampleApramycin, pretreatment a novel aminoglycoside with anion exchange antibiotic SPE on removes bacterial interference protein synthesis. of sulfate Eur. ion A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside AntibioticsSCX Using-UV method Charged Aerosol Detection – FIGURE 1. FIGURE 2. Effect of SPE Pre-treatment of Apramycin Sulfate Sample. 4 12.61 4.2 - 6.4 - andJ. Biochem. allows for1979, more 99, accurate 623 628. determination of impurities. Structure of Apramycin 200 μ 5.8 g on column 5 13.32 24.4 19.4 - 1 2 1 1 2 2 3 14 6 14.10 19.6 19.5 3.9 - 2.• ThisBritish method Pharmacopoeia can also be (Veterinary): used for the 2013. analysis London: of many Her other Majesty's aminoglycoside Stationery AZhen Sensitive Long , QiMethod Zhang , Yanfor JinDirect, Lina Analysis Liang , Bruce of Impurities Bailey , Ian Acworth in Apramycin, Deepali Mohindra and Other Aminoglycoside Antibiotics50 Using Charged Aerosol15 Detection 7 14.76 29.0 44.6 6.2 - antibiotics.Office, v.4, p. 38-40, 122-125, 2013. 0 11 1 2 3 13 8 15.78 15.0 14.0 3.2 - 1 2 1 Methods1 2 2 3 7 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in ™ ™ ™ 5 6 8 9 10 mV 9 17.79 18.6 22.9 - ZhenThermo Long ,Fisher Qi Zhang Scientific,, Yan Jin Shanghai,, Lina Liang China,Thermo, Bruce ScientificThermoDionex Bailey UltiMFisherate, 3000Ian Scientific,RSLC Acworth system with: Chelmsford,, Deepali Mohindra MA, USA and Thermo Fisher Scientific, Sunnyvale, CA, USA1 2 3 4 12 16 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD min 10 18.47 10.1 12.3 - - oral soluble powder by a HPLC method using pre-column derivatization with o- • Pump: LPG-3400SD -10 -200 1 2 3 11 18.85 51.7 137.9 11.8 3.7 phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. • Auto Sampler: WPS-3000TSL 0.0 5.0 10.0 15.0 20.0 25.1 Thermo Fisher Scientific, Shanghai, China, Thermo Fisher Scientific, Chelmsford, MA, USA and Thermo Fisher Scientific, Sunnyvale, CA, USA 12 19.34 4.8 - - • Column Compartment :TCC-3000RS Peak 13 Retention21.55 Time (min) 12.7 19.3 S/N - • Diode Array Detector: DAD-3000RS -400 Number SO - 14 21.87 79.549 μ g on column293.9 2021.3 µg on column5.6 © 2015 Thermo Fisher Scientific Inc. All trademarks are the property of Thermo Fisher Scientific and its 4 • Charged aerasol detector: Corona Veo RS FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. 15 22.52 77.0 238.2 17.9 4.1 subsidiaries. • CAD ELSD CAD ELSD References Varian ELSD 385-LC 0 10 20 161 23.5210.30 6.94.9 6.4- - - - This information is not intended to encourage use of these products in any manners that might infringe the - 400 time (min) 1. Perzynski, S.; Cannon, M.; Cundliffe, E.; Chahwala, S.B.; Davies, J. Effects of SO4 2 11.27 10.4 8.8 - intellectual property rights of others. A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside AntibioticsComparison of Using CAD and ELSD DetectionCharged Aerosol DetectionAnalysis of3 Other Aminoglycoside11.94 Antibiotics3.0 - - Apramycin, a novel aminoglycoside antibiotic on bacterial protein synthesis. Eur. Overview Results SCX-UV method – FIGURE 1. 4 12.61 4.2 - 6.4 - J. Biochem. 1979, 99, 623 628. Purpose: To develop a sensitive non-derivatization method for impurity assessment of Sample preStructure-treatment of Apramycin with SPE 200 μ 1 2 1 1 2 2 3 5.8 g on column This method5 has also been13.32 applied to impurity24.4 analysis19.4 of an additional eleven- Aapramycin Sensitive sulfate and other aminoglycoside Method antibiotics. for Direct Analysis of Impurities in Apramycin andSample pre Other-treatment with SPEAminoglycoside AntibioticsComparison of CAD Using and ELSD Detection Charged Aerosol DetectionAnalysis of Other Aminoglycoside Antibiotics 2. British Pharmacopoeia (Veterinary): 2013. London: Her Majesty's Stationery OverviewZhen Long , Qi Zhang , Yan Jin , Lina LiangSPE, Column:Bruce BaileyDionex OnGuard, Ian II A Acworth , Deepali MohindraResults CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside6 antibiotics,14.10 including neomycin,19.6 gentamicin,19.5 kanamycin,3.9 streptomycin,- 7 14.76 29.0 44.6 6.2 - Office, v.4, p. 38-40, 122-125, 2013. Purpose:1 To develop a sensitive non-derivatization method for impurity assessment of SampleSample pre2 -treatmentsolvent: with SPE80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for3 apramycin impurity assessment with a HILIC method. Comparison0 between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient1 method using hydrophilic2 interaction liquid chromatography1 1 2 2 Sample pre-treatment3 with SPE This method has8 also been applied15.78 to impurity15.0 analysis of14.0 an additional3.2 eleven - apramycinZhenThermo sulfate Long and other Fisher , aminoglycoside Qi Zhang Scientific, antibiotics., Yan Shanghai, Jin , Lina China,LiangSample: , BruceThermo Bailey 220.6Fisher mg/mL, in Ian 2Scientific, mL sample Acworth solvent Chelmsford,, Deepali Mohindra MA,Without USAsample cleanup, and some earlyThermo eluting impurities Fisher were found to Scientific, be masked under Sunnyvale,demonstrated that CAD CA, is much USA more sensitive than ELSD. As shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in with charged aerosol detection (HILIC-CAD) was developed for direct analysis of SPE Column: Dionex OnGuard II A CADmV and ELSD are both nebulization-based universal detection technologies. aminoglycoside9 antibiotics, including17.79 neomycin,18.6 gentamicin, 22.9 kanamycin, streptomycin,- SPE procedure: Condition the SPE cartridge with 6 mL sample solvent, then the huge sulfate peak and could not be detected. A Dionex anion exchange SPE chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the 10HILIC-CAD method.18.47 For some other10.1 aminoglycoside12.3 antibiotics,- modification- oral soluble powder by a HPLC method using pre-column derivatization with o- apramycin1 sulfate . Samples were pretreated with solid phase extraction (SPE) to Sample 2solvent: 80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin3 impurity assessment with a HILIC method. Comparison-200 between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient method using hydrophilic interaction liquid chromatography pass the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE detected with CAD at an injected amount of 49.6 μg apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. removeThermo sulfate ion for Fishermore accurate determination Scientific, of impurities. Shanghai, The same sample was China, Sample: Thermo220.6 Fisher mg/mL in 2 mL Scientific,sample solvent Chelmsford, WithoutMA, sample USA cleanup, and some early Thermo eluting impurities wereFisher found to be Scientific, masked under Sunnyvale,demonstrated that CAD isCA, much more USA sensitive than ELSD. As shown in the Figure 5 shows11 impurity analysis18.85 of kenamycin,51.7 etimicin, 137.9 ribostamycin11.8 and paromomycin3.7 with charged aerosol detection (HILIC-CAD) was developed for direct analysis of only 12 impurities were detected with ELSD at this level. When injection amount cartridge with additional 2 mL sample solvent . Combine cartridge while the apramycin and impurities passed through the cartridge and collected chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC12 -CAD method.19.34 For some other4.8 aminoglycoside - antibiotics, modification- also analyzed with the SCX-UV method recommended by British pharmacopoeia SPE procedure: Condition the SPE cartridge with 6 mL sample solvent, then the huge sulfate peak and could not be detected. A Dionex anion exchange SPE μ apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to collected loading eluent and wash solution for analysis. The for further analysis. Sulfate was replaced by bicarbonate after SPE, which has little decreased-400 to 20 g on column, 7 impurity peaks were detected with CAD at S/N > 3, 13 21.55 12.7 19.3 - (veterinary) 2013, which requires post column derivatization. pass the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE detected with CAD at an injected amount of 49.6 μg apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. 14 21.87 79.5 293.9 21.3 5.6 © 2015 Thermo Fisher Scientific Inc. All trademarks are the property of Thermo Fisher Scientific and its final concentration of apramycin sample was 49 mg/mL. interference with apramycin analysis, since it is volatile and elutes earlier than the peaks only 12 impurities were detected with ELSD at this level. When injection amount cartridge with additional 2 mL sample solvent . Combine cartridge while the apramycin and impurities passed through the cartridge and collected FIGURE 5.15 Chromatograms22.52 for impurity analysis77.0 of kenamycin,238.2 etimicin,17.9 ribostamycin4.1 and subsidiaries. also analyzed with the SCX-UV method recommended by British pharmacopoeia A 2 mL volume of sample solvent was treated with the same of interest. As seen in Figure 2, after removing sulfate, more impurities can now be μ Results: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD collected loading eluent and wash solution for analysis. The for further analysis. Sulfate was replaced by bicarbonate after SPE, which has little decreased0 to 20 g on column, 7 impurity10 peaks were detected with20 CAD at S/N > 3, 16 23.52 6.9 - - - (veterinary) 2013, which requires post column derivatization. procedure and used as blank. detected. Recovery of three impurity peaks, labeled as peak 11, 14 and 15 in Figure 3a, The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. This information is not intended to encourage use of these products in any manners that might infringe the method. The SCX-UV method recommended by British Pharmacopoeia only detected just final concentration of apramycin sample was 49 mg/mL. interference with apramycin analysis, since it is volatile and elutes earlier than the peaks while only 3 peaks were detected with ELSDtime with (min) much lower S/N compared to CAD. intellectual property rights of others. Comparisonto the sigmoidal of CAD nature and of ELSDits response Detection curve, resulting in much lower sensitivity. Analysis of Other Aminoglycoside Antibiotics Overviewseven impurities. The HILIC-CAD method is much more sensitive than ELSD. With Resultswas calculated to be 107%, 92% and 93%, respectively. FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and Results: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD A 2 mL volume of sample solvent was treated with the same of interest. As seen in Figure 2, after removing sulfate, more impurities can now be Purpose:20 μg apramycinTo develop sulfate a sensitive on column, non- derivatization7 impurities were method detected for impurity by CAD assessment at S/N > 3, ofwhile Sample pre-treatment withprocedure SPE and used as blank. The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. method. The SCX-UV method recommended by British Pharmacopoeia only detected just HILIC-CAD/ELSD method detected.Sample Recoverypre-treatment of three with impurity SPE peaks, labeled as peak 11, 14 and 15 in Figure 3a, This method has also been applied to impurity analysis of an additional eleven apramycinonly 3 impurities sulfate andwere other detected aminoglycoside by ELSD. This antibiotics. method, with or without slight modification, toComparison the sigmoidal of nature CAD of and its responseELSD Detection curve, resulting in much lower sensitivity. Analysis of Other Aminoglycoside Antibiotics sevenOverview impurities. The HILIC-CAD method is much more sensitive than ELSD. With SPE Column: Dionex OnGuard II A wasResults calculated to be 107%, 92% and 93%, respectively. CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, was also used for impurity measurement of an additional eleven aminoglycoside Comparison of HILIC-CAD method with SCX-UV method 20Purpose: μg apramycinTo develop sulfate a on sensitive column, non- 7 impuritiesderivatization were methoddetected for by impurity CAD at assessmentS/N > 3, while of SampleSample pre solvent:-treatment with 80% SPE 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin impurity assessment with a HILIC method. Comparison between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient method using hydrophilic interaction liquid chromatography HILIC-Column:CAD/ELSD methodACCHROM (Beijing, China), Click XIon, 4.6 x 150mm, 5µm Sample pre-treatment with SPE This method has also been applied to impurity analysis of an additional eleven apramycinantibiotics, sulfate including and neomycin,other aminoglycoside gentamicin, kanamycin, antibiotics. streptomycin, tobramycin, Sample: 220.6 mg/mL in 2 mL sample solvent FIGUREdemonstrated 4. Comparison that CAD of isDetection much more Sensitivity sensitive B etweenthan ELSD. CAD As and shown ELSD. in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin onlywith 3 chargedimpurities aerosol were detecteddetection by (HILIC ELSD.-CAD) This wasmethod, developed with or for without direct slight analysis modification, of SPETemperature: Column: Dionex30 ºC OnGuard II A WithoutThe HILIC- sampleCAD cleanup, method some was comparedearly eluting to impuritiesthe SCX-UV were method found recommended to be masked byunder British CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Comparison of HILIC-CAD method with SCX-UV method chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC-CAD method. For some other aminoglycoside antibiotics, modification was also used for impurity measurement of an additional eleven aminoglycoside SPEFlow procedure:rate: Condition1 mL/min the SPE cartridge with 6 mL sample solvent, then thePharmacopoeia huge sulfate peak (veterinary) and could 2013 not version be detected. (BP2013) A Dionex . As shown anion exchange in Figure 3,SPE the number apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to Column:Sample solvent: AC 80%CHROM 5mM (Beijing, ammonium China), formate, Click 20%XIon acetonitrile, 4.6 x 150mm, 5µm Sulfate is a major interference for apramycin impurity assessment with a HILIC method. Comparison between CAD and ELSD under the μsame chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. antibiotics,Methods: includingA 30min neomycin,gradient method gentamicin, using kanamycin,hydrophilic interactionstreptomycin, liquid tobramycin, chromatography Mobile Phase A: passAcetonitrile the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE detected with CAD at an injected amount of 49.6 g apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was Temperature:Sample: 30 220.6ºC mg/mL in 2 mL sample solvent Withoutof impurity sample peaks cleanup, resolved some and early detected eluting was impurities greatly increasedwere found with to bethe masked HILIC-CAD under FIGUREdemonstrated 4. Comparison that CAD of Detection is much Sensitivity more sensitive Between than CAD ELSD. and As ELSD. shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin with charged aerosol detection (HILIC-CAD) was developed for direct analysis of cartridge with additional 2 mL sample solvent . Combine Thecartridge HILIC- CADwhile the method apramycin was compared and impurities to the passedSCX-UV through method the recommended cartridge and by collected British only 12 impurities were detected with ELSD at this level. When injection amount amikacin,also analyzed etimicin, with netilmicin, the SCX -UVsisomicin, method ribostamycin recommended and by paromomycin. British pharmacopoeia SPEMobile procedure: Phase B: Condition500 mM ammonium the SPE cartridge formate, withpH 2.96 mL sample solvent, then approach. About 16 impurity peaks were detected with the HILIC-CAD method at chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC-CAD method. For some other aminoglycoside antibiotics, modification apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to Flow rate: 1 mL/min Pharmacopoeiathe huge sulfate (veterinary) peak and could2013 versionnot be detected. (BP2013) A . Dionex As shown anion in Figureexchange 3, the SPE number decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, Mobile phase C: collectedWater loading eluent and wash solution for analysis. The forS/N further > 3 (Figureanalysis. 3A). Sulfate The SCXwas-UV replaced method by onlybicarbonate detected afterseven SPE, impurities, which has as notlittle all of μ (veterinary) 2013, which requires post column derivatization. Mobile Phase A: Acetonitrilepass the sample solution through the cartridge and wash the ofcartridge impurity peaksOn Guard resolved II A was and used detected to remove was greatlysulfate. increased Sulfate was with retained the HILIC-CAD on the SPE detected with CAD at an injected amount of 49.6 g apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was Gradient final0 min., concentration 70 %A, 20%B, of apramycin 10% C sample was 49 mg/mL. interference with apramycin analysis, since it is volatile and elutes earlier than the peaks while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. Mobile Phase B: 500cartridge mM ammonium with additional formate, 2 mLpH sample2.9 solvent . Combine cartridgethem could while be the derivatized apramycin by and the impuritiesSCX-UV approach passed through (Figure the3B). cartridge Furthermore, and collected the only 12 impurities were detected with ELSD at this level. When injection amount alsoIntroduction analyzed with the SCX-UV method recommended by British pharmacopoeia A 2 mL volume of sample solvent was treated with the same approach.of interest. About As seen 16 impurity in Figure peaks 2, after were removing detected sulfate, with the more HILIC impurities-CAD method can now at be FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and Results: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD collected30 min., 21% loading A, 20%B, eluent 59% and Cwash solution for analysis. The improved chromatographic resolution and peak shape allows for a higher sample load decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, (veterinary) 2013, which requires post column derivatization. Mobile phase C: Water S/Nfor > further 3 (Figure analysis. 3A). The Sulfate SCXwas-UV replacedmethod only by bicarbonate detected seven after impurities, SPE, which as hasnot alllittle of The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. method.Aminoglycosides The SCX- UVare methoda group recommended of structurally similarby British antibiotics Pharmacopoeia used to treat only infectionsdetected just Injection volume: procedure1 μL and used as blank. detected.with the RecoveryHILIC-CAD of method three impurity enabling peaks, detection labeled of low as level peak impurities. 11, 14 and 15 in Figure 3a, while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. 1 Gradient 0 min.,final concentration70 %A, 20%B, of 10% apramycin C sample was 49 mg/mL. theminterference could be withderivatized apramycin by theanalysis, SCX-UV since approach it is volatile (Figure and 3B). elutes Furthermore, earlier than the the peaks Introductioncaused by aerobic gram-negative bacteria . Analytical methods are required for rapid Corona Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, was calculated to be 107%, 92% and 93%, respectively. to the sigmoidal nature of its response curve, resulting in much lower sensitivity. FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and seven impurities.16 impurities The HILICof apramycin-CAD method were detectedis much moreat S/N sensitive ≥ 3 with thanthe HILIC ELSD. With 30 Amin., 2 mL 21%volume A, 20%B, of sample 59% C solvent was treated with the same improvedof interest. chromatographic As seen in Figure resolution 2, after and removing peak shape sulfate, allows more for impurities a higher cansample now load be Results:assessment of drug purity and detection of minor degredants. As they lack -CADa strong ELSD: Nebulizer temp. 50 °C , evaporation temp. 70 °C Aminoglycosides20 μg apramycin are sulfate a group on column,of structurally 7 impurities similar were antibiotics detected used by to CAD treat at infections S/N > 3, while Injection volume: 1 μprocedureL and used as blank. detected.The effect Recovery of mobile of bufferthree impuritystrength peaks,and pH labeled on separation as peak and 11, peak 14 and shape 15 inwas Figure 3a, The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. method.chromophore, The SCX- theseUV compoundsmethod recommended are not amenable by British to UVPharmacopoeia detection. only detected just HILIC-CAD/ELSD method with the HILIC-CAD method enabling detection of low level impurities. causedonly 3 byimpurities aerobic weregram -detectednegative bybacteria ELSD.1. ThisAnalytical method, methods with or are without required slight for modification, rapid Corona Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, wasinvestigated. calculated The to be method 107%, was 92% optimized and 93%, with respectively. 100mM ammonium formate at pH 2.9. The to the sigmoidal nature of its response curve, resulting in much lower sensitivity. seven impurities. The HILIC-CAD method is much more sensitive than ELSD. With Comparison of HILIC-CAD method with SCX-UV method assessmentwas also used of drug for impuritypurity and measurement detection of ofminor an additional degredants. eleven As they aminoglycoside lack a strong ELSD: Nebulizer temp. 50 °C , evaporation temp. 70 °C sample load was increased to 48.9 μg for the HILIC-CAD method and still maintains good 20Apramycin μg apramycin is an sulfateantibiotic on usedcolumn, in veterinary 7 impurities medicine. were detected Reported by methods CAD at S/N for >apramycin 3, while Column: ACCHROM (Beijing, China), Click XIon, 4.6 x 150mm, 5µm The effect of mobile buffer strength and pH on separation and peak shape was chromophore,antibiotics, including these compounds neomycin, are gentamicin, not amenable kanamycin, to UV detection.streptomycin, tobramycin, HILIC-SCX-UVCAD/ELSD method method peak shape with half peak width W0.5 = 0.77min. While further increase of sample loading onlyand 3 impurities impurities analysis were detected usually by involves ELSD. pre This- or method, post column with or derivatization without slight followed modification, by UV Temperature: 30 ºC investigated.The HILIC- CADThe method method was was optimized compared with to the 100mM SCX- ammoniumUV method formaterecommended at pH 2.9. by BritishThe FIGURE 4. Comparison of Detection Sensitivity Between CAD and ELSD. amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Column: Venusil SCX-F 4.6 x 150 mm, 5 µm Comparisonamount with ofthe HILIC SCX-CAD-UV method methodμ caused with SCX- significantUV method peak broadening and results in wasdetection also used2,3. Such for impurity approaches measurement are tedious of andan additional time consuming, eleven aminoglycosideand may not be able to Flow rate: 1 mL/min samplePharmacopoeia load was increased(veterinary) to 201348.9 versiong for the (BP2013) HILIC-CAD . As method shown and in Figure still maintains 3, the number good Apramycin is an antibiotic used in veterinary medicine. Reported methods for apramycin Column:Temperature: AC30CHROM ºC (Beijing, China), Click XIon, 4.6 x 150mm, 5µm decreased resolution between apramycin and impurity peaks. antibiotics,detect all impurities. including neomycin, gentamicin, kanamycin, streptomycin, tobramycin, SCX-UVMobile Phasemethod A: Acetonitrile peakof impurity shape peakswith half resolved peak width and Wdetected0.5 = 0.77min. was greatly While increasedfurther increase with the of HILIC-CAD sample loading and impurities analysis usually involves pre- or post column derivatization followed by UV Temperature:UV Detector: 30568 ºC nm The HILIC-CAD method was compared to the SCX-UV method recommended by British FIGURE 4. Comparison of Detection Sensitivity Between CAD and ELSD. amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Column:Mobile Phase B: Venusil500 mM SCX- ammoniumF 4.6 x 150formate, mm, 5pH µm 2.9 amountapproach. with Aboutthe SCX 16- UVimpurity method peaks caused were significant detected peakwith the broadening HILIC-CAD and method results atin detection2,3. Such approaches are tedious and time consuming, and may not be able to FlowSample: rate: 1 0.28mL/min mg/mL Pharmacopoeia (veterinary) 2013 version (BP2013) . As shown in Figure 3, the number Aminoglycosides can be measured directly by charged aerosol detection without Temperature:Mobile phase C: 30Water ºC decreasedS/N > 3 (Figure resolution 3A). between The SCX apramycin-UV method and only impurity detected peaks. seven impurities, as not all of Conclusions detect all impurities. MobileInjection Phase volume: A: Acetonitrile20 μL ofFIGURE impurity peaks3A. Impurity resolved Analysis and detected of Apramycin was greatly with increased the HILIC with-CAD the Method. HILIC-CAD derivatization. Corona Veo is a universal mass-sensitive detector, Its response is UVGradient Detector: 5680 min., nm 70 %A, 20%B, 10% C them could be derivatized by the SCX-UV approach (Figure 3B). Furthermore, the Introduction Mobile Phase B: 500 mM ammonium formate, pH 2.9 approach. About 16 impurity peaks were detected with the HILIC-CAD method at • The described HILIC-CAD method for apramycin enables more accurate impurity independent of chemical structure, and does not require the presence of chromophores. Sample: 0.2830 min.,mg/mL 21% A, 20%B, 59% C improved chromatographic resolution and peak shape allows for a higher sample load Conclusionsassessment, due to the universal detection of CAD and improved sample loading AminoglycosidesAminoglycosides can are be a groupmeasured of structurally directly by similar charged antibiotics aerosol useddetection to treat without infections MobileChromatographic phase C: conditionWaterμμ and post-column derivatization procedure are same as in 200S/N > 3 (Figure 3A). The SCX-UV method only detected seven impurities, as not all of Capable of measuring any nonvolatile and many semi-volatile analytes, charged aerosol InjectionInjection volume: volume: 201 LL FIGUREwithpA the 3A.HILICImpurity-CAD method Analysis enabling of Apramycin detection with of low the level HILIC impurities.-CAD Method. capacity. More than 16 impurities were detected. The SCX-UV method derivatization.caused by aerobic Corona gram Veo-negative is a universal bacteria mass-1. Analyticalsensitive methodsdetector, areIts responserequired foris rapid GradientBritish Veterinary Pharmacopoeia0 min.,° 70 2013 %A, 20%B, 10% C them could be derivatized by the SCX-UV approach (Figure 3B). Furthermore, the • The described HILIC-CAD method for apramycin enables more accurate impurity Introductiondetection enables accurate degradation studies and improved assessments of product Corona Veo RS: 55 C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, recommended by British Pharmacopoeia only detected seven impurities. independent of chemical structure, and does not require the presence of chromophores. 30 min., 21% A, 20%B, 59% C improved chromatographic resolution and peak shape allows for a higher sample load assessment, due to the universal detection of CAD and improved sample loading assessmentpurity. The Coronaof drug purityVeo is and much detection more sensitive of minor than degredants. other universal As they detectors lack a strong like ELSD ChromatographicELSD: conditionNebulizer and post -temp.column 50 derivatization°C , evaporation procedure temp. 70are ° Csame as in 200 The effectHILIC of mobile-CAD methodbuffer strength and pH on separation and peak shape was Aminoglycosides are a group of structurally similar antibiotics used to treat infections Injection volume: 1 μL pA Capablechromophore, of measuring these compounds any nonvolatile are andnot amenablemany semi to-volatile UV detection. analytes, charged aerosol British Veterinary Pharmacopoeia 2013 150with the HILIC-CAD method enabling detection of low level impurities. capacity. • Comparison More than between 16 impurities Corona Veowere and detected. ELSD detectionThe SCX -showedUV method that CAD is much causedand RI, by offering aerobic low gram nanogram-negative quantitation. bacteria1. Analytical This poster methods presents are a required sensitive for HILIC rapid-CAD CoronaData Analysis Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, investigated.48.9 Theµg onmethod column was optimized with 100mM ammonium formate at pH 2.9. The detection enables accurate degradation studies and improved assessments of product ™ ™ ™ recommendedmore sensitive by Britishthan ELSD. Pharmacopoeia With 20µg sampleonly detected on column, seven 7 impurities. impurities were detected assessmentmethod for ofdirect drug analysis purity and of apramycin detection andof minor other degredants. aminoglycosides. As they Method lack a strongperformance Thermo Scientific Dionex Chromeleon Chromatog° raphy Data System° software, 7.2 sample load was increased to 48.9 μg for the HILIC-CAD method and still maintains good purity.Apramycin The Corona is an antibiotic Veo is much used more in veterinary sensitive medicine.than other Reported universal methods detectors for like apramycin ELSD ELSD: Nebulizer temp. 50 C , evaporation temp. 70 C The HILICeffect of-CAD mobile method buffer strength and pH on separation and peak shape was at S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. chromophore,was compared these to the compounds British Pharmacopoeia are not amenable HPLC to-UV UV method detection. and ELSD detection. SCX-UV method 150 peak shape with half peak width W0.5 = 0.77min. While further increase of sample loading • Comparison between Corona Veo and ELSD detection showed that CAD is much andand RI, impurities offering lowanalysis nanogram usually quantitation. involves pre This- or posterpost column presents derivatization a sensitive followed HILIC-CAD by UV Data Analysis investigated.48.9 µg Theon column method was optimized with 100mM ammonium formate at pH 2.9. The Column: ™ ™Venusil SCX-F™ 4.6 x 150 mm, 5 µm 100amount with the SCX-UV method caused significant peak broadening and results in more• Sample sensitive pretreatment than ELSD. with With anion 20µg exchange sample onSPE column, removes 7 impurities interference were of detectedsulfate ion methoddetection for2,3 direct. Such analysis approaches of apramycin are tedious and andother time aminoglycosides. consuming, and Method may not performance be able to Thermo Scientific Dionex Chromeleon Chromatography Data System software, 7.2 sample load was increased to 48.9 μg for the HILIC-CAD method and still maintains good Apramycin is an antibiotic used in veterinary medicine. Reported methods for apramycin Temperature:FIGURE 2. Effect of SPE30 ºCPre-treatment of Apramycin Sulfate Sample. decreased resolution between apramycin and impurity peaks. at S/Nand ≥ allows3 with for CAD, more while accurate only 3 determinationpeeks at S/N ≥of 3 impurities. were detected with ELSD. wasdetect compared all impurities. to the British Pharmacopoeia HPLC-UV method and ELSD detection. SCX-UV method peak shape with half peak width W0.5 = 0.77min. While further increase of sample loading and impurities analysis usually involves pre- or post column derivatization followed by UV UV Detector: 568 nm 100 Column: Venusil SCX-F 4.6 x 150 mm, 5 µm amount with the SCX-UV method caused significant peak broadening and results14 in • Sample• This methodpretreatment can also with be anion used exchange for the analysis SPE removes of many interference other aminoglycoside of sulfate ion detection2,3. Such approaches are tedious and time consuming, and may not be able to Sample: 0.28 mg/mL 50 15 Aminoglycosides can be measured directly by charged aerosol detection without FIGURETemperature: 2. Effect of SPE Pre-30 ºCtreatment of Apramycin Sulfate Sample. decreased resolution between apramycin and impurity peaks. Conclusionsandantibiotics. allows for more accurate determination of impurities. detect all impurities. Injection volume: 20 μL 11 UV Detector: 568 nm FIGURE 3A. Impurity Analysis of Apramycin with the HILIC-CAD Method.13 derivatization.Methods Corona Veo is a universal mass-sensitive detector, Its response is 7 • • The described HILIC-CAD method for apramycin enables more accurate impurity ™ ™ ™ 50 5 6 8 9 10 14 This method can also be used for the analysis of many other aminoglycoside independentThermo Scientific of chemicalDionex structure, UltiM ate and does3000 notRSLC require system the with:presence of chromophores. Sample: 0.28 mg/mL 1 2 3 4 12 15 16 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD min Conclusionsassessment, due to the universal detection of CAD and improved sample loading Aminoglycosides• Pump: can be measured directly by charged aerosol detection without Chromatographic condition and post-column derivatization procedure are same as in 200 11 antibiotics. Capable of measuring any nonvolatileLPG-3400SD and many semi-volatile analytes, charged aerosol Injection volume: 20 μL -10FIGUREpA 3A. Impurity Analysis of Apramycin with the HILIC-CAD Method. Methodsderivatization. Corona Veo is a universal mass-sensitive detector, Its response is British Veterinary Pharmacopoeia 2013 13 • capacity. More than 16 impurities were detected. The SCX-UV method • Auto Sampler:™ ™ WPS-3000TSL™ 0.0 5.0 10.0 5 6 7 15.0 9 10 20.0 25.1 The described HILIC-CAD method for apramycin enables more accurate impurity Thermodetection Scientific enablesDionex accurate UltiM degradationate 3000 studies RSLC and system improved with: assessments of product 2 8 12 16 recommended by British Pharmacopoeia only detected seven impurities. independent• Column of chemical Compartment structure, :TCC and does not require the presence of chromophores. 1 3 4 Table 1. ComparisonPeak of Detection Sensitivity Between CAD and ELSD assessment, due to the universal detection of CAD and improved sample loading purity.• Pump:The Corona Veo is much more -sensitive3000RS than other universal detectors like ELSD Chromatographic condition and post-column derivatization procedure are same as in 200 HILIC-CAD method min Retention Time (min) S/N LPG-3400SD -10 pA Capable• Diode of measuring Array Detector: any nonvolatile DAD-3000RS and many semi-volatile analytes, charged aerosol British Veterinary Pharmacopoeia 2013 150 Number • Comparisoncapacity. More between than 16 Corona impurities Veo were and ELSDdetected. detection The SCX showed-UV methodthat CAD is much and RI,• Auto offering Sampler: low nanogramWPS-3000TSL quantitation. This poster presents a sensitive HILIC-CAD Data Analysis 0.0 48.9 µg5.0 on column 10.0 15.0 20.0 25.1 49 μg on column 20 µg on column detection• Charged enables aerasolaccurate detector: degradation Corona studies Veo andRS improved assessments of product ™ ™ ™ FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. morerecommended sensitive thanby British ELSD. Pharmacopoeia With 20µg sample only ondetected column, seven 7 impurities impurities. were detected method• Column for direct Compartment analysis of :TCCapramycin-3000RS and other aminoglycosides. Method performance Thermo Scientific Dionex Chromeleon Chromatography Data System software, 7.2 Peak CAD ELSD CAD ELSD References purity. The• Varian Corona ELSD Veo 385- is muchLC more sensitive than other universal detectors like ELSD HILIC-CAD method Retention Time (min) S/N at S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. was •compared Diode Array to the Detector: British DADPharmacopoeia-3000RS HPLC-UV method and ELSD detection. 150 Number1 10.30 4.9 6.4 - • Comparison between Corona Veo and ELSD detection showed that CAD is much and RI, offering low nanogram quantitation. This poster presents a sensitive HILIC-CAD Data Analysis 100 400 48.9 µg on column 49 μg on column 20 µg on column 1. Perzynski, S.; Cannon, M.; Cundliffe, E.; Chahwala, S.B.; Davies, J. Effects of • ™ ™ ™ 2 11.27 10.4 8.8 - • methodCharged for direct aerasol analysis detector: of apramycin Corona andVeo other RS aminoglycosides. Method performance Thermo Scientific Dionex Chromeleon Chromatography Data System software, 7.2 FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. SamplemoreApramycin, sensitive pretreatment thana novel ELSD. with aminoglycoside anion With exchange20µg sample antibiotic SPE on removes oncolumn, bacterial interference7 impurities protein synthesis. ofwere sulfate detected ion Eur. • 3 11.94 CAD 3.0 ELSD - CAD ELSD - Referencesat S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. was comparedVarian ELSD to the 385- BritishLC Pharmacopoeia HPLC-UV method and ELSD detection. FIGURE 2. Effect of SPE Pre-treatment of Apramycin Sulfate Sample. SCX-UV method 1 10.30 4.9 6.4 - and J. allows Biochem. for more1979, accurate 99, 623 determination–628. of impurities. FIGURE 1. Structure of Apramycin 4 12.61 4.2 - 6.4 - 1. Perzynski, S.; Cannon, M.; Cundliffe, E.; Chahwala, S.B.; Davies, J. Effects of 100 400200 5.8 μg on column 2 11.27 10.4 8.8 - 14 5 13.32 24.4 19.4 - •• ThisSample method pretreatment can also withbe used anion for exchange the analysis SPE of removes many other interference aminoglycoside of sulfate ion 50 15 3 11.94 3.0 - - 2.Apramycin,British Pharmacopoeia a novel aminoglycoside (Veterinary): antibiotic 2013. onLondon: bacterial Her protein Majesty's synthesis. Stationery Eur. FIGURE 2. Effect of SPE Pre-treatment of Apramycin Sulfate Sample. SCX-UV method 6 14.10 19.6 19.5 3.9 - antibiotics.and allows for more accurate– determination of impurities. FIGURE 1. Structure of Apramycin 11 4 7 12.6114.76 4.229.0 -44.6 6.4 6.2 - - J. Biochem.Office, v.4,1979, p. 38 99,-40, 623 122-628.125, 2013. Methods 200 0 5.8 μg on column 13 ™ ™ ™ 5 7 9 10 5 13.32 24.4 19.4 - • Thermo Scientific Dionex UltiMate 3000 RSLC system with: 50 2 6 8 12 14 8 15.78 15.0 14.0 3.2 - This method can also be used for the analysis of many other aminoglycoside 1 3 4 15 16 Table 1. Comparison6 of 14.10Detection Sensitivity19.6 Between19.5 CAD and ELSD3.9 - 2. 3.BritishBarbosa, Pharmacopoeia E.A.; Lourenco, (Veterinary): F. R.; Terezinha 2013. London: Pinto. HerDetermination Majesty's Stationery of apramycin in • Pump: mV min 9 17.79 18.6 22.9 - antibiotics. LPG-3400SD -10 11 7 14.76 29.0 44.6 6.2 - Office,oral v.4,soluble p. 38 powder-40, 122 by-125, a HPLC 2013. method using pre-column derivatization with o- 0 13 10 18.47 10.1 12.3 - - Methods• Auto Sampler: 0.0 -200 5.0 10.0 15.07 20.0 25.1 ™ WPS-3000TSL™ ™ 5 6 8 9 10 8 11 15.7818.85 15.051.7 14.0137.9 3.211.8 - 3.7 phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. Thermo• Column Scientific CompartmentDionex UltiM :TCCate 3000 RSLC system with: 1 2 3 4 12 16 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in -3000RS mV P9eak 17.79 18.6 22.9 - • Pump: LPG-3400SD min 12 Retention19.34 Time (min) 4.8 S/N- - oral soluble powder by a HPLC method using pre-column derivatization with o- • Diode Array Detector: DAD-3000RS -10 N10umber13 18.4721.55 10.112.7 12.319.3 - - - • Auto Sampler: WPS-3000TSL -2000.0 -400 5.0 10.0 15.0 20.0 25.1 μ phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. • Charged aerasol detector: Corona Veo RS FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. 11 14 18.8521.87 51.749 79.5 g on column137.9293.9 2011.8 µg21.3on column3.7 5.6 © 2015 Thermo Fisher Scientific Inc. All trademarks are the property of Thermo Fisher Scientific and its • Column Compartment :TCC Peak • -3000RS 12 15 Retention19.3422.52 Time (min) 4.8CAD77.0 ELSD238.2- S/N CAD17.9 ELSD- 4.1 Referencessubsidiaries. • VarianDiode ArrayELSD Detector:385-LC DAD Number1 10.30 4.9 6.4 - -3000RS -400 0 10 20 13 16 21.5523.52 12.7 6.9 19.3 - - - - 400 μ 1.ThisPerzynski,information is S.;not intendedCannon, to encourageM.; Cundliffe, use of theseE.; Chahwala, products in any S.B.; manners Davies, that might J. Effects infringe theof • 142 21.8711.27 79.54910.4 g on column293.98.8 2021.3 µg on column5.6 - © 2015 Thermo Fisher Scientific Inc. All trademarks are the property of Thermo Fisher Scientific and its Charged aerasol detector: Corona Veo RS FIGURE 3B. Impurity Analysis of Apramycintime (min) with the SCX-UV Method. intellectualApramycin, property a rights novel of others. aminoglycoside antibiotic on bacterial protein synthesis. Eur. • 153 22.5211.94 77.0CAD3.0 238.2ELSD- 17.9CAD ELSD4.1 - subsidiaries.References Varian ELSD 385-LC SCX-UV method 1 10.30 4.9 6.4 - J. Biochem. 1979, 99, 623–628. FIGURE 1. 0 10 20 164 23.5212.61 6.94.2 - - 6.4- - - Structure of Apramycin 200400 μ This1.informationPerzynski, is not intendedS.; Cannon, to encourage M.; Cundliffe, use of these E.; products Chahwala, in any manners S.B.; thatDavies, might infringeJ. Effects the of 5.8 g on column 52 13.3211.27 24.410.4 19.48.8 -- time (min) intellectualApramycin, property rights a ofnovel others. aminoglycoside antibiotic on bacterial protein synthesis. Eur. 63 14.1011.94 19.63.0 19.5- 3.9 -- 2. British Pharmacopoeia (Veterinary): 2013. London: Her Majesty's Stationery SCX-UV method – FIGURE 1. 74 14.7612.61 29.04.2 44.6- 6.26.4 -- Office,J. Biochem. v.4, p. 1979,38-40, 99, 122 623-125,628. 2013. Structure of Apramycin 2000 μ 5.8 g on column 85 15.7813.32 15.024.4 14.019.4 3.2 -- 6 14.10 19.6 19.5 3.9 - 3.2. Barbosa,British Pharmacopoeia E.A.; Lourenco, (Veterinary): F. R.; Terezinha 2013. Pinto.London: Determination Her Majesty's of apramycinStationery in mV 9 17.79 18.6 22.9 - 107 18.4714.76 10.129.0 12.344.6 6.2- -- oralOffice, soluble v.4, p. powder 38-40, by 122 a -HPLC125, 2013. method using pre-column derivatization with o- 2 -2000 A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside Antibiotics Using Charged Aerosol Detection phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. 118 18.8515.78 51.715.0 137.914.0 11.83.2 3.7- 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in

mV 129 19.3417.79 18.64.8 22.9- -- 10 18.47 10.1 12.3 - - oral soluble powder by a HPLC method using pre-column derivatization with o- -400-200 13 21.55 12.7 19.3 - 1411 21.8718.85 79.551.7 293.9137.9 21.311.8 5.63.7 © 2015phthalaldehyde Thermo Fisher Scientific and Inc. UV All detection. trademarks areBraz. the propertyJ. Pharm. of Thermo Sci. Fisher2011, Scientific 47, 261 and-268. its 1512 22.5219.34 77.04.8 238.2- 17.9 4.1- subsidiaries. -400 0 10 20 1613 23.5221.55 12.76.9 19.3- - -- This information is not intended to encourage use of these products in any manners that might infringe the time (min) 14 21.87 79.5 293.9 21.3 5.6 intellectual© 2015 Thermo property Fisher rights Scientific of others. Inc. All trademarks are the property of Thermo Fisher Scientific and its 15 22.52 77.0 238.2 17.9 4.1 subsidiaries. 0 10 20 16 23.52 6.9 - - - This information is not intended to encourage use of these products in any manners that might infringe the time (min) intellectual property rights of others. - SO4 A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside Antibiotics Using Charged Aerosol Detection Zhen Long1, Qi Zhang2, Yan Jin1, Lina Liang1, Bruce Bailey2, Ian Acworth2, Deepali Mohindra3 1Thermo Fisher Scientific, Shanghai, China, 2Thermo Fisher Scientific, Chelmsford, MA, USA and 3Thermo Fisher Scientific, Sunnyvale, CA, USA

Overview Results Comparison of CAD and ELSD Detection Analysis of Other Aminoglycoside Antibiotics Purpose: To develop a sensitive non-derivatization method for impurity assessment of Sample pre-treatment with SPE This method has also been applied to impurity analysis of an additional eleven apramycin sulfate and other aminoglycoside antibiotics. Sample pre-treatment with SPE SPE Column: Dionex OnGuard II A CAD and ELSD are both nebulization-based universal detection technologies. aminoglycoside antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, Comparison between CAD and ELSD under the same chromatographic conditions tobramycin, amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. Methods: A 30min gradient method using hydrophilic interaction liquid chromatography Sample solvent: 80% 5mM ammonium formate, 20% acetonitrile Sulfate is a major interference for apramycin impurity assessment with a HILIC method. demonstrated that CAD is much more sensitive than ELSD. As shown in the Figure 5 shows impurity analysis of kenamycin, etimicin, ribostamycin and paromomycin with charged aerosol detection (HILIC-CAD) was developed for direct analysis of Sample: 220.6 mg/mL in 2 mL sample solvent Without sample cleanup, some early eluting impurities were found to be masked under chromatograms in Figure 4 and data summarized in Table 1, 16 impurities (S/N >3) were using the HILIC-CAD method. For some other aminoglycoside antibiotics, modification apramycin sulfate . Samples were pretreated with solid phase extraction (SPE) to SPE procedure: Condition the SPE cartridge with 6 mL sample solvent, then the huge sulfate peak and could not be detected. A Dionex anion exchange SPE detected with CAD at an injected amount of 49.6 μg apramycin sulfate on column, while of the gradient may be required for optimized separation and resolution. remove sulfate ion for more accurate determination of impurities. The same sample was pass the sample solution through the cartridge and wash the cartridge On Guard II A was used to remove sulfate. Sulfate was retained on the SPE only 12 impurities were detected with ELSD at this level. When injection amount also analyzed with the SCX-UV method recommended by British pharmacopoeia cartridge with additional 2 mL sample solvent . Combine cartridge while the apramycin and impurities passed through the cartridge and collected decreased to 20 μg on column, 7 impurity peaks were detected with CAD at S/N > 3, (veterinary) 2013, which requires post column derivatization. collected loading eluent and wash solution for analysis. The for further analysis. Sulfate was replaced by bicarbonate after SPE, which has little final concentration of apramycin sample was 49 mg/mL. interference with apramycin analysis, since it is volatile and elutes earlier than the peaks while only 3 peaks were detected with ELSD with much lower S/N compared to CAD. FIGURE 5. Chromatograms for impurity analysis of kenamycin, etimicin, ribostamycin and Results: 16 impurities of apramycin were detected at S/N ≥ 3 with the HILIC-CAD A 2 mL volume of sample solvent was treated with the same of interest. As seen in Figure 2, after removing sulfate, more impurities can now be The rapid decrease in analyte response at lower concentration found with ELSD is due paromomycin using the HILIC-CAD method. method. The SCX-UV method recommended by British Pharmacopoeia only detected just procedure and used as blank. detected. Recovery of three impurity peaks, labeled as peak 11, 14 and 15 in Figure 3a, to the sigmoidal nature of its response curve, resulting in much lower sensitivity. seven impurities. The HILIC-CAD method is much more sensitive than ELSD. With was calculated to be 107%, 92% and 93%, respectively. 20 μg apramycin sulfate on column, 7 impurities were detected by CAD at S/N > 3, while HILIC-CAD/ELSD method only 3 impurities were detected by ELSD. This method, with or without slight modification, was also used for impurity measurement of an additional eleven aminoglycoside Comparison of HILIC-CAD method with SCX-UV method Column: ACCHROM (Beijing, China), Click XIon, 4.6 x 150mm, 5µm antibiotics, including neomycin, gentamicin, kanamycin, streptomycin, tobramycin, Temperature: 30 ºC FIGURE 4. Comparison of Detection Sensitivity Between CAD and ELSD. amikacin, etimicin, netilmicin, sisomicin, ribostamycin and paromomycin. The HILIC-CAD method was compared to the SCX-UV method recommended by British Flow rate: 1 mL/min Pharmacopoeia (veterinary) 2013 version (BP2013) . As shown in Figure 3, the number Mobile Phase A: Acetonitrile of impurity peaks resolved and detected was greatly increased with the HILIC-CAD Mobile Phase B: 500 mM ammonium formate, pH 2.9 approach. About 16 impurity peaks were detected with the HILIC-CAD method at Mobile phase C: Water S/N > 3 (Figure 3A). The SCX-UV method only detected seven impurities, as not all of Introduction Gradient 0 min., 70 %A, 20%B, 10% C them could be derivatized by the SCX-UV approach (Figure 3B). Furthermore, the 30 min., 21% A, 20%B, 59% C improved chromatographic resolution and peak shape allows for a higher sample load Aminoglycosides are a group of structurally similar antibiotics used to treat infections Injection volume: 1 μL with the HILIC-CAD method enabling detection of low level impurities. caused by aerobic gram-negative bacteria1. Analytical methods are required for rapid Corona Veo RS: 55 °C evaporation temp., PFV 1.00, data rate 10 Hz, filter 5 s, assessment of drug purity and detection of minor degredants. As they lack a strong ELSD: Nebulizer temp. 50 °C , evaporation temp. 70 °C The effect of mobile buffer strength and pH on separation and peak shape was chromophore, these compounds are not amenable to UV detection. investigated. The method was optimized with 100mM ammonium formate at pH 2.9. The sample load was increased to 48.9 μg for the HILIC-CAD method and still maintains good Apramycin is an antibiotic used in veterinary medicine. Reported methods for apramycin SCX-UV method peak shape with half peak width W0.5 = 0.77min. While further increase of sample loading and impurities analysis usually involves pre- or post column derivatization followed by UV amount with the SCX-UV method caused significant peak broadening and results in 2,3 Column: Venusil SCX-F 4.6 x 150 mm, 5 µm detection . Such approaches are tedious and time consuming, and may not be able to Temperature: 30 ºC decreased resolution between apramycin and impurity peaks. detect all impurities. UV Detector: 568 nm Sample: 0.28 mg/mL Aminoglycosides can be measured directly by charged aerosol detection without Conclusions Injection volume: 20 μL FIGURE 3A. Impurity Analysis of Apramycin with the HILIC-CAD Method. derivatization. Corona Veo is a universal mass-sensitive detector, Its response is • The described HILIC-CAD method for apramycin enables more accurate impurity independent of chemical structure, and does not require the presence of chromophores. assessment, due to the universal detection of CAD and improved sample loading Chromatographic condition and post-column derivatization procedure are same as in 200 Capable of measuring any nonvolatile and many semi-volatile analytes, charged aerosol British Veterinary Pharmacopoeia 2013 pA capacity. More than 16 impurities were detected. The SCX-UV method detection enables accurate degradation studies and improved assessments of product recommended by British Pharmacopoeia only detected seven impurities. purity. The Corona Veo is much more sensitive than other universal detectors like ELSD HILIC-CAD method • and RI, offering low nanogram quantitation. This poster presents a sensitive HILIC-CAD Data Analysis 150 48.9 µg on column Comparison between Corona Veo and ELSD detection showed that CAD is much method for direct analysis of apramycin and other aminoglycosides. Method performance Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System software, 7.2 more sensitive than ELSD. With 20µg sample on column, 7 impurities were detected at S/N ≥ 3 with CAD, while only 3 peeks at S/N ≥ 3 were detected with ELSD. was compared to the British Pharmacopoeia HPLC-UV method and ELSD detection. 100 • Sample pretreatment with anion exchange SPE removes interference of sulfate ion FIGURE 2. Effect of SPE Pre-treatment of Apramycin Sulfate Sample. and allows for more accurate determination of impurities.

14 • This method can also be used for the analysis of many other aminoglycoside 50 15 11 antibiotics. 13 Methods 7 ™ ™ ™ 5 6 8 9 10 Thermo Scientific Dionex UltiMate 3000 RSLC system with: 1 2 3 4 12 16 Table 1. Comparison of Detection Sensitivity Between CAD and ELSD min • Pump: LPG-3400SD -10 • Auto Sampler: WPS-3000TSL 0.0 5.0 10.0 15.0 20.0 25.1 • Column Compartment :TCC Peak -3000RS Retention Time (min) S/N • Diode Array Detector: DAD-3000RS Number μ • Charged aerasol detector: Corona Veo RS FIGURE 3B. Impurity Analysis of Apramycin with the SCX-UV Method. 49 g on column 20 µg on column • CAD ELSD CAD ELSD References Varian ELSD 385-LC 1 10.30 4.9 6.4 - 1. Perzynski, S.; Cannon, M.; Cundliffe, E.; Chahwala, S.B.; Davies, J. Effects of 400 2 11.27 10.4 8.8 - 3 11.94 3.0 - - Apramycin, a novel aminoglycoside antibiotic on bacterial protein synthesis. Eur. SCX-UV method – FIGURE 1. 4 12.61 4.2 - 6.4 - J. Biochem. 1979, 99, 623 628. Structure of Apramycin 200 μ 5.8 g on column 5 13.32 24.4 19.4 - 6 14.10 19.6 19.5 3.9 - 2. British Pharmacopoeia (Veterinary): 2013. London: Her Majesty's Stationery 7 14.76 29.0 44.6 6.2 - Office, v.4, p. 38-40, 122-125, 2013. 0 8 15.78 15.0 14.0 3.2 - 3. Barbosa, E.A.; Lourenco, F. R.; Terezinha Pinto. Determination of apramycin in

mV 9 17.79 18.6 22.9 - 10 18.47 10.1 12.3 - - oral soluble powder by a HPLC method using pre-column derivatization with o- -200 11 18.85 51.7 137.9 11.8 3.7 phthalaldehyde and UV detection. Braz. J. Pharm. Sci. 2011, 47, 261-268. 12 19.34 4.8 - -

-400 13 21.55 12.7 19.3 - 14 21.87 79.5 293.9 21.3 5.6 © 2015 Thermo Fisher Scientific Inc. All trademarks are the property of Thermo Fisher Scientific and its 15 22.52 77.0 238.2 17.9 4.1 subsidiaries. 0 10 20 16 23.52 6.9 - - - This information is not intended to encourage use of these products in any manners that might infringe the time (min) intellectual property rights of others.

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