Validation of a quantitative HPLC method for bacitracin and bacitracin zinc using EDTA as a mobile

Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 619–623Contents lists available at SciVerse ScienceDirectJournal of Pharmaceutical and BiomedicalAnalysisj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j p baShort communicationValidation of a quantitative HPLC method for bacitracin and bacitracin zinc using EDTA as a mobile-phase modifierAlan R.Potts ∗,Tatiana Psurek,Cassandra Jones,Luba Parris,Ahalya WiseUS Pharmacopeial Convention,Rockville,MD 20852-1790,USAa r t i c l ei n f oArticle history:Received 24April 2012Received in revised form 7June 2012Accepted 9June 2012Available online 28 June 2012Keywords:BacitracinBacitracin zinc Metal chelation Method validation EDTA HPLCa b s t r a c tAny product marketed in the United States and labeled bacitracin must comply with the tests,proce-dures,and acceptance criteria in the relevant monograph published in the US Pharmacopeia (USP ).The test procedure relies on accurate recovery of Bacitracin A and many other bacitracin components.The authors determined that the current USP procedure does not recover Bacitracin A at low concentration levels.They postulate that this low recovery is the result of bacitracin’s known ability to chelate metal ions,e.g.in bacitracin–zinc complexes.Thus the ubiquitous metal ions in HPLC systems may be respon-sible for sequestering bacitracin and giving low recoveries.Addition of edetate disodium (EDTA)to the mobile phase improved the recovery.The method validation results include precision,accuracy,linearity,specificity,and robustness.© 2012 Elsevier B.V. All rights reserved.1.IntroductionBacitracin is a Gram-positive antibacterial agent expressed by Bacillus subtilis and Bacillus licheniformis .Bacitracin is part of a class of antibiotics called polymyxins that are characterized by cyclic peptidic structures with hydrophobic side chains (Fig.1a).There are many bacitracins,but forms A,B1,B2,and B3are of major therapeutic importance.Bacitracin is bactericidal for a vari-ety of Gram-positive organisms.It interferes with bacterial cell wall synthesis by inhibition of the regeneration of phospholipid receptors involved in peptidoglycan synthesis.This interaction is stimulated by divalent metal cations.Two active sites on baci-tracin have been postulated for involvement with the chelation of metal ions (Fig.1b).Analysts believe that the zinc salt of bacitracin,for instance,coordinates zinc and Bacitracin A via the imidazole and the peptide nitrogen on the histidine residue.Another site for divalent metal cation binding is thought to be between the thiazo-line ring and the free amine at the N-terminal end of the peptide [1].Researchers have provided evidence of this site’s importance by examining the effect of several divalent metal cations,includ-ing Mg 2+,Ca 2+,Cu 2+,Zn 2+,Fe 2+,as well as others.The metal cations inhibit the enzymatic dephosphorylation of C 55-isoprenyl pyrophosphate [2].These researchers proposed that a divalent metal cation forms a bridge between bacitracin and C 55-isoprenyl∗Corresponding author.E-mail address:arp@ (A.R.Potts).pyrophosphate,preventing dephosphorylation.Furthermore,addi-tion of a small amount of edetate disodium (EDTA)mitigated this effect.HPLC methods have been developed for the characterization and quantification of different forms of bacitracin,and there is a long history of separation techniques for the various forms [3–18].Early isocratic methods have been replaced by more sophisticated gra-dient methods over the years.Although high-performance liquid chromatography (HPLC)gradient elution and more recently micel-lar electrokinetic chromatography methods prove to be superior in the separation of about 50bacitracin forms,isocratic HPLC is still preferred for its simplicity and robustness for drug quality testing.The forms of major therapeutic use are A and B,while Bacitracin F is a degradation product that shows nephrotoxicitiy [19].The US Pharmacopeia (USP )monographs for bacitracin [20]and baci-tracin zinc [21]include a Composition Test that employs an isocratic HPLC method and requires not less than 40%of Bacitracin A,not less than 70%of the sum of Bacitracins A,B1,B2,and B3,not more than 20%of the early-eluting peptides,and not more than 6.0%of the degradation product Bacitracin F.The composition limits for these bacitracin forms are consistent with commercially marketed products and maintain a standard of safety,purity,and quality.The current procedure was first published in the second supplement to USP 31and has served as the drug quality standard for bacitracin and bacitracin zinc composition since 2008[20,21].USP has recently received several complaints that the test is problematic for recovering Bacitracin A from the Reporting Thresh-old Solution ,which is a 0.5%concentration solution of the Test0731-7085/$–see front matter © 2012 Elsevier B.V. All rights reserved./10.1016/j.jpba.2012.06.016620 A.R.Potts et al./Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 619–623Fig.1.(a)Chemical structures for Bacitracins A,B1,B2,and B3.(b)Peptide sequence of Bacitracins A,B1,B2,B3,C1,C2,C3,and F (from reference [18]).Solution .The purpose of the Reporting Threshold Solution is to estab-lish a peak area limit for the calculation of total peak area.Method development studies reported here have determined that addition of EDTA to the mobile phase resolved the problematic recovery of the Reporting Threshold Solution .To our knowledge,there have been no other published reports of the need for or the importance of sequestering low levels of divalent metal cations during HPLC anal-ysis of bacitracins.EDTA is used in the preparation of the diluent for bacitracin zinc USP in order to sequester Zn 2+from the complex,but the concentration of EDTA is too dilute after injection to have much effect in the mobile phase.Divalent metal cations such as Fe 2+and Cu 2+are commonly known to be present in HPLC systems at low concentrations,and various passivation methods are readily available (Waters Corporation,oral communication,October 2011).These metal cations could interact with bacitracin and may explain loss of bacitracin at low concentrations.In this article,the authors report the use of EDTA as a mobile phase additive to accurately recover low concentrations of Bacitracins A,B1,B2,and B3.The column and system metal content,therefore,can be considered a critical quality attribute,and thus for its test procedures USP employs a control strategy that minimizes the risk that different results will be obtained on different brands or models of columns and systems.2.Materials and methods2.1.Reagents and materialsThe following reagents were used as received from the supplier:Potassium phosphate dibasic (VWR International,Radnor,PA,USA),potassium phosphate monobasic (Fisher Scientific,Pittsburgh,PA,USA),sodium hydroxide (50%,w/w,Fisher Scientific,Pittsburgh,PA,USA),EDTA (ACS grade,99.2%purity;Sigma–Aldrich,St.Louis,MO,USA),methanol (Honeywell Burdick &Jackson,Morristown,NJ,USA),acetonitrile (Honeywell Burdick &Jackson,Morristown,NJ,USA).Milli-Q water (resistivity:18.2M cm;Millipore,Billerica,MA,USA)was generated locally.A Waters HPLC Separation Module 2695with Waters PDA detector 996was used for method development and validation experiments.Chromatograms were collected and analyzed with Empower 2software.The HPLC column used is the L1column spec-ified in the monograph supporting information:Thermo Scientific,Hypersil ODS,250mm ×4.6mm,5␮m (Thermo Fisher Scientific,Inc.,Waltham,MA,USA).USP Bacitracin Zinc Reference Standard (RS)and USP Bacitracin RS (USP,Rockville,MD,USA)were used for all development and validation studies.2.2.Mobile phase preparationThe USP monograph for bacitracin specifies a mobile phase mixture of methanol,water,buffer,and acetonitrile (26:15:5:2).General chapter <621>Chromatography permits adjustment of the organic and aqueous parts of the mobile phase as a flexible option to account for differences in HPLC hardware.Thus,the mobile phase ratio can be stated as (acetonitrile:methanol)and (buffer:water)at 58:42.Experiences in the authors’laboratory suggest that it is beneficial to change the mobile phase ratio from the mono-graph conditions to (acetonitrile:methanol)and (buffer:water)at 63:37because the adjusted ratio results in notable shortening of run time without degradation of specificity or peak integration.The monograph specifies recording the chromatograph for about three times the retention time of Bacitracin A,which is observed at about 39min in our experience and thus would result in a 120-min run time.At the modified conditions [(acetonitrile:methanol)and (buffer:water)at 63:37]the Bacitracin A retention time was about 18min and the total run time was about 50min.Fig.2presents a typical chromatogram of a Test Solution (or System Suitability Solu-tion at 2mg/mL)obtained at these modified conditions.The main bacitracin forms (A,B1,B2,B3,C1,C2,and C3)and the degradation impurity,Bacitracin F,are labeled.The organic phase consists of a ratio of 26:2methanol:acetonitrile.A slight improvement of the peak resolution was obtained by adjusting this ratio to 27:2,and thus all method vali-dation work was done at the 27:2ratio.2.3.Mobile phase preparationThe mobile phase was prepared in two parts,aqueous and organic.The mobile phase was combined in situ in the HPLC pumpand Biomedical Analysis 70 (2012) 619–623621Fig.2.Typical chromatogram of bacitracin zinc Test Solution .capable of mixing up to 4different solutions.The aqueous portion of the mobile phase was prepared by dissolving 34.8g of potassium phosphate dibasic in 1L of deionized water,followed by dissolving 27.2g of potassium phosphate monobasic in 1L of deionized water and combining these two solutions in a ratio of 1:4.5.The result-ing solution was diluted with deionized water in a ratio of 1:3.To this solution,EDTA was added to give a concentration of 0.1mM.The organic portion of the mobile phase was prepared by combin-ing methanol and acetonitrile in a ratio of 27:2.The ratio between aqueous and organic portions is set by the HPLC mixing valve to be 63:37.Adjustments made to the mixing ratio between the aqueous and organic portions of the mobile phase were necessary during the validation testing of method robustness.2.4.Preparation of the reporting threshold solutionThe calculation in the Composition Test requires the determi-nation of total detected area.The Reporting Threshold Solution concentration is 0.01mg/mL and is prepared by dilution of the Sys-tem Suitability Solution ,2mg/mL,with deionized water.The peak area of the Bacitracin A peak is used to set a peak area threshold for calculation of total peak area of the Test Solution chromatogram.2.5.System suitability solution preparationThe System Suitability Solution preparations in the bacitracin USP and bacitracin zinc USP monographs differ slightly.For the bac-itracin monograph,bacitracin RS is dissolved in deionized water and diluted hydrochloric acid using about 2%of the final volume to give a final concentration of 2.0mg/mL.For the bacitracin zinc monograph,bacitracin zinc RS is dissolved in diluent to give a final concentration of 2.0mg/mL.2.6.Diluent preparationEDTA is required in the preparation of diluent for bacitracin zinc USP.The normality of sodium hydroxide used to adjust pH “with dilute sodium hydroxide”is not specified.The authors found that in order to reduce the volume of sodium hydroxide solutionrequiredFig.3.Chromatogram at a dilution of 2%Test Solution concentration.to adjust the pH of the diluent in the bacitracin zinc monograph,an 8N sodium hydroxide solution was needed.Therefore,the diluent was prepared by dissolving 40g of EDTA in 1L of water,and the pH was adjusted to 7.0with 8N sodium hydroxide.3.Results and discussion3.1.Detection of the reporting threshold solutionIn the initial experiments,the Reporting Threshold Solution chro-matogram was indistinguishable from the control blank injection.To investigate,analysts made serial dilutions of the Test Solution to determine the detection threshold.Interestingly,at levels where Bacitracin A was no longer visible,Bacitracins B1,B2,B3,C1,C2,and C3peaks were still detectable.A chromatogram of a dilution representing 2%of the Test Solution concentration is presented in Fig.3,and the peak areas and percent recoveries from the serial dilutions are shown in Table 1.The response of each of the bacitracin peaks was found to be lin-ear but had a notable negative y -intercept as a result of a consistent loss of analyte upon each injection.Also,the bacitracin components were unequally affected.The authors found a loss of approximately 2%of Bacitracin A,which prevented them from evaluating the peak area of the Threshold Solution.This result is concerning because relative percentages of Bacitracins A,B1,B2,and B3were not accu-rately determined.The differences in recovery may be caused by differences in the complexation and stability of each bacitracin with metal cations in the HPLC system.This may be typical of HPLC sys-tems that are not routinely passivated or otherwise controlled for sources of divalent metal cations.3.2.Mobile phase modificationTo investigate if the low recovery of bacitracins was originat-ing from chelation with metal ions,analysts introduced 0.1mM EDTA into the aqueous portion of the mobile phase.EDTA com-monly is used as a strong metal-chelating agent,and it is used here to complex any divalent metal cations present in the HPLC system.622 A.R.Potts et al./Journal of Pharmaceutical and Biomedical Analysis70 (2012) 619–623Table1Peak areas and recoveries of a Test Solution of bacitracin zinc.Bacitracin component A B1B2B3Sum of Bacitracin C1,C2,C3Peak area100%Test Solution concentration(2mg/mL)8,889,6671,824,8261,743,3601,320,0091,176,711Peak area2%Test Solution concentration(0.04mg/mL)Not observed28,53928,52918,22214,892%recovery0%78%82%69%63%Fig.4.Linear regressions of serial dilutions of a Test Solution of bacitracin zinc with and without EDTA as a mobile phase modifier.The Bacitracin A peak response was measured at0.5%,1%,5%, 10%,50%,75%,and100%Test Solution concentration levels.An R2 of0.9999and a y-intercept of−0.25%were observed.This showed that addition of EDTA to the mobile phase improved the y-intercept to an acceptable level.The linear regression with and without EDTA as a mobile phase modifier is presented in Fig.4.Thisfinding suggests that the loss in recovery was caused by bacitracin’s interacting with divalent metal cations in the HPLC sys-tem.Fig.5shows the chromatogram of Reporting ThresholdSolutionFig.5.Reporting Threshold Solution for bacitracin zinc with EDTA as a mobile phase modifier.(0.5%Test Solution concentration)tested under the new conditions with the addition of EDTA to mobile phase.Bacitracins A,B1,B2, and B3peaks were detected with confidence and were recovered at approximately100%.The modified mobile phase was success-ful in detecting bacitracins at all concentrations required in the Composition Test.The sequestration does not appear to be completely reversible, and running the system post EDTA in the mobile phase results in better detection limits of Bacitracin A than does a system that has not been passivated with a chelating agent.3.3.Method evaluationThe performance of the modified Composition Test was assessed according to USP34general chapter Validation of Compendial Procedures<1225>for both the bacitracin and bacitracin zinc mono-graphs.The assessment included tests for precision,accuracy, linearity,specificity,limit of quantitation,limit of detection,and mobile phase robustness.4.ConclusionThe current USP Composition Test for bacitracin and bacitracin zinc may result in low recoveries of Bacitracins A,B1,B2,and B3 caused by the ability of bacitracins to form complexes with ubiqui-tous metal ions in the HPLC system.Addition of a small amount of EDTA to the mobile phase was shown to avoid this phenomenon.The authors are unaware of the potential impact this conclusion has on the assessment of the quality of drugs in the P is in the process of updating these monographs.The authors suggest that addition of EDTA as a mobile phase modifier is simple and inexpensive and permits assay performance with the proper level of quality.Other possibilities for addressing this issue do exist,e.g. passivating the plumbing in the HPLC system,but this would be both more time consuming and more difficult to validate. 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