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Tech TipPeak Integration, Part 3:Common Integration Errors

In the first two parts of this series (HPLC Solutions #127 and #128), welooked at how HPLC data systems integrate chromatograms and someof the adjustments that can be made if you don’t like the way the defaultsettings perform. Even with proper adjustment of the settings, you maystill observe occasional or regular problems with integration. Here we’lllook at several common problems and how to correct them.

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Featured ApplicationsVeterinary Drug Analysis with Supercritical FluidChromatography and Triple Quadrupole MS Analysis

Using Longer Aeris PEPTIDE Core-Shell HPLC/UHPLCColumns for Improved Peptide Mapping

A High-Throughput SPE Method to Support theBiomonitoring of Phthalate Metabolites in Human UrineUsing ISOLUTE® ENV+ Columns Prior to LC-MS/MS

HPLC Solutions on ChromForumYou can now comment on or submit questions aboutHPLC Solutions articles on Chromatography Forum.A specific topic category has been set up...Click here to learn more

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Peak Integration, Part 3: Common Integration Errors

In the first two parts of this series (HPLC Solutions #127 and #128), we

looked at how HPLC data systems integrate chromatograms and someof the adjustments that can be made if you don’t like the way the defaultsettings perform. Even with proper adjustment of the settings, you may stillobserve occasional or regular problems with integration. Here we’ll look atseveral common problems and how to correct them. 

Figure 1 shows three examples of problems that are commonly encounteredin integrated chromatograms. In each case, the solid red line shows how thedata system selected the baseline for integration and the dashed lines are thecorrected baseline. Normally the baseline is determined by drawing a straightline connecting the baseline before the peak to that after the peak. In example(a) the baseline has a negative peak or dip before the peak of interest is eluted. The data system mistakenly identified this as the low point in the baseline aheadof the peak with an obvious error in determining the peak area. The problem wascorrected by moving the baseline to the position of the dashed red line.  The second chromatogram, (b), shows a dilemma that is often encountered. The default integration assigned a perpendicular drop from the valley betweenthe two peaks, so the peak areas for the first and second peaks are divided asshown. In this case, the proper integration is to skim the smaller peak off the tailof the larger one. You can see that this treatment may not make much differencein the area of the larger peak, but the original integration assigns more than twicethe appropriate area to the minor peak. This is especially critical in reporting

pharmaceutical impurities, where a batch of product may fail quality testing if

the impurity levels are reported to be higher than they actually are. In making adecision on this, I use what I call the “10% Rule,” which tells me that if the minorpeak is less than 10% of the peak height of the major one, it should be skimmed(dashed line), whereas if it is more than 10% of the major peak, a perpendiculardrop should be used (solid line). You can see that, even though the top of thelarger peak is not visible, the larger peak is at least ten times as tall as the minorone. Another example of the application of the 10% Rule was given in HPLCSolutions #115.  In the last example, the data system determined that the peak in chromatogram(c) returned to the baseline too early. This is a particularly common problem withsmall peaks on a noisy or drifting baseline. In this case, the correct baseline wasdrawn with the dashed line to the point where the peak actually returned to thetrue baseline.  In each of these cases, manual integration was required. I regularly getquestions in the training classes I teach about this subject. In some laboratories,workers are not allowed to manually integrate peaks because managementis afraid of negative feedback from regulatory authorities. This is being falselyconservative. There are clear guidelines for this published in the US Code ofFederal Regulations, CFR 21 Part 11. The essential rules for manual reintegrationare (a) the person doing the integration must be identified, (b) the date and timemust be noted, (c) a copy of the original (raw) data must be preserved, and (d)a reason for the change must be recorded. Most modern data systems include

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an “audit” function that can be turned on; this forces compliance with these

requirements.Yes, it may be possible to have every chromatogram integrated properly, butthis is only likely when all the peaks are large and the baseline noise is minimal.With small peaks on a noisy or drifting baseline, it is very likely that manualintegration will be required to get high quality results.

John Dolan is best known as one of the world’s foremost HPLC troubleshootingauthorities. He is also known for his ongoing research with Lloyd Snyder,resulting in more than 100 technical publications and three books.Contact John at TechTips@sepscience.com

Figure 1

Figure 1: Three examples of problems that are commonly encountered in integrated chromatograms.

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FEATURED APPLICATIONS

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VeterinaryDrug Analysis with

SupercriticalFluid Chromatographyand TripleQuadrupoleLC/MSThe Agilent1260 Infinity AnalyticalSFCSolution and

Agilent6490 Triple Quadrupole LC/MS

Application Note

Authors

Edgar Naegele,Joachim Thiemann,

and ThomasGlauner

Agilent Technologies, Inc.

Waldbronn,Germany

FoodTesting and Agriculture

Abstract

ThisApplication Notedescribes the combination ofthe Agilent 1260Infinity

Analytical SFC Solutionand an Agilent 6490TripleQuadrupoleLC/MS for

the measurement ofveterinarydrugs.The connectionofthe SFC tothe triple

quadrupoleMS was made through the Agilent Jet Stream Technology electrospra y

ionizationsource. The configurationofthe systemisdescribed,including a split

fromthe SFC tothe MSand a separatemake-up flowfor improvedionization,

togetherwiththe methodparameters. The datashowlimitsofquantitation

(LOQs)and limitsofdetection (LODs)in the lower ppbrange for all measured

veterinarydrugs.

Veterinary Drug Analysis with Supercritical Fluid Chromatography and Triple Quadrupole MS Analys isCompany: Agilent TechnologiesThis application note describes the combination of the Agilent 1260 Infinity Analytical SFC Solution and an Agilent6490 Triple Quadrupole LC/MS for the measurement of veterinary drugs. 

Using Longer Aeris PEPTIDE Core-Shell HPLC/UHPLC Columns for Improved Peptide MappingCompany: PhenomenexA new 3.6 µm 100 Å HPLC/UHPLC column (Aeris PEPTIDE) has been introduced that is specifically designed to improveseparations of peptide and peptide mapping applications. The Aeris PEPTIDE XB-C18 column was developed tocomplement Aeris WIDEPORE XB-C18 core-shell columns for protein characterization.

A High-Throughput SPE Method to Support the Biomonitoring of Phthalate Metabolites in Human Urine UsingISOLUTE® ENV+ Columns Prior to LC-MS/MS

Company: BiotagePhthalates are plasticizers used in industry to adjust the mechanical (and sometimes barrier) properties of plastics inconsumer products and packaging. This application note describes the extraction of nine phthalate metabolites fromhuman urine using ISOLUTE® ENV+ solid phase extraction columns.

Excellent LC-MS Separation of Penicillins and Cephalosporins Using Ultra IBD ColumnsCompany: Restek Antibiotics are the most widely used medications in the world. Whether by prescription, addition to animal feedstocks, or use of cleaning agents, everyone in the civilized world is either directly or indirectly exposed to antibioticsin daily life. This application note describes the LC-MS separation of penicillins and cephalosporins using Ultra IBDcolumns.

Separation of Statistic MMA-MAA Copolymers using Gradient SECCompany: PSS PolymerIsocratic GPC/SEC is a powerful tool to separate macromolecules based on their hydro-dynamic volume. In the caseof homopolymers GPC/SEC allows the fast, precise and easy determination of the complete molar mass distribution.Here SEC-gradients or polymer HPLC is the method of choice.

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PharmaceuticalApplications

ExcellentLC-MSSeparationofPenicillinsandCephalosporinsUsingUltraIBDColumns

  www.restek.comInnovativeChromatography Products

Introduction

Antibioticsarethemost widely used medicationsin theworld.Whetherby prescription, addition to animalfeed stocks, oruseof cleaningagents, everyonein thecivilized world iseitherdirectlyorindirectly exposed to antibioticsin daily life. Teoveruseof antibiotics, however, hasallowed resistant bacteria to thrive. Tedeath of12,500 peoplein Guatemala froman episodeofShigellafevercan betraced to a simplemutation ofthebacterialstrain.Research indicated that thebacteriumincorporated a singleplas-mid into itsRNA sequenceand resultantly becameresistant tofourdifferent antibiotics. Tisillustratesthedangerofresistancecaused by adaptation. o combat resistant bacteria, new antibi-oticderivativesmust becreated to overcomethebacteria’snewdefensemechanisms. ypically, HPLC columnscan beused toanalyzepenicillinsand theirstructurally related cephalosporins.However, thesimilarityofmany derivativesmay requireaddition-alinteractionsto effectively separaterelated compounds. Restek’ sUltra IBDcolumn isbetterableto resolvethesecompoundsusingpolarand hydrophobicinteractions.

Background

Penicillinsand cephalosporinsrepresent nearly sixty percent of antibioticsworldwide. Teseantibioticspossessa sulfuratomwithin a five-orsix-membered ring, attached to a fourmemberß-lactamring. Tey areproduced by fermentation processesusingeitherselected fungiorspeciesof Streptomyces bacteria. Deriva-tivesareproduced in two fashions:

1.Biosyntheticprocess—Tefungusorbacteriaaregeneti-callyengineeredtoproduceanewderivative,orthestartingmaterialsarealteredtoproducebiosyntheticvariantsduringfermentation.

2.Semi-syntheticprocesses—Tematerialsfromabiosyntheticprocessareconvertedtochemicalderivatives.Penicillinderiv-ativesarecreatedfrompenicillinGorV,whilecephalosporinderivativesarecreatedfromcephalosporinCorcephamycinC.

Figure 1: Ultra IBDcolumn separates penicillin Vfromfermentation impurities.

0 1

1

2 3 4 5 6 7 8

Time(min ) L C_ 0096

Co lu mn Ultra IBD(c a t.#91 7 5565)Dim ensions:1 50m m x 4.6 mmIDPa rtic leS iz e : 5 µ mP o r e Si z e : 1 0 0 ÅT e m p. : 3 0 ° CS amp le

Dil u e nt : a c e to n itrile:wa ter (1 0:90,v /v )Conc .: 1 . 2 m g / mLInj.Vol.: 2 . 5µ LMo b ile P h ase 10mM ammoniumform a te, pH2 .5:a c etonitrile(95:5,v /v )F l o w: 1 . 2 m L /m i nDe te cto r  UV/Vis@2 7 0nm

  P e ak  1 .Penic illinV

Click torequest copy

1

Biomonitoringof Phthalate Metabolitesin H uman U rine usingISOLU TE® EN V+ ColumnsPriorto LC-MS/MS | Page 1

Application NoteAN

©B iotage

This application note describes the extraction of nine phthalate metabolites from human urine using

ISOLUTE® ENV+ solid phase extraction columns.

Application N oteAN

Figure1.Struc turesofthetargetanalytes inthephthalatemetabolitespanel.

MMP

MBzP

MEHP

M E CP P M i NP

MEHHP

MHxP

M E P M B P

 A High-Throughput SPEMethodto Support theBiomonitoringof PhthalateMetabolites inHumanUrineUsingISOLUTE ® ENV+Columns Prior toLC-MS/MSRoy Gerona , Frank Kero   Matthew Friesen , Victor Vandell  , Michael Yu   

. UCSF De pa rtm e nt of  La bora tory Me dicine , Pa rna ssusAve ,Me dica l  Scie nce s Bldg S Sa n Fra ncisco,CA, USA  

. Bi ota g e , Ha rri s Oa k sBlvd., Suite C, Cha rlotte ,NC USA 

 

AnalytesMonomethyl phthalate (MMP); Monoethyl phthalate (MEP);Monobutyl phthalate (MBP); Monobenzyl phthalate (MBzP);

Monohexyl phthalate (MH xP); Mono (-ethylhexyl)phthalate

(MEH P); Mono(-ethyl--hydroxyhexyl)phthalate (MEH H P);

Mono (-ethyl--carboxypentyl) phthalate (MECPP);

Monoisononyl phthalate (MiN P)

IntroductionPhthalates are plasticizers used in industry to adjust the

mechanical (andsometimes barrier) properties of plastics

in consumer products and packaging. Their ubiquitous

presence in our everyday lives constantly presents threats

of low level exposure through inhalation or ingestion. Thus,large biomonitoring studies including the U S N ational H ealth

and N utrition ExaminationSurvey (N H AN ES) have screened

for phthalatessince . Because phthalates themselves

are difficultto eliminate from sampling and processing

materials, including laboratory ware and instruments,

analysis of phthalates in human samples have focused ontheir monoestermetabolites. Monoethylphthalate (MEP),

monobutyl phthalate (MBP), monobenzyl phthalate (MBzP)

and mono (-ethyl--hydroxyhexyl) phthalate (MEH H P) havebeen constantly detected in human urine since the first

N H AN ES survey of phthalates in . Phthalates exposure

has beenassociated with decreased anogenital distance,

lower spermcount, cryptorchidism and hypospadias amongother clinical endpoints in humans.

To facilitatethe high throughput population screening

of phthalate metabolites, sample preparation methodsneed to be simple, sensitive and robust to mitigate matrix

suppression and instrument down time commonto many

dilute and shoot approaches to mass spectrometry. Thus, a

solid phase extraction procedure was developed for these

analytes. This application note details the optimization

strategy for nine phthalate metabolites. Proof-of-concept for 

this samplepreparation method wasdetermined on a setof real patient samples (n=). The results were in general

agreement with previously reported concentration ranges for 

these compounds. It is anticipated that this method will have

significant impact in environmental biomonitoring strategies

for these analytes.

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 APPLICATIONSTN-1124

Foradditionaltechnicalnotes ,v is i  twww.phenomenex .com Page1of2

UsingLonger Aeris™  PEPTIDE Core-ShellHPLC/UHPLCColumnsfor ImprovedPeptideMappingMichaelMcGinley ,DeborahJarrett,andJeffLaynePhenomenex,Inc.,411MadridAvenue,Torrance,CA90501USA 

backpressure amenable to usi ng standard HPLC systems (  200bar at 1.2 mL/min). These results demonstrate the performanceadvantage and utility of the Aeris PEPTIDE 3.6µm XB-C18 mediafor highly complex peptide mapping mixtures where one can uti-lize different column lengths to optimize resolution and separa-tion time based on the needs of a specific application.

ConclusionMaximizing resolution between proteins and their modifiedimpurities is critical i n obtaining useful quanti tation of post-translational modifications of biogeneric proteins.  The differentapplications in this technical note show the utility of AerisWIDEPORE columns for obtaining accurate data for intact protei napplications. The optimized geometry of the core-shell   AerisWIDEPORE columns, as well as good selectivities of the threeseparate phases offered (XB-C18, XB-C8 and C4), deliver betterresolution and recovery than existing fully porous 300 Å columnsforintact protein analysis. Finally, thelarge particle sizeof the AerisWIDEPORE column delivers a significantly lower backpressurethan sub-2 µm 300 Å col umns which allows for more flexibility i ninstrument used (HPLC or UHPLC) as well  as column length indeveloping biogeneric protein applications.

 A new 3.6 µm 100 Å HPLC/UHPLC col umn (Aeris PEPTIDE) has been introduced that is specificallydesi  gned to improve separationsof pepti de and peptide mapping appli  cations. The Aeris PEPTIDE XB-C18 column was developed to complement Aeris WIDEPORE XB-C18 core-shell  columns for protein characteri  zati on. When one l ooks at peptide mappi  ng applications, performance requirements are signi ficantl  y different versus i  ntact protein separati ons, as i  ncreased retenti on and sel ectivity are required to separate the large number of peptides generated i  n pepti de mapping applications.Because increased resolution is a hi  gher pri ority versus speed, a l  arger particle (3.6 µm) core-shell   parti cle was developed all owingthe use of l onger columns at lower backpressures. I n this appl  icationthe increased resolution that longer Aeris PEPTIDE 3.6 µm XB-C18 provide will be demonstrated.

Materialsand Methods

 All chemicals, standards andantibodies were obtained from SigmaChemical ( St.Louis,Missouri). Solvents were purchased from EMD(San Diego, California). Core-shell Aeris PEPTIDE 3.6µm XB-C18columns (150 × 4.6 mm and 250 × 4.6 mm)  were obtained fromPhenomenex (Torrance, California). Bovine serum albumin wasdigestedwithtrypsin andanalyzedon an Agilent 1200 HPLC systemwith autosampler, column oven, solvent degasser, andUV detectorset at 214 nm. Data was collected using ChemStation software(Agilent, Santa Clara, California). Mobile phases used were 0.1 %Formic acid in water (A) and 0.1 % Formic acid in acetonitrile (B)with a gradient from 3 to 65 % B at a flow rate of 1.2 mL/min.Gradient times were adjusted based on column l ength (33 to 55minutes respectively). Column was maintained at 40 °C.

Resultsand Discussion Aeris PEPTIDE 3.6µm XB-C18 core-shell particles demonstratesimilaror better performance than sub-2 µm fully-porous columnsat a fraction of the backpressure, allowing the use of longer col-umns at backpressures compatible with existing HPLC systems.The 3.6 µm core-shell mediais of particular utili ty for peptide mapapplications where the increased resolution of longer col umns isdesired (for high-speed UHPLC applications the Aeris PEPTIDE1.7µm XB-C18 can be used instead). An example of the uti lity isdemonstrated in Figure 1 where 150 × 4.6 mm and 250 × 4.6 mm Aeris PEPTIDE 3.6µm XB-C18 columns were compared for apeptide map of BSA. The 150 × 4.6 mm column provides excel-lent separation of the peptide mixture at a low column backpres-sure (140 bar at 1.2 mL/min)  such that a longer column coul d beused to achieve additional resolution if required. When the 250× 4.6 mm Aeris PEPTIDE 3.6 µm XB-C18 col umn was used forthe separation, additional  peptides were resolved while stil l at a

Figure 1.

BSA Tryptic map separated on di fferent length Aeris PEPTIDE 3. 6µmXB-C18 columns (150 × 4.6 mmtop,250 × 4.6mm bottom). Notethegood separation on the shorter Aeris PEPTIDE column and the increasedresolutionprov ided by the longer Aeri sPEPTIDE (250 ×4.6mm) column.Because backpressure for the Aeri s 3.6µm column is so low, onecanoptimiz e column lengths based on their separationtime and resolutionrequirements .

7 8 9 1 0 1 1 1 2 m in

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19880

   A  p  p   I   D 

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20

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Click torequest copy

Separation ofstatisticMMA-MAACopolymersusingGradientSEC

 Application NotePharmaceutical Analysis

 Author Dr.WolfgangRadke

contact:WRadke@pss-polymer .com

Isocratic GPC/SEC is a powerful tool to separate macromolecules based on their hydro-dynamic volume. In case of homopoly mers GPC/SEC allows the fast, precise and easydetermination of the complete molar mass distribution.Unfortunatel y many modern polymeric materials are copolymers and isocratic GPC/SEC doesnot provide any information about the chemical composition. Here SEC-gradients or polymer HPLC is the method of choice.

IntroductionStatistic copolymers of methylmethacrylate(MMA) andmethacrylic acid(MAA) arewidely usedin pharmaceutical applications. Besides the molar mass distribution the chemical compositionand the amount of comonomers in the copolymer is of importance. Separ ations by convention algradient HPLC failed, since the polar eluents required to dissolve polymers of high acid contentprevent adsorptionontothestationary phase, resulting in pronounced breakthrough peaks. Theproblem can be avoided applying SEC-gradients, resulting in the desired separation accordingto the amount of methacrylic acid. The system can be calibrated using reference materials of known composition. This allows determining the average copolymer composition as well as thecompositional heterogeneity.

SystemRequirementsConditions

Pump PSSSECcur ityGPC1260binarypump•fl owrate[mL/min]: 1.0•mobilephase:

Gradient:Chloroform/DMAc

In jectionsystemPSSSECcur ityGPC1260Autosampler  In jection in terva l:32min

C o l u m ns • P S S P R O T EE M A p r e co l u m n ( 8* 5 0 m m )•PSSPROTEEMA,3µm,100 Å(8x300mm)Temperature: 60/C

Calibration PSSMMA-MAAcopolymersofd ifferentacidcontent(MAA:9%,25%,31%,42% ,48%wt)

Loading SamplesdissolvedinDMAc•1mg/mL, 100µLinjectionvolume

D e t e c to r • P S S S E C cu r i t y EL S 1 0 0 0Gasflow:1.5SL/mi n

Nebulizer temperature:100/CEvaporator temperature:200/C.

Software PSSWinGPCUniChromwithChromPilo tandChemica lHeterogeneity module

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