The Process Pharma Special

2

Preparative LC and LCMS »3

Together we are strong »7

Lead Generation

Method transfer and vali-dation according to Qualityby Design requirements –DryLab®2010 and Nexera –

a powerful combination »20

Method Development

Speed beyond comparison –High speed drug screening

and quantification »23

Residual Solvents – U.S.

Pharmacopoeia – revised

General Chapter Monograph

<467> »25

Drug Testing

Extrusion testing made easy – Testing machine for

blister packaging »27

Big potential with proteinbased drugs –Quality control of recombinant

proteins by N-terminal Edman

sequencing »28

Analysis of residual catalystsin pharmaceuticals –ICP-OES technology quantifies

14 elements according to

EMEA guidelines »30

How cleaning validation prevents contamination »32

Determination of sodium,potassium and calcium –

Analysis of Na, K and Ca

with flame atomic absorption

spectrometry in microsam-

pling mode »34

Quality control of specialpharmaceutical organics –Identification with UV spec-

troscopy »35

Quality control of ultra pure water in pharmaceuticalmanufacturing – Two TOC

instruments offering different

oxidation methods »38

How heavy metals canmigrate into pharmaceu-ticals – EDX series detects

elements from C to U –

carbon and uranium »40

Chemistry/Manufacturing/Control

From Crude to Powder –Solid-Phase Trapping as a

new approach to shorten and

improve purification process

»10

Drug Discovery

Bringing light into darkness – 2-D LC and

LCMS-IT-TOF – Better drug

impurity ID efficiency »12

Drug Development

Future-proof analysis –UHPLC 3.0 – Nexera per-

fectly serves all needs of the

Pharmaceutical Industry »15

From powder to pill – qualitycontrol of pharmaceuticalgoods – Active agent analysis

using FTIR spectroscopy »18

Drug Optimization

Lead GenerationPharma Special

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Preparative LC and LCMS

Preparative LC as well as thecombination of preparativeLC with MS detection is

used in several steps in drug de-velopment.

A preparative system can be usedto recover high purity compo-nents from some types of liquidextract obtained either from a syn-thesis reaction or from a naturalsubstance by separating and puri-fying the target compounds.Obtaining these target compoundsat high purity enables their struc-tural analysis, permitting evalu-ation and analysis of their variousfunctions while allowing theirsubsequent processing to be con-ducted more reliably. Since apreparative system must separatethe target components from thecoexisting substances in a sample,the basic system configurationcomprises of solvent deliverypumps, sample injector, columnand detector, as in a typical LCsystem. Additional is a mechanismfor collecting fractions consistingof the target substances, therebycompleting the basic configurationof a preparative system.

Efficient collection of targetcompounds is key

The objectives of a typical LCsystem are to conduct quantitativeand qualitative analysis, but apreparative system is used morefor so-called “pretreatment” withthe objective of actually obtainingthe necessary compounds for evaluation and analysis, as well asfor subsequent processing. It istherefore important that targetcompounds are obtained quicklyand at high purity. Just as with atypical pretreatment process, pro-ductivity is the key point in apreparative system. To ensure thatthe intended evaluation and analy-sis operations can be started assoon as possible following thepreparative process to achieve thehighest possible productivity, anappropriate system should be able

lem, most of the column eluate isdirected to the fraction collector,and the remaining micro volumeis introduced into the ELSD orMS. To accomplish this, an activeAPV splitter (Automated Propor-tioning Valve) is used to split theflow line. In addition, if the vol-ume of eluate to be introducedinto the ELSD or MS detector isultra small, a make-up pump isused to augment the mobile phaseflow and maintain steady detec-tion by the ELSD or MS.

Determination of the optimum preparative system

Once the total amount of fractionnecessary for evaluation and anal-ysis of the target compounds andsubsequent processing is calcu-lated and the detector has beenselected, the next step is to deter-mine what scale of fractionation isrequired. The amount of samplethat can be injected at one time(load with respect to column) tomaximize productivity must alsobe determined. Once the maxi-mum sample injection volume isknown, the number of requiredfractionation runs can be calculat-ed based on the target fractionquantity. This allows determina-tion of the optimum preparativesystem based on time (total timeto collect the required fraction)and cost (purchase price ofpreparative system and column,and solvent expense). This type ofexamination can be conductedefficiently by first using a generalpurpose LC (conventional LC).

If the concentration of the frac-tionation target compound in thesample is known, the absoluteinjection quantity of the fractiona-tion target compound can be ob-tained from this concentration andthe sample injection volume. �

bance, detectors based on princi-ples other than optical absorbancedetection such as ELSD (evapora-tive light scattering detector) andMS (mass spectrometer) can beused as supplementary detectorsto improve purification efficiency,thereby improving productivity.

Since ELSD and MS detectors relyin principle on nebulization of thecolumn eluate inside the detector,the target substances emergingfrom these detectors cannot berecovered. To resolve this prob-

to separate the quantity of com-pounds and integrate a detectionprinciple that can most effectivelyensure collection of the all possi-ble target compounds.

What is the optimal detector?

Most simple solutions are basedon UV detectors (ideally multi-wavelength or photo diode array).If the target compound or thecoexisting impurities in the samplesolvent have no optical absor-

Figure 1: Preparative LC systems with automatic sample injection

and fraction collection

Figure 3: Configuration schema of prep LCMS system

Lead Generation Pharma Special

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To efficiently use the preparativeLCMS for fractionation – MassDirected Purification signals fromUV detector as well as m/z valuesobtained with the LCMS areapplied. The following example(see box on page 4) shows theadvantage of combining both sig-nals.

Transfer analytical condition and separation to preparative scale

After examining conventional-sizecolumn conditions, the next stepis to scale up from analysis topreparative level. To smoothenthis transition, a preparative col-umn is selected using the samepacking material as in the analyti-cal column. With the same pack-ing material, the mobile phase

flow rate and sample injectionvolume need only be increasedaccording to the analytical columnand preparative column cross-sec-tional area ratio to obtain nearlythe same chromatographic pattern (see table 1).

An example in which the scale-upwas conducted using the samepacking material is shown in figure 6. Here, the 4.6 mm I.D.column used for the analyticalscale was exchanged with 20 mmI.D. column for the preparativescale. Since the cross-sectionalarea of the 20 mm I.D. column isabout 19 times that of the 4.6 mmI.D. column, the solvent deliveryflow rate was scaled up from 0.8 mL/min to 15 mL/min, whilethe sample injection volume wasscaled up from 50 μL to 1 mL.

<< Analytical Scale>>Column I.D. : 4.6 mmSolvent delivery flow rate : 0.8 mL/minSample injection volume : 50 μL

<<Preparative Scale>> Column I.D. : 20 mmSolvent delivery flow rate : 15 mL/minSample Injection volume : 1 μL

Figure 6: Example for scale-up

Figure 7: Website Shimadzu ( Internet Explorer)

Figure 4: Fraction tiggered by MS only

When an UV trace is used to trigger fractions and both slope

and threshold are triggered, the inflection in the UV trace is

detected where the by-product starts to elute and the fraction

tube being collected to is automatically changed.

The same amount of fraction is still collected but this time

into two tubes. The first tube contains the pure target pro-

duct. Therefore purifi-cation has been achieved.

Figure 5: Fraction tiggered by combination UV and MS signal

With MS, only a specific product can be targeted. In order to

trigger a fraction on the target product the trace for target

product ions m/z is extracted and the collection of fractions

using this single mass’s trace is triggered. Co-eluting substan-

ces may also be present. Therefore, with a partial co-elution

the target product continues to collect although the by-product

has started to elute. This leads to contamination of the target

product's fraction with the by-product. Purification has

therefore not been achieved.

It is recommended that the triggering option for the UV trace

be set to slope due to the excellent peak shape achieved by

the UV detector; the MS should trigger on an intensity thres-

hold since the MS only considers the target ion and is very

sensitive. The AND logic is used to combine the two triggers

i.e., both the MS and UV trace must trigger together for the

fraction to be collected. This enables narrowing the collection

down to the product of interest, reducing fraction width and

therefore helping to remove the problem of contamination

from other products eluting close to the product of interest.

Combining the power of slope and threshold along with the MS

and UV trace both being considered, a target compound can

be isolated even in a complex mixture with many products elu-

ting close to each other.

Lead GenerationPharma Special

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Open Solution is a newly devel-oped software application whichachieves this open access environ-ment.

Open Solution is a software en-abling control of the Shimadzuhigh performance liquid chro-matograph prominence and theultra high performance liquidchromatograph (Nexera andprominence XR), in addition tothe LCMS-2020 mass spectrome-ter – not through direct operationof their control software, but viaopen access.

Open Solution utilizes the Inter-net Explorer, thereby eliminatingboth the need to install specialsoftware on every PC terminaland the requirement for additionalsoftware licenses. By simply in-stalling Open Solution on the PCdirectly connected to the analyti-cal instrument, operations such asviewing analysis results and out-putting reports can be conductedfrom any PC on the network.

Open Solution pushes the limit

Elimination of every unnecessarymouse click is a wish shared by alllab personnel. Analysts believe,

correctly, that their job is to con-duct analysis and not operateinstruments. There is justifiablefrustration with the complicatedPC operations which actuallyimpede the efficiency with whichanalysis might otherwise be con-ducted.

Open Solution pushes the limit toachieve a simplified level of PCoperation. From login to analysisstart, the process is reduced to alevel equivalent to operating asimple lab device such as a pHmeter or electronic balance. ThePC operations are reduced toOne-Two-Three: (Step 1) Log in /(Step 2) Specify number of vials,and set vials in place / (Step 3)Start.

The rest can be left to the instru-ment. The analysis may even becompleted by the time the analystreturns to the office. �

Explorer environment, since thesoftware operations would remainbasically unchanged. In general,the following criteria would needto be satisfied for Internet Explor-er to be effectively employed forcontrolling analysis: the ability to

(1) construct an open access envi-ronment

(2) display sufficient informationin the Internet Explorer win-dow.

(3) output information in printedas well as electronic format(PDF output) Note 1.

Once these problems are resolved,analysis using Internet Explorerwill become a reality. Further-more, since Open Solution runsonly on the PC that is directlyconnected to the analytical instru-ment, it can also run in standalone mode, so any future lab network construction could beaccomplished without wastingpast investments.

A true open-access environment

The ability to construct an openaccess environment within theInternet Explorer environment, asindicated above, is critical. “Openaccess” refers to access by whoev-er, whenever, wherever, with res-pect to any data, and for whateverreason, while satisfying a given setof security conditions. The possi-bilities become greatly extended ifthis open access environment isachieved using an Ethernet net-work, as this overcomes positionaland geographical restrictions toallow information sharing fromany PC on the network. This de-fines a true open access environ-ment.

As a result, nearly equivalentchromatographic patterns wereobtained.

At this point, further handling ofthe fractionated compounds usual-ly determines the next steps. If the target purity is not achievedbecause of insufficient separation,the sample injection volume andcolumn length etc. must be reex-amined. The same applies withgradient conditions.

If high sensitivity is requiredwhile using a small injection vol-ume in this purity confirmationanalysis, the usage of an analyticalsystem for verification rather thana preparative system is suitable.

These reprocessing steps are close-ly related to easy-to-use softwareto provide a fast overview of theresults.

Open Solution software

There is a growing demand formultifunctional software that canbe used to set conditions and per-form high-level analysis in an easyand intuitive way. But, whatwould be the ideal software whichcould easily be used by anyone?From the view of day-to-dayoperations, access to the internet ismeanwhile considered as essentialto communicate by e-mail and toaccess readily available web-basedinformation.

If this effortless Internet Explorercould be used for LC and LC/MSanalysis, a reduction or even elimi-nation of the cost of training toimpart the expertise for softwareoperation could be avoided. Fur-thermore, there would be no needfor software licensing for each PCor user, thereby reducing even fur-ther the overall costs associatedwith analysis related operations.

Might it be possible to useInternet Explorer for LC andLC/MS analysis?

At this time, there is probably asmall advantage to be gained byrunning the currently used LCand LC/MS software in their cur-rent states under the Internet

Column

Cross-Sectional

Area Based on

‘1’ on 4.6 mm ID

cross sectional

area

1

19

115

Flow rate based

on ‘1’ as 4.6 mm

ID flow rate

(mL/min)

1

19

115

Injection vol-

ume based on

‘10’ as 4.6 mm

ID injection

volume (μL)

10

190

1150

Cross Sectional

Area (mm2)

17

314

1963

Internal

Diameter (mm)

4.60

20

50

Figure 8: Open Solutions – How to setup samples for analysis

Table 1: Relationship between Column Cross-sectional area and Flow rate /

Injection volume (out of Technical Report 31)

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In addition to simplifying PCoperation to the greatest extentpossible, the ease with which anal-ysis results can be checked is alsoimportant to increase analysisthroughput. Since analysis resultsare displayed in the Internet Ex-plorer window when using OpenSolution, the information to bedisplayed can be limited to theminimum required without per-forming complicated operations.

The information in this window is organized in a simple, easy-to-understand manner. For example,the sample rack and loaded vialsare displayed graphically, so aquick glance allows a comparisonof the samples (vials) with theanalysis results. The window lay-out can of course be changed tosuit individual preferences (seefigure 9 and 10).

Moreover, when outputting theanalysis results, the informationdisplayed in this window can beprinted and outputted easily in apredefined format.

The ability to perform these oper-ations from any PC on the net-work is certainly one of the bigadvantages of open access.

Ease of data retrieval is also im-portant for improving analysisthroughput. Where data from aprevious analysis is required for

verification of suitability of condi-tions in the current analysis, thisdata may be located among hugeamounts of analysis data. Thetime needed to retrieve this datamay counterbalance any time ben-efit achieved up to then.

Hyperlink function for high-speed access to analysis results

In such situations, the Windowssearch function would typically beused, but this may be very timeconsuming depending on the filestructure and other factors. OpenSolution addresses this limitationusing the hyperlink function toachieve high-speed access to anal-ysis results. For example, an e-mail message is generated andtransmitted to inform that ananalysis is complete, and the URLspecifying the location of thestored results is included. Justclicking on the URL hyperlinkwill display the analysis results inthe Internet Explorer window.Furthermore, by clicking on a vialin the sample rack diagram, thedisplay switches to the analysisresults obtained from that sample.In this way, the analysis resultsobtained from another vial in thesame rack can also be viewedinstantly.

Open Solution supports promi-nence / Nexera system configura-

tions including the pumps, auto-sampler, column oven and alldetectors, and because of this sup-ports measurement using multipledetectors simultaneously. Further-more, rather than just a single sys-tem, multiple systems can be usedin the open access environmentprovided by Open Solution. Ifcollective data management is alsorequired, use of a server PC issupported.

Open Solution provides anopen access environment

Open Solution can effectively fillthe requirements of a preparativeLC or preparative LC/MS openaccess environment. In the case ofLC/MS, for example, the mass ofa target compound can be used toobtain a purity fraction higherthan possible with a UV detector,but the seamless synchronizationof the LC/MS and fraction collec-tor provided with Open Solutionallows fractions to be easily veri-fied and reliably collected at veryhigh speed. As described above,there are great advantages to berealized in an open access systemcomprising a closed network in alaboratory that is linked to anopen network in a separate officeenvironment. The introduction ofOpen Solution provides an openaccess environment which can beutilized to its maximum to achievegreat efficiencies in LC and

LC/MS analysis in the analyticallaboratory.

Remark

Further information can be found

in technical reports No. 29 (Open

Solution: Open Access Environment

by Web Browser) and 31 Basic

techniques of preparative Liquid

Chromatography and Apporaches

to Efficency Improvement (1)

Figure 9: Display of the Analysis results with Open Solution Figure 10: Peak purity analysis

Lead GenerationPharma Special

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Together we are strong

The demand to identify andcharacterize unknown sub-stances occurs in many

application fields in the pharma-ceutical industry, for exampleanalysis of impurities of synthe-sized compounds, degradationproducts in stability studies, meta-bolite identification to detectexpected and unexpected metabo-lites and metabolite profilingexperiment to search for differ-ences in mass profiles in largersample sets and their identifica-tion. In general, identification isan important part, because furtheractions are based on this identifi-cation. The ability to deliver ananswer in a reasonable time or toefficiently support the decisionprocess for further charac-teriza-tion steps defines the strength of atotal solution.

LCMS-IT-TOF with its manyunique software options is one ofsuch powerful solutions.

Combining two different massspectrometric tools (ion trap andTOF in one instrument) results incapabilities beyond any singlemass analyzing technology (Fig-ure 1). Ion trap mass spectrometryallows target molecules to beselected and fragmented easily(MSn; MS/MS, MS/MS/MS, MS/MS/MS/MS…) while time-of-flight MS provides high mass res-olution and accurate mass deter-mination.

The LCMS-IT-TOF can be equip-ped with different ionization

unknown substance. Recent publi-cations have highlighted that massaccuracy, while extremely impor-tant, is not sufficient for confidentcompound confirmation and iden-tification of unknowns. Accurateisotopic abundances of measuredions are critical as well. The rela-tive isotope abundance error hasto be below 5 % for effectivemolecular formula generation forunknowns, helping to eliminate alarge percentage of possible com-pounds.

“Formula Predictor” software

To determine the elemental com-position of an unknown sample,Shimadzu’s Formula Predictorsoftware uses all generated accu-rate masses of the compound,including the parent mass as wellas generated fragment masses fromthe MSn experiments to calculateand validate the total elementalcomposition of the molecule ofinterest, and generates a candidatelist. Additionally, the softwareuses isotope pattern informationto compare the measured samplewith the theoretical pattern ofpotential candidates which eitherincreases the probability score orexcludes certain candidates fromthe ranking. This dramaticallydecreases the number of false pos-itive candidates and makes deter-mination much easier for the ope-rator. Shimadzu holds the patentfor using the fragment �

sources (ESI, APCI, APPI, ESI/APCI combi source as well as anano spray source) and can easilybe combined with fast chroma-tographic separation delivering therequired results with 100 msecpolarity switching time in one run.In general, it delivers a mass accu-racy of around 3 ppm externallycalibrated, independent of the MSmodus. A flight tube constantlykept at 40° Celsius supports massstability even during analysis oflarger sample set studies.

The easy and reliable generation ofhigh quality data is an importantissue for identifying unknowncompounds. However, the soft-ware tools supporting interpreta-tion of the data are equally ofimportance.

In addition to the fragment infor-mation, the elemental compositionand the isotopic pattern of a com-pound are important informationfor the characterization of an

Figure 1: Schematic of the LCMS-IT-TOF

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different approaches to determineexpected as well as unexpectedmetabolites.

First Workflow – TraditionalPeak Picking

One or more time-series analytefiles (i.e. subject’s biofluid at inter-vals after taking drug) are screenedagainst a control file (i.e. subject’sbiofluid just after taking drug).The common peaks are excluded,the remaining peaks contain meta-bolites.

There are published biotransfor-mations for which parent drugfunctional groups can be metabo-

of MSn data can be utilized to firstbuild a ‘fingerprint’ of the parentcompound or drug (Y) and thenacquire MSn data for each peakduring analysis to get metabolite‘fingerprints’ (X’s).

Partial least squares regressionscores show how similar each X isto Y and since metabolites sharefunctional groups with the parent,many unexpected peaks can bescreened rapidly for potentialmetabolites and also to find newbiotransformations.

Profiling Solution software

The comparison of large data setsand the detection of difference isthe strength of the Profiling Solu-tion software package. Fast andreliable, it uses the generatedchromatographic and MS data ofmultiple sample measurements torecognize a difference (Figure 5).

The software supports and offersthe following steps:• In a patented way, it aligns mul-

tiple data sets using a uniquespectral binning technique andgenerates an aligned data arrayusing spectrum data (mass reso-lution independent) and chro-matogram data for multiple datastreams (supports polarityswitching within a run)

• Integrated filters for pooled QCanalysis (filtering out idiosyn-cratic ion behaviour based onpeak area, retention time)

• Integrated statistical tools

lized (added to or replaced by) inthe body. These peaks are calledexpected transformations and canbe screened for by generatingaccurate mass XIC’s (ExtractedIon Chromatogram) using themetabolized formula.

Remaining peaks that cannot becategorized are called unexpectedand are manually checked formetabolic likelihood. This can belaborious, but the Formula Pre-dictor software reduces the effort.

Second workflow – IsotopeLabelling

To aid confirmation of metabo-lites, discovery labs can label theparent using rare isotopes (whichmay or not be radioactive).

MetID automatically handleslabelling in the Peak Pickingworkflow and can also use peaksin a radiolabel detector chroma-togram.

Additionally, it can synthesize anIsotope Filtered Chromatogramfrom the MS chromatogram tolocate these labelled metabolites(which helps to locate compoundscontaining distinctive isotope pat-terns such as halogens; schematicshown in figure 3).

Third workflow – PLS regression analysis (Partialleast square)

With the LCMS-IT-TOF the con-stant mass accuracy and resolution

information of MSn experimentsto predict the elemental composi-tion of the molecular ion.

MetID Solution identifiesmetabolites in time-seriesanalyses of bio fluids

The MetID Solution is used in thefield of toxicity assessment/toxi-cology, where pharmaceuticalcompanies test the toxicity ofpotential drug candidates; it isapplied in the field of functionalgenomics where it determines thephenotype caused by a geneticmanipulation, such as gene dele-tion or insertion. The MetIDSolution software combines three

Figure 2: Formula Predictor software window showing one hit after using MS3 spectra

Figure 4: Real example of extracted isotope filtered chromatogram of Nefazodone,

based on the chlorine isotope distribution

Figure 3: Schematic of the Isotope Filtering Chromatogram procedure

Lead GenerationPharma Special

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(p-value, normalisation, de-isotoping)

• Export to statistical packages(SIMCA-P, MatLab as a textfile)

• Hot links to web based datasearch engines (LipidMaps,KEGG, Metlin, ChemSpider)

Open Solution ComponentID

In many synthesis laboratorieschemists get access to a singlequadrupole system using “OpenAccess” software. However, inorder to get an accurate massmeasurement for a target com-pound the mass spectrometrydepartment in general have to ana-lyze the samples. This can create abig overhead for this mass spec-trometry service departments.Open Solution Component ID isa software tool to automate theformula prediction workflow(Figure 6a). Its combination withthe reliability of the LCMS-IT-TOF makes it to a powerful solu-tion for molecular weight confir-mation and impurities profiling.

ComponentID provides a simpleand easy to understand user inter-face for logging samples (and run-ning analyses) and printing re-ports of results using the LCMS-IT-TOF (Figure 6b). This makes iteasy for synthesis researchers andother laboratory users to analyzetheir own samples. It provides aneffective tool for customers, suchas those described below, that liketo use the instrument in a mannergenerally referred to as “openaccess.”

5. On completion of an analysis,obtained results are printed as aPDF file, attached to an emailand sent to a pre-specified e-mail address. The layout ofthe output can be customizedby the system administrator, sothe PDF file can include notonly formula prediction soft-ware results, but also MS chro-matograms, spectra, LC chro-matograms or other informa-tion desired by the user.

6. In addition, ComponentIDincludes many other functionsdeveloped from actual opera-tion of OpenSolution and Psi-Port systems, such as a desktoplock feature, overnight analysisfeature and priority settings.

accurately predict the composi-tion of synthesized substancesand impurities. These results arethen output automatically in areport.

3. ComponentID supports high-throughput LC/MS analysisusing prominence systems, andalso supports flow injectionanalysis which does not use acolumn. Using flow injectionallows the processing of a largernumber of samples in a shorttime.

4. Data entry time can be reducedby importing a list of sampleinformation, such as the com-position of synthesized com-pounds, from an Excel spread-sheet or directly copying andpasting from Excel. This is alsoconvenient for synthetic sub-stances in plate format.

• Customers who intend to sharea single LCMS-IT-TOF systemamong many researchers, soeach can confirm the molecularweight of synthesized com-pounds or confirm their puritylevel or contaminants

• Users that want to utilize for-mula prediction software toconfirm synthesis and improvethe accuracy of such determina-tions

• Users with their desks locatedaway from the instruments,which involves time-consumingmovement before they can viewthe results.

ComponentID offers the follow-ing features to address the cus-tomers’ needs described above.

1. After logging in to the software,it takes only five steps to startan analysis: enter the number ofsamples, select a method, enterthe target chemical composi-tion, place the samples in theautosampler, and click the[Start] button. A report of theresults can be received viaemail. Training is not thereforenecessary for general users.LC/MS system administrationand method development isperformed by the system ad-ministrator. The sample loggingfunction is the same as that usedin the highly acclaimed OpenSolution and PsiPort softwarefor LCMS-2020/LCMS-2010series systems.

2. MSn accurate mass measure-ment results from the LCMS-IT-TOF system are utilized to

Figure 5: Array of different samples where significant variations are highlighted Figure 6a: Workflow to generate accurate mass data in an open access environment

Figure 6b: Login Window of ComponentID Solution

Drug Discovery Pharma Special

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Figure 1: Vials with powder after pruification and solvent evaporation

Figure 2: Rack Changer P with tray for trap cartridge and vials

From Crude to PowderSolid-Phase Trapping as a new approach to shorten and

improve purification process

Pharmaceutical research anddrug development are time-consuming processes. After

target finding and assay develop-ment it may take 10 - 15 yearsuntil a new drug can be released.Development costs often reach500 - 1000 million Euro. New andmore efficient technologies indrug discovery can help to accel-erate the development process anddecrease the costs.

During the drug discovery pro-cess, auto purification systems uti-lizing preparative LC/MS havebeen utilized widely for purifyingnewly synthesized compoundsahead of screening. Reversed-phase gradient HPLC methodsare often used as ‘generic’ or ‘uni-versal’ methods for a wide rangeof compounds due to their adapt-ability and scalability in additionto ease-of-use.

Fractionation and evapora-tion determine the speed ofthe purification process

For further structural identifica-tion and more detailed testing it isrequired, which needs fractioningthe compounds and transfering

them from solution into solidstage. Fractionation and evapora-tion are key steps after the purifi-cation as they determine the speedof the whole process and the qual-ity of the sample for subsequentanalytical methods.

Typically, the evaporation is seenas the bottleneck, as soft dryingand solvent evaporation take along time compared to the rela-tively fast separation. This processis even more problematic whenmobile phase additives such asTFA (Trifluoroacetic Acid) remainand may cause false results inscreening. Eliminating or shorten-ing the evaporation process isdesired by many researchers forimproving the throughput andquality of the auto purificationprocess.

Modern ways of purification (e.g.LC/MS) inevitably produce ‘solu-tions’ of pure sample (normallysolid) in a liquid (mobile phase).Recovery of these solids is gener-ally attempted by evaporativeremoval of the liquid (N2 blow-down, vacuum centrifuge or acombination of both). This pro-cess is generic but unfortunately

has some potentially ‘unmeasured’associated errors.

Any substance which is not suffi-ciently volatile/evaporable in themobile phase/background willconcentrate together with thesample, leading to loss of mass-purity and potential for significantsample contamination. This canlead to ‘weighing errors’ if weigh-ing is the quantification method,which propagate into preparationof a known-concentration solu-tion. Furthermore, commonmobile phase additives such asFormic acid/TFA/TEA etc. canform salts with specific chemicalfunctionalities leading to saltform/stoichiometry issues. In gen-eral, HPLC or HPLC-MS basedtechniques are increasingly thepharmaceutical industry’s methodof choice in order to obtain veryhigh purity compounds.

Trapping concept as an alter-native to evaporation

An alternative to evaporation uti-lizes a trapping concept wherebyfractions are transferred onto atrap column, washed and precon-centrated and in a second stepextracted from the trap as powder.

The eluate from the preparativeseparation is transferred to thetrapping module and flushed ontoa trap column. During the transferthe ‘sample’ can be diluted, whilemodifiers can be applied to ensurethat either a salt form or the freebase is trapped.

Why ‘Trapping’?

Trapping technology offers severaladvantages:• removing chromatographic frac-

tion volume scale dependence• removal of certain salt forms

(e.g. TFA)• removing (large volume) evapo-

ration as a standard approach.

One removal process for all, independent of sampletrapping method

Trapping allows as an alternativemeans the selective removal of justthe sample onto a solid support,enabling everything else to passthrough or be washed out. Thesalt form of the sample can thenbe manipulated prior to recoveryin a convenient solvent system(i.e. one that is very easy to re-move). This trapping task can beperformed on-the-fly as part of

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your purification process as wellas from already collected frac-tions.

How does trapping actually work?

Trapping works by transferringpure compound ‘fraction’ (ob-tained from Mass directed auto-preparation or similar), with addi-tion of a diluent, onto a trap (20 x30 mm dimension) which containsa specific resin based on PS-DVB(polystyrene-divinylbenzene). Thecompound is immobilized whilethe background solvent/additivesetc. are not. The compound can beconverted to the free base on-trap.

Compound recovery

The trap can then be transferredto a recovery module where thecompound can be retrieved as a

Instrumentation

Task-specific instrumentationdeveloped by Shimadzu performstrapping and recovery operationsin dedicated hardware modules.This enables automation of thewhole process detailed in an OpenAccess (OA) mode with a dedicat-ed software wizard. Ultimately,the system can be combined withthe LC-MS front-end to functionas a complete ‘crude-to-pure’ sys-tem purifying up to 150 mg massof sample in a single trap.

Summary

A new compound isolation andhandling process based on ‘SolidPhase Trapping’ has been devel-oped providing a new and muchimproved workflow to deliverpure, largely dry compounds. Theoffline, two step process will bepossible in new, purpose designedinstrumentation. Ultimately, thiscan be combined with the purifi-cation system to form a full on-line system.

• high chemical purity (very lowlevels of structurally relatedcontaminants) giving confidentspecificity

• high ‘weight purity’ (no detec-tor-invisible or non-structurallyrelated process contaminants orsolvents) giving confidentpotency

Ideally, 1 mg of compound weight contains 1 mg of com-pound (100 % weight purity).

Accurate weights mean accurateconcentrations. This in turnmeans testing and ranking com-pounds correctly. Generally, stor-ing compounds as dry solids givesthe highest confidence that com-pound integrity is maintained.

Sample and Workspace Management

Each user (individual chemist) cansubmit his/her own fractionsmanage their own traps with ulti-mately dry recovered samples bymanagement of their own work-space. This takes the form of a setof up to four traps and correspon-ding recovery (haystack) vialscontained within an MTP-foot-print workspace (see below).

very concentrated solution in avolatile organic solvent, or direct-ly as the dry, powdered material.

Technically, the trap column isflushed in the opposite directionusing a non-water miscible solventwhich washes out the water fromthe trap column. The compound isreleased with this solvent (dichlo-romethane being used in practiceat many sites). The dissolved com-pound is sprayed with nitrogeninto a pre-heated vial and trans-ferred back to the solid stage,remaining as powder in the vial.

The recovery module is availableas manual dual channel as well assix channel parallel fully automat-ed version.

It is important for subsequent ana-lysis steps, that the trapped mole-cule is of high quality, meaning:

Figure 3: Schematic of the flow line to the trapping systems /Trapping Module

Figure 4: Schematic of the recovery module

Figure 5: Trapping System with Needle in Trap position (fraction load on cartridge 4)

Drug Development Pharma Special

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Bringing light into darkne

The analytical method de-velopment section in CMC(Chemistry, Manufacturing

and Control) sections of pharma-ceutical companies constitute partof the pharmaceutical develop-mental process that deals specifi-cally with the physical nature of adrug substance and the drug prod-uct, for example how it is made,and the control of the manufac-turing process to provide a reli-able and reproducible product.HPLC systems in these areas areutilized for the specification testsof active pharmaceutical ingredi-ents (API) to ensure the quality ofthe product as well as the level ofimpurities.

Traditionally, non-volatile mobilephases containing phosphate buf-fer solutions have been used in theHPLC test methods for QA/QC.When LC/MS analysis is per-formed, it is necessary to switchfrom a mobile phase containingnon-volatile additives to a com-pletely volatile mobile phase suit-able for atmospheric pressure ion-ization techniques.

This alteration in analytical condi-tions may cause changes in theelution order of analytes as well asintroduce the possibility ofobscuring impurities due to their

Desalting is executed using adivert valve installed just beforethe MS inlet. The addition of theUV detector to the second dimen-sion greatly facilitates the detec-tion of impurities. In an actualsample workflow, both impuritydata and the corresponding blankdata are acquired, and the impuri-ty peaks are identified throughcomparison of the respectivechromatograms. Peak isolationand fractionation is conductedapplying multiple valves in spe-cific combinations. Creating thepeak isolation program is easyusing a provided macro program.The macro automatically specifiesthe best valve sequence, demand-ing only that the user adds theretention times of the first dimen-sion separation. To make opera-tion even easier, a separate macro-program which automates theconstruction of a batch schedulefor acquiring multiple impurityand blank data, is also provided.The individually collected impuri-ties are then analyzed with theLCMS-IT-TOF. In a very fast andefficient way fragment spectra canbe generated and used with itsaccurate mass to determine theelemental composition of impuri-ties. Additional software packages,like MS fragmenter from ACDlabs, can use this accurate mass

backdrop, it is no wonder that anew analytical system is eagerlyanticipated.

To address the demand for LC/MS analysis allowing utilization ofexisting non-volatile mobile phaseanalytical conditions, Shimadzuhas developed a new kind of setup,working independently of a trap-ping column approach. The advan-tage of simplifying the flow path isthat the time and effort requiredfor optimizing the conditions canbe shortened. This overall analysisof the system led to the construc-tion of the Co-Sense LC/MS sys-tem with a modified flow path.

close proximity to the chromato-graphic peaks of the major con-stituents. The change in methodconditions requires attention todetail and a great deal of effort bythe user. Furthermore, recentmodifications in regulation ofimpurities along with globaliza-tion of the supply chain have ledto even greater demand for impu-rity identification. Of course, thiscauses a bottleneck due to thenecessity to modify HPLC condi-tions conforming to the existingvalidated test methods, and theneed to satisfy demands from themanufacturing section for identifi-cation of impurities. Against this

Figure 1: Basic flow diagram of Trap free 2D LCMS impurity system

Figure 2: Used HPLC modules and system flow diagram

Drug DevelopmentPharma Special

13

ess 2-D LC and LCMS-IT-TOF – Better drug impurity ID efficiency

fragment pattern information andsearch for homologies betweenthe impurity and the main com-pound.

Analysis example

A sample consisting of sulfadi-methoxine (Figure 4) as the prin-ciple compound, and four sulfadrugs having a similar structure toserve as impurities is used for thisexample (Figure 5). The impuritieswere each mixed with the princi-ple compound at percentages of0.1 % relative to the principlecompound which had a concen-tration of 500 μg/L. The impuri-ties were selected and fractionatedinto the loops based on their elu-tion times (Figure 6). The struc-tures of the respective substancesare shown below, and the meas-urement results are shown in fig-

ure 7. All of the “impurities” wereconfirmed in the results.

Analytical conditions LC first dimension

Column: Shim-pack VP-ODS, 150 mm L. x 4.6 mm I.D., 5 μmMobile phase: 0.01 mol/L phos-phate buffer, solution (pH 2.6),methanol mixture (7 :3)Mobile phase flow rate:1 mL/minColumn temperature: 40 °CSample injection volume:10 μLPDA detection wavelength:200 - 350 nm (270 nm monitored)

Analytical conditions LC second dimension

Column: Shim-pack XR-ODS 75 mm L. x 2.0 mm I.D., 2.2 μm

Mobile phase A: Shim-pack XR-ODS 75 mm L. x 2.0mm I.D.,2.2 μmMobile phase B: MethanolMobile phase ratio: 10 % B (0min) -50 % B (10 min) -10 % B(10.01 - 20 min)Mobile phase flow rate:0.3 mL/minColumn temperature: 40 °CSample injection volume:0.3 mL/minUV detection wavelength:270 nm

MS conditions

Ionization mode: ESI+Nebulizer gas flow rate:1.5 L/minDrying gas pressure: 0.15 MPaImpressed voltage: 4.5 kVCDL temperature: 200 °CBH temperature: 200 °CScan range: m/z 100 - 1000

Utilizing the data browser

The data browser provided inLCMSsolution software offersadditional functionality enhanc-ing the efficiency of impurityanalysis-related tasks. The layouttemplate for picturing data can becustomized and saved beforehand.As shown in figure 8, the storedlayout was constructed so that the

Figure 3: Fractionating loops available in 5 μL, 10 μL, 20 μL and 50 μL

Figure 5: Compounds of similar structure like Sulfadimethoxine serving as impurities

for this example

Figure 6: First Dimension PDA Chromatogram: The blue coloured trace displays the

system pressure and the black coloured trace the UV 270 nm signal. These correlate

perfectly, indicating the elution peaks are reliable fractioned in the loop.

Figure 4: Structure of Sulfadimethoxine

upper tier is reserved for display-ing the UV chromatogram and MSspectrum of impurity-related data(2-D LC), and the lower tier forshowing the UV chromatogramand MS spectrum of the blankdata corresponding to the impuri-ty peak (2-D LC). In this case, theUnk-1 (sulfamerazine) data andthe respective blank data are pre-sented using the “drag and drop”function from the data explorerfor display in the data browser.

The differences in retention timesbetween those obtained with theUV detector and those obtainedwith MS were synchronized usingthe retention time correctionfunction provided in the browser.The retention times are stored inthe method file, and since thisretention time averaging functioncan be set up beforehand to be �

Drug Development Pharma Special

14

executed automatically, it does notneed to be executed manually eachtime a new set of data is dis-played.

Furthermore, utilization of thisfunction permits automatic detec-tion of the impurity peak from theUV chromatogram and the corre-sponding display of the MS spec-trum. In addition, since all of thedata files are processed automati-cally, identification of the impuri-ty on the UV chromatogramallows not only synchronous dis-play of the corresponding impuri-ty spectrum, but also synchronouspresentation of the blank MS

required otherwise. The ability toconfirm impurities through com-parison with blank data shouldcontribute greatly to the efficiencyof CMC impurity identification.

cannot be applied unless retentiontimes are consistent.

However, it is reasonable to as-sume that the repeatability ofretention times has already beendetermined since retention timerepeatability is fundamental to thedevelopment of a specific testmethod. If the separation tech-nique for the first dimension sepa-ration is already established, meas-urement can be conducted with-out re-evaluation of the techniqueintroduced here, eliminating theneed for LC/MS measurementusing complex volatility condi-tions that would have been

spectrum as well. The actual oper-ation is conducted using just themouse to drag from the start ofthe impurity peak in the impurityUV chromatogram to the end ofthe peak. Upon releasing themouse button, the averaged corre-sponding impurity spectrum andblank spectrum will be shown.

When using the two dimensionalLC system introduced here, re-peatability of retention time in thefirst dimension is extremely im-portant because impurity peakfractionation is conducted withinspecific time-controlled intervals.In other words, this technique

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5

100 200 300 400 500 600 700 800 900 m/z

-0.250.00

1.00

0.75

0.50

0.25

7.5

5.0

2.5

0.0

0.00

0.250.500.751.001.251.50

Second D-LC Unk-1uV (x 1,000)

(x 1,000,000)

Inten. (x 1,000,000)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5

100 200 300 400 500 600 700 800 900 m/z

-0.25

0.00

1.00

0.75

0.50

0.25

2.0

1.5

0.0

0.00

0.25

0.50

0.75

1.00

Second D-LC Unk-3uV (x 1,000)

(x 100,000,000)

Inten. (x 1,000,000)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5

100 200 300 400 500 600 700 800 900 m/z

-0.25

0.00

1.00

0.50

0.75

0.25

7.5

5.0

2.5

0.0

0.00

0.25

0.50

0.75

1.00

Second D-LC Unk-2uV (x 1,000)

(x 100,000,000)

(x 10,000,000)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5

100 200 300 400 500 600 700 800 900 m/z

-2.0-1.0

1.00

0.50

0.75

0.25

4.03.02.0

0.01.0

0.00

0.01.02.03.04.05.06.07.0

Second D-LC Unk-4uV (x 100)

(x 100,000,000)

(x 1,000,000)

Figure 7: UV Chromatograms, MS Chromatograms and MS spectra of four fractionated impurities

Figure 8: Unk-1 and Blank Data

Displayed in Data Browser

Drug OptimizationPharma Special

15

Future-proof analysis – UHPLC 3.0

No matter whether DrugDiscovery, Pre-Clinicaland Clinical Studies,

Active Ingredients or FinishedProducts – HPLC is the tech-nique of choice in the Pharmaceu-tical Industry. Depending on thestage, e.g. drug development orproduction of finished pharma-ceuticals, there are many demandson HPLC system properties.

Whether in drug detection orquality control, the choice of ded-icated HPLC systems should notbe limited by the software plat-form controlling the systems andprocessing the data. �

zero carry over. Furthermore,they should be capable of han-dling various plate and vial for-mats as well as large sample quan-tities. The systems need to per-form consistently with a mini-mum of variations from system tosystem so that poor precision doesnot obstruct data pooling later on.Since sample amounts are oftenlimited, there is a high demand forsystems dealing with smallestsample quantities while still pro-viding high sensitivity measure-ments.

At a later stage, testing for identi-fication, quantification/impurity,stability/content uniformity, dis-solution as well as cleaning valida-tion requires different and addi-tional options. HPLC determina-tions in cleaning validation de-mand high sensitivity and univer-sal detection using large samplequantities at low concentrations,while HPLC determinations indissolution testing require specificaccurate detection of large andsmall quantities within a limitedtime. Quality Control usuallycalls for precise and robust HPLCsystems. High sensitivity isimportant for stability and impu-rity testing, since degradationproducts usually appear in muchsmaller concentrations.

High system precision enablesearly trend recognition and inter-vention before out-of-specifica-tion results, thereby offeringmajor advantages over medium-precision HPLC systems.

Nowadays, screening for activesubstances as well as the chemicaloptimization is unthinkable with-out the use of LCMS. There aredifferent needs and priorities forthe MS detectors applied in thisstage, but the requirements forfront-end HPLCs are more or lessthe same for all. The systems areexpected to perform high resolu-tion analysis at high speed with

Figure 1: Nexera System with Rackchanger II for large sample capacity

Figure 3: Inside view of CTO-30A.

First figure: Heating block and separated

heating cartridges. Second figure:

Low volume low/dispersion pre-heater.

Third figure: Column compartment.

Figure 2: SIL-30AC Flow Line DIIMS. Sample suction line is part of the flow line and

the metering pump is isolated via a low pressure valve.

Nexera perfectly serves all

needs of the Pharmaceutical

Industry

Drug Optimization Pharma Special

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In development of the NexeraUHPLC system, Shimadzu notonly focussed on high through-put, high resolution capable ofhandling modern sub 2 μm parti-cle columns with system pressuresover the 1,000 bar limit of mostcurrent UHPLC systems. Shimadzu also aimed at a versatilesystem with almost zero carry-over which could be utilized inany area based on its comprehen-sive pressure flow range and scala-ble injection volume range. Shimadzu’s Nexera provides aflexible all-round HPLC systemwith a precision and robustnesssuited for daily routine analysis,as well as groundbreaking highspeed and high resolution separa-tions. Nexera can be controlled bythe LabSolutions software andother software platforms such asWaters EmpowerTM, DionexChromeleonTM and AB Sciex Ana-lystTM.

the market. For critical samplesand matrixes, carry-over can beeliminated by the use of threerinsing solvents (plus one externalneedle wash solvent) in combina-tion with various rinse modesbefore and after sample aspiration.

In addition to the well-establishedand precise DIIMS technique(Direct Injection with IsolatedMetering System), the autosam-pler also offers fixed-loop injec-tion for ultra-fast separation withminimized peak width and gradi-ent delay. An overlapping injec-tion function is standard withNexera enabling maximum reduc-tion in cycle time.

For high-temperature LC analysisup to 150 °C, Nexera offers effi-cient pre-heating for solvents toenable stable and reliable condi-tions. The advanced IntelligentHeat Balancer (IHB) minimizesband-broadening during high-temperature analysis through useof independently controlled heatzones within the column compart-ment, ensuring accurate columntemperature regardless of flowrate. The post-column cooler canreduce detector noise whenequipped for high-temperatureanalysis.

Why select a system exceed-ing requirements?

The following example shows aseries of runs to determine thecontent of an active ingredient.The User Requirement Specifica-tion for Peak Area Precision

The LC-30AD solvent deliverymodule is a high resolution microvolume dual piston pump bring-ing new performance in precisionand accuracy to modern solventdelivery pumps. Its flow raterange covers 0.0001 mL/min to 10 mL/min with a pressure rangeup to 130 MPa. The LC-30ADallows setting up of binary or ter-nary high pressure gradient sys-tems as well as quaternary lowpressure gradient systems.

Two types of multilayer solventblending reactors (Shimadzupatent) allow solvent mixing withminimized system dwell volumes.Depending on requirements, 180 μL or 20 μL reactors can beutilized.

In terms of carryover and repro-ducibility, the autosampler SIL-30AC exceeds the performance ofany other HPLC autosampler on

Nexera is a modular and flexibleUHPLC system consisting of • LC-30AD high precision

solvent delivery pumps • fast and flexible SIL-30AC

autosampler with lowest carry-over marketwide

• Rackchanger II for extra-largesample quantities

• standard column thermostathandling column switchingvalves, mixing reactors and largecolumns as well as the new dedi-cated high temperature columnthermostat allowing columnheating up to 150 °C.

With the low dispersion semi-micro flow cells and the 100 Hzsampling rate, Shimadzu HPLCdetectors are fit for high speed/high resolution separations.

In the following text, the mainNexera modules are described inmore detail.

0.0

Minutes

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000uV

0.6850

Minutes

0.69750.69500.69250.69000.6875

57,500

60,000

62,500

65,000

67,500

uV

Figure 4: Chromatogram Overlay of 10 subsequent runs (Right Part: Zoomed Peaks)

Figure 5: Trend Plot of Peak Areas

0 5 10 15 20 250

20,000

40,000

60,000

80,000

110,000

120,000

Area 01: 98323,99726

Area 02: 98135,49221

Area 03: 97628,27635

Area 04: 97381,93631

Area 05: 96906,66735

Area 06: 96620,62883

Area 07: 96201,38288

Area 08: 96002,34449

Area 09: 95753,05462

Area 10: 95521,59548

Area 11: 94819,36395

Area 12: 94734,80582

Area 13: 94527,49133

Area 14: 94327,95764

Area 15: 93789,9226

Area 16: 93728,87015

Area 17: 93156,98504

Area 18: 92646,18933

Area 19: 92465,57713

Area 20: 92576,1354

AV 95262,43371

SD 1874,015569

% RSD 1,967214

Drug OptimizationPharma Special

17

Figure 6: Carryover of chlorohexidine without injection port rinse, needle internal rinse

Figure 7: Carryover of chlorohexidine with injection port rinse, needle internal and outer surface rinse

0.50.0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5---

---

0.0

50

60

70

80

90

100

110

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Minutes

mV MPa

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5-1

0

50

60

70

80

90

100

110

1

2

3

4

Minutes

mV MPa

(injection volume = 5 μL) is ≤ 2 %RSD.

The overlay of the chromatograms(Figure 4) shows good precision,but in the zoomed view a cleartrend towards decreasing peakarea is apparent. The % RSD forpeak area precision is within thelimit of 2 % RSD, but the trend(Figure 5) indicates a problem. Itis therefore possible to interveneand avoid out-of-specificationresults.

Carryover works against productivity:

Particularly in drug discovery,sample carryover may cause de-lays and additional costs, sincefalse positive candidates passthrough the entire process. WithDIIMS (Direct Injection with Iso-lated Metering System) construc-tion and well selected materials ofthe SIL-30AC autosampler, carry-over is reduced to a minimum. Byapplying special rinse modes sam-ple carryover can be reduced to anundetectable amount. The figurebelow shows 0.0006 % carryoverfor a Chlorhexidine injection.

By applying additional rinsingsteps, carryover can be eliminatedcompletely as shown in figure 7.

These are just a few examples de-monstrating the advantages of ahigh end UHPLC system. TheNexera system provides the fea-tures and flexibility to cope withall demands expected of a modernUHPLC used in the Pharmaceuti-cal Industry. In the final analysis,system reliability and long main-tenance interval defines the realvalue in daily routine use. Thiswas taken into account whendesigning the Nexera. The resultis a reliable, robust and precisefuture-proof UHPLC system.

Carryover

Chlorhexidine

2,000 ng/μL

Post-blank 1

Post-blank 2

Post-blank 3

----

0.0006 %

0.0003 %

0.0004 %

Column : ODS (2.0 mm I.D.�100 mm L., 1.8 μm)

Mobile Phase : Methanol, Water = 2 /8

Flow rate : 0.4 mL/min

Temperature : 30 °C

Injection volume : 5 μL

Rinse solution : 0.05 % formic acid in methanol

Needle wash : outer surface flush by rinse pump (1 second flush)

: needle soak wash (0 second)

Carryover

Chlorhexidine

2,000 ng/μL

Post-blank 1

----

N.D.

Column : ODS (2.0 mm I.D.�100 mm L., 1.8 μm)

Mobile Phase : Methanol, Water = 2 /8

Flow rate : 0.4 mL/min

Temperature : 30 °C

Injection volume : 5 μL

Rinse solution : 0.05 % formic acid in methanol

Needle wash : outer surface flush by rinse pump (1 second flush)

: needle soak wash (0 second)

: needle internal rinse

Injection port rinse : performed

Drug Optimization Pharma Special

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From powder to pill – quality cont

In analytical instrumentation,FTIR measurement is theonly technique providing

non-destructive analysis of sub-stances. It identifies powder, solidor liquid samples within shortesttime, needing just milligrams ofmaterials.

FTIR spectroscopy’s advantage isthe ability to verify substanceswithin 1 minute. Proof of identityis guided by documents preparedby the pharmaceutical industry aswell as the national or EuropeanPharmacopoeia. The EuropeanPharmacopoeia set up a range ofcomments for each of Pharma-copoeia’s relevant substances. Thesame set of rules helps in its phys-ical observations in qualificationof the instruments. Some of thepharmaceuticals prepared for theexport business are regulated byUSP or FDA rules. The task of amodern FTIR is to be equippedwith a validation concept whichaccounts for all cases.

More and more, a concept ofeasy-to-use and result-orientedanalysis is required. A selection offunctions is therefore neededwhich will select the requested

goods. It can be the pill capsulemade from gelatin, sugar, andstarch or it can be the blister car-rying the pills and providing easyhandling for the end-user. Theseblisters, metal foils as well as met-al and plastic tubes for paste andcreams can be analyzed withFTIR.

One sample of analysis will bepresented here: the control ofMetoprolol succinate, an activeagent reducing blood pressure.Identification was carried outusing FTIR-Spectroscopy.

Next to the active agent, othersubstances are contained in atablet (Figure 1), determining col-or or contributing to stability.These fillers and accompanyingmaterials including dye enable aregulated reception of the activeagent (dissolution) by the humanorganism.

High blood pressure is a creepingillness that can occur slowly overthe years. It can cause heart dis-ease, circulation problems andstrokes. Pharmaceutical activeagents based on Metoprolol repre-sent an effective means to reduce

product, e.g. a tablet. In case ofvery hard substances, a diamond-based version of single reflectionsupports the application, pressingthe substance onto a measurementwindow. A soft sample gives easyaccess to the IR spectrum becauseof a good contact between sampleand window. In case of hard mate-rials, more force is needed toestablish a good contact.

The measurement technique isATR (attenuated horizontal reflec-tion). Depending on the opticalelements used the analysis beamwill penetrate the sample surfaceby approximately 2 μm. In thecase of ready-to-use pills, the sur-face cover can be hard and blocksthe pharmaceutical agent below.Such pills need only a cut. Cap-sules have to be opened, whereasfilm-styled surfaces can be cut.For example, capsules made forliquids can be cut and the liquidtransferred onto the measurementwindow.

Active agent analysis with FTIR

A different aspect in pharmaceuti-cal analysis is the packaging of the

comparison analysis based onPharmacopoeia. A comparison can be:• a simple visual comparison• search in a library• purity check• comparison of dedicated

signals• contaminant analysis.

The cycle of measurement

The highly accurate measurementtechnique should still be fast.FTIR analysis with non-destruc-tive approach supports these im-portant aspects. A cycle of meas-urement can be described easily:• take the sample• place it in the measurement

window• press the sample (solids only)• measure• remove and clean the instru-

ment’s window.

A time frame of one minute is arough approximation for this han-dling cycle. Such simple and quicksolutions must be supported byaccessories of the same class, suchas single reflection accessoriesenabling a simple quality controlfrom raw material up to the final

Active agent analysis using FTIR spectroscopy

Figure 1/2: A tablet with beta blocker based on Metoprolol succinate with 47.5 mg

active agent out of 447.5 mg total weight / Structure of Metropolol

4000

1/cm

% T

3500 3000 2500 2000 1750 1500 1250 1000 750

Figure 2: IR-spectrum of the surface measurement of the tablet using a single

reflection unit (blue) and IR-reflection-spectrum of aparticle from the inside of the

tablet (red)

Drug OptimizationPharma Special

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trol of pharmaceutical goods

high blood pressure. Commonvariants are Metoprolol tartrateand Metoprolol succinate. Tartrateand succinate are salts belongingto the class of bicarbonic acids,derived respectively from wineacid and amber acid. In the Phar-macopoeia, both materials arecontained.

Composition declared

Active agent: Metoprolol succi-nate (PH. Eur) of beta blocker47.5 mg per tablet Contents: Cellulose (E460),Polyvinyl pyrrolidone, glucose,Lactose-Monohydrate, Macrogol4000 (Polyethylenglycol), magne-sium stearate (PH. Eur), cornstarch, Polyacrylate, Silicon diox-ide, Sucrose, talcum, titaniumdioxide as a dye (E171)

The active agent Metroprolol isdescribed as follows: (Metoprolol)2 ‘HO2C-CH2-CH2-CO2H, sumformula C34H56N2O10; molecularweight of the racemic mixture Mr = 653 g/mol. The active agentmakes up approx. 10 % of thetotal weight of the tablet, whichshould be detectable in the com-plex mixture.

Figure 3: Infrared spectra of tablets with low and high dosage agent

(blue = low, green = high dosage)

Table 1: Coordination of the vibration signals

4500

1/cm

% T

4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500

40

50

60

70

80

90

100

Measurement• The tablet was first tested in its

as-is state.• A single-reflection-ATR-unit

was used. • The IR-spectrum of the surfaces

measured determined starch andother filling materials (Figure 2).

The tablet was then divided intwo, and a cross-section was tak-en. This powder granular materialwas then measured (Figure 2, redspectrum).

When comparing the spectra, cleardifferences are apparent. The sur-face spectrum (penetration depthof the light beam approx. 2 μm) ofthe filler material highlights astrong signal around 1,100 cm-1,covering many of the componentsin the contents list. Starch, cellu-lose and sugar (in the form ofLactose and Sucrose) influencethis signal.

When observing the second spec-trum in figure 2, filler materialswith their infrared signals arepresent, but the spectrum is locat-ed around some dissolved signals.Interestingly, a secondary Aminomolecular group appears which is

from the active agent Metoprolol;as do oxygen atoms in the form ofEthoxy-, Phenoxy- or Hydroxy-groups, as well as a 6-atom ringmolecule.

For filler materials, interpretationof the signal in the IR-spectrum ismore complex than with analysisof the pure materials. However,some significant signal vibrationscan be recognized unambiguously:Metroprolol and Succinate (Table1). The vibrations representingSuccinate could not be found inthe spectra of the filler materialsor in the spectra of the pure mate-rials of Metoprolol and Metopro-lol tartrate (Sadtler Crime/Foren-sic Libraries).

An intensive CO-signal volume isnoticeable in 1,726 cm-1, thatcould be identified as Succinate.Furthermore, a signal doublet wasfound similar to Succinate in 1,111and 1,157 cm-1 with a differenceof 46 cm-1, corresponding to adoublet of the amber acid (1,084

and 1,132, with a difference of 48 cm-1). The signal deviation canbe explained by the differentmolecule interactions in the salt incomparison to the acid (approx.20 cm-1).

IR-spectra of tablets with 47.5 mgand 95 mg are shown in figure 3.The active agent’s increase in con-centration can be recognized by areduction in the single signalheights.

Instruments

FTIR-instrument: IRprestige-21 Software: IRsolution includingsearch functionAccessories: DuraSamplIRIILibraries: Shimadzu Pharmaceuti-cal, Sadtler

Accessory

A single-reflection-ATR-unit wasused. The measurement was car-ried out by simple dropping andpressing of the sample.

Wavenumbers [cm-1] Vibration Material

-C-C-

C-O-C asy. Stretch

-NH

Ether C-O Stretch

C = C Stretch Ring, aldehyde

C = C Stretch Ring

NH secund. Amine

C = C Stretch Ring

-C = O Aldehyd

Combination band

Combination bands + NH

fine structure

-CH asy. Stretch influences

through -O-CH3

-OH

-NH secund. Amine

Filler and

active agent

Metoprolol

+ Succinate

Metoprolol

Metoprolol

Metoprolol

Succinate

1014.56

1049.28

1070.49

1111.00

1157.29

1240.23

1332.81

1444.68

1516.05

1558.48

1614.42

1726.29

1977.04

2029.11

2916.37

3332.99

3398.57

Method Development Pharma Special

20

Method transfer and validation accDesign requirements

In 2004, the FDA issued thePAT initiative (process ana-lytical technology), intending

to change development processesalso in the pharmaceutical indus-try. Nowadays, the Quality byDesign concept as a logical con-tinuation is discussed and influ-ences current and future actions.Guidelines issued by ICH (Inter-national Conference on Harmo-nization) and other regulatoryauthorities request the applica-tion of QbD principles.

The complexity of parameters ina HPLC method makes it verydifficult to change and modifysome of the parameters once thismethod is validated. Adapting aseparation from conventional toultra-high pressure LC and therelated effect are difficult to des-cribe and predict. DryLab2010 isa valuable tool to simplify thisprocess, based on designed exper-iments to predict method chang-ing effect and describe the ‘De-sign Space.’

Shimadzu and the Molnar Insti-tute in Berlin cooperated inapplying well-known Dry-Lab2010 software with the newgeneration of UHPLC instru-ments.

simultaneously. It finds the bestseparation with a mouse click andhelps to find the optimum solventcomposition (e.g. Ratio betweenAcetonitril / Methanol). The nec-essary input data for these 3Dmodels is depicted in figure 4:three tG-T models at three differ-ent values of a third parameterare needed.

Design of Experiments

Preliminary evaluation deter-mined that the most influentialexperimental variables on criticalresolution (separation betweencritical peak pair) were• the gradient time (tG)• the temperature (T)• the pH value• the ternary eluent composition.

Overnight, these experiments canbe run automatically and unat-tended using the Shimadzu Lab-Solution software.

Over 30 years of solvophobictheory [1] makes the continuouschange in reversed-phase chro-matography selectivity under-standable. Retention forces areprimarily based on the highlyorganized water structure and onthe desire of water to reduce thecavity interfaces towards nonpo-lar molecules. Lipophobicity ofwater can be reduced by dilutionwith the strong eluent: MeOH orACN.

method are replaced with instant-ly generated chromatograms cor-responding to conditions that canbe selected individually.

DryLab is useful in almost allchromatography applications inthe lab. The software helps to• quickly find a good set of sepa-

ration conditions• develop complete method in

minimal time• evaluate method robustness• shorten run times• transfer methods to better and

more modern instruments suchas Shimadzu Nexera

• easily carry out validation ofyour methods

• find the best separation condi-tions for any component in amixture

• fine tune or troubleshoot exist-ing methods

• make the method fit to its pur-pose

• adjust methods for ageingcolumns.

The Cube

The cube represents over a mil-lion simulated chromatogramsand evaluates three parameters

With Drylab2010 it is now possi-ble to simultaneously optimizeseveral measured experimentalparameters (tG, T, pH or ternarycomposition of eluent B) with sixother derived factors (columnlength, -ID, dp, flowrate, dwellvolume, extra column volume) tofurther define the Design Space ofa separation. DryLab2010 usestwo-dimensional tG-T resolutionmaps [2]. By applying three ofthem, three-dimensional resolu-tion spaces can be created inwhich the combined influence ofthree parameters can be assessedand optimized.

DryLab applies QbD principlesince 20 years with a systematicevaluation of parameters influenc-ing method performance (selec-tivity, resolution).

DryLab simplifies and speeds upthe process of developing excel-lent chromatographic separationsby enabling users to model chan-ges in separation conditions usingtheir personal computer. Thetime-consuming laboratory runsthat are typically required toachieve a satisfactory separationor development of a complete

DryLab®2010 and Nexer

Fig. 1: Cube generate by DryLab2010 –

the read area describes the robust area

of the method

Figure 2: Shimadzu Nexera with

LCMS 2020

Figure 3: Interface of Peak Match with Shimadzu’s LabSolution software parameters

Method DevelopmentPharma Special

21

The following example of anoptimization of a separation ofsulfonamides and additionaldrugs underlines the power of theDryLab2010 and Nexera combi-nation.

Sulfonamides are the basis of sev-eral groups of drugs. The originalantibacterial sulfonamides aresynthetic antimicrobial agentscontaining the sulfonamidegroup.

Standard configuration, promi-nence with LPGE, column ovenand PDA detector.

Chemicals:A: Buffer 25 mM phosphoric acidB: Acetonitril

C: Buffer 25 mM Sodium Dihy-drogen Phosphate

D: MethanolColumn: Gemini-NX® Pheno-menex (150 x 4.6 mm, 3 μm)Flow rate: 1.00 mLWavelength: 280 nmTemperature: 40 °Celsius

Test mixture including followingsubstances (see table 1).

Optimization Step with DryLab 2010 3D Cube

Optimization on Nexera andShim-Pack XR column with PDAdetector

Flow rate: 1.30 mL/minTemperature: 48 °Celsius

cording to Quality by ra – a powerful combination

Figure 4: Experimental design for a three-dimensional HPLC method optimization Figure 5: Design Space Visualization with DryLab®2010. Each point in these 3D

resolution spaces corresponds to a highly accurate chromatogram

Figure 6: DryLab 3D model. The best chromatogram by one mouse click. Figure 7: Gradient Editor

Figure 8: Sulfonamide group

Wavelength: 280 nmColumn: Shim-pack XR-ODSIII (75 x 2 x 1.6 μ)Buffer A: 0.05 mol/L, pH = 2.3Potassium dihydrogen phosphate(KH2PO4)Buffer B: 50 % Methanol 50 %Acetonitril

Conclusion

With the support of the MolnarInstitute and the DryLab 2010software the group of drugs wasseparated from starting condi-tions with a conventional �

Method Development Pharma Special

22

HPLC and a runtime of more than 30 min by switching to a moderncolumn, here the Shim-pack XR ODS III (75 x 2 x 1.6 μ) and theUHPLC System Nexera, with binary gradient to a runtime of less thanthree minutes. The critical peak pair (peak 6 and peak 7) has a resolu-tion of 1.65; the pressure on the system is 1089 bar.

Quality by Design

The Quality by Design (QbD) concept was outlined by quality expertJoseph Juran (1904-2008), stating that quality can be planned, and thatmost quality problems relate to the way it was planned originally.

Molnár-Institute for applied chromatography

Founded in 1981, the Institute brings 30 years of experience in high perfor-

mance liquid chromatography (HPLC). Dr. Imre Molnár, former coworker of

Prof. Csaba Horváth from Yale and of Lloyd Snyder and John Dolan of LCRe-

sources USA, is a well known expert in HPLC method development.

The Molnár-Institute plays a small but essential role in the improvement of

worldwide healthcare, ensuring safe products in pharmaceutical, life science-

and food industries, while supporting research and development at universi-

ties. The Molnár-Institute continuously serves companies all over the world in

successfully designing and shaping HPLC method conditions. By offering soft-

ware solutions, courses and development services, the Molnár-Institute

defines its mission in applying modern software tools such as DryLab2010 to

increase efficiency in HPLC laborato-ries and to help to find the best separa-

tion as soon as possible.

The ideal state for pharmaceutical manufacturing in the 21st centurymean:• understanding the product and its production processes • understanding the methodologies (HPLC, etc.) which generate

data to support the product quality.

[1] Solvophobic Theory Cs. Horvath, W. Melander, I.Molnàr J. Chromatogr.

125 (1976) 129.

[2] Aspects of the “Design Space” in high pressure liquid chromato-graphy

method development; I.Molnàr; H.-J. Rieger, K. E: Monks J. Chromato-

gr. A, 1217 (2010) 3193-3200

5

1 280 nm 4 nm

mAU

10 15 20 25

0

50

100

150

200

Figure 9: Chromatogram of scouting run

Figure 10: 3D Cube from DryLab®2010 Figure 11: Prediction and Analytical Conditions with Shimadzu Nexera UHPLC

Table 1: List of compounds

Peak Name Formula

1

2

3

4

5

6

7

8

9

10

11

12

Acetaminophene

Sulfathiazole

Caffeine

Sulfadimidine

Sulfamerazine

Sulfamethoxypyridazine

Sulfamethoxazaole

Sulfafurazole

Sulfaquinoxaline

Propyhenazone

MPPH (5-(p-Methylphenyl)-5-phenylhydantoin)

Oxazepam (N-Ethyl)

C8H9NO2

C9H9N3O2S2

C8H10N4O2

C12H14N4O2S

C11H12N4O2S

C11H12N4O3S

C10H11N3O3S

C14H13N3O3S

C14H12N4O2S

C14H18N2O

C16H14N2O2

C15H11ClN2O2

In the different phases of drugdevelopment, metabolitesneed to be screened on a reg-

ular basis. In the early stagewhere the number of samplesscreened is still relatively low, thefocus is mainly on sensitivity.However, it shifts in general tohigher throughput with the

tion, resulting in narrow peakwidth, requires the acquisition ofenough data points over a peak tobe able to generate accurateinformation for quantification aswell as generating product ionscans for further confirmation. Inaddition to the required fast scanspeed the polarity switching is animportant feature as well toshorten run time without sacrific-ing the level of information gen-erated. The demand of low dwelland pause time may be importantif a large number of MRM transi-tions (Multiple Reaction Moni-toring) are relevant to screen. Inthis context, a low cross talkbetween the different MRMs isimportant to avoid contaminationbetween the different identifica-tion steps as well as an error inthe quantification.

Highest speed in its class

The LCMS-8030 has the highestspeed in its class, even in Multi-ple Reaction Monitoring modefor the triple quadrupole LC/MS/MS. In combination with the high performance NexeraUHPLC system with the lowestcarryover market-wide andfastest auto sampler, speed can bereached which is beyond compa-rison.

In order to minimize cross talk inthe collision cell which is a bott-leneck in high-speed analysisusing triple quadrupole LC/MS/MS, Shimadzu has developed itsunique UFsweeperTM collisioncell (patent pending). As a result,the LCMS-8030 can obtain sta-ble, highly-reliable data evenduring ultra fast measurement. �

demand for moderate sensitivityat a later stage.

In order to be able to fulfill thisdemand, the analytical solutionneeds to be reliable on the HPLCas well on the MS whereas a onehand supplier solution has a num-ber of advantages. Fast separa-

Speed beyond comparisonHigh speed drug screening and quantification

Nexera/LCMS-8030 a perfect combination of high resolution UHPLC

with MS/MS detection

Figure 1: The fast scan speed enables generation of product ion scans without the risk

of losing too many data points.

Drug TestingPharma Special

23

Atenolol

Procaine

Lidocaine

Atropine

Yohimbine

Chlorpheniramine

Propranolol

Alprenolol

Tetracaine

Diphenhydramine

Doxepin

Desipramine

Imipramine

Nortriptyline

Amitriptyline

Verapamil

Carbamazepine

Isopropylantipyrine

Alprazolam

Triazolam

Cilostazol

Nifedipine

Diazepam

Warfarin

Chloramphenicol

Nitrendipine

Compound MW Detection

266.16

236.15

234.17

289.17

354.19

274.12

259.16

249.17

264.18

255.16

279.16

266.18

280.19

263.17

277.18

454.28

236.09

230.14

308.08

342.04

369.22

346.12

284.07

308.10

322.01

360.13

Positive

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Negative

No

No

No

No

Yes

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

Yes

Yes

No

Yes

Yes

Yes

Drug Testing Pharma Special

24

Here up to 500 channels/sec arepossible with minimum dwelltime of 1 msec as well as a pausetime of 1 msec respectively.

Best conditions for a certaincompound are defined within10 minutes

The LCMS-8030 system enablesthe operator to optimize instru-ment parameters such as massaccuracy of the precursor ion andfragment ions as well as collisionenergy for compounds of interestby flow injection. The instrumentuses multiple injections of a stan-dard to optimize the individualinstrument parameters so that inless than 10 min the best condi-tions for a certain compound aredefined. This process can be setup easily for overnight runs sothat larger sample sets can also beconveniently used for optimiza-tion.

26 pharmaceutical compoundswere analyzed using Shimadzu’sSynchronized Survey Scan (SSS)shown in figure 1. In this mode,full scan measurement is rapidlyfollowed by automated production scanning. High-speed polarityswitching (15 msec) and rapidscan rates (15,000 u/sec) allowmultiple collision energies to beemployed for unknowns evenwith narrow peak widths. Thisenables molecular weight confir-mation from the Q3 scan dataand also generates structural frag-mentation information from thesame peak.

A total of 26 pharmaceuticalcompounds were evaluated. Figu-re 3 shows the analytical resultfor Cilostazol and Warfarin. Asillustrated in table 1, all com-pounds were detected in eitherpositive mode, negative mode orboth, demonstrating the LCMS-8030’s effectiveness for drugdiscovery and synthesis confirma-tion.

Chromatographic condition

Column: Shim-pack XR-ODSII(2.0 mm I.D. x 50 mm L.)Mobile phase A: 5 mmol/Lammonium formate-water

Figure 2: MS method using Synchronized

Survey Scan function

Mobile phase B: acetonitrileGradient program: 5 %B (0min) –> 95 %B (3-3.50 min) –> 5% B (3.51 - 5 min)

Table 1 : List of screened compounds

Figure 3: Analytical results (Cilostazol, Warfarin)

Flow rate: 0.3 mL/minInjection volume: 5 μL CDL temperature: 250 °CBlock heater temperature:400 °C

Nebulizing gas flow: 1.5 L/minDrying gas flow: 10 L/minColumn temperature: 40 °CCollision energy: pos -20V, -40V;neg 20V, 40VQ1/Q3 resolution: Unit/UnitScan type: Q3 scan – product ionscanScan range: m/z 200 - 500Ionization mode: ESI positiveand ESI negativeScan speed: 3333 u/sec

The Nexera/LCMS-8030 triplequadrupole solution enables set-ting up of screening experimentsin a very efficient way and standsout due to its highest flexibilityin pressure range, flow rate andscan speed paired with ease of useand maintenance.

Residual Solvents

Satisfy new method require-ments using the ShimadzuGC-2014 and HT200H

PResidual solvents are trace-level chemical residues indrug substances and drug

products that are byproducts ofmanufacturing, or that form dur-ing packaging and storage. It isthe drug manufacturers’ responsi-bility to ensure that these resid-ues are removed, or are presentonly in limited concentrations.

The United States Pharmacopeia(USP) recently revised GeneralChapter <467> on residual sol-vent analysis by adopting theInternational Committee on Har-monization (ICH) Q3C guide-lines. The new revisions alsoinclude analytical methods forthe identification, control andquantification of residual solventsthat are adopted from the Euro-pean Pharmacopoeia (EP). Thisrevision, effective July 1, 2008,replaces previous methods and

2, the method calls for a gaschromatograph analysis withflame ionization detection (FID)and a headspace injection fromeither water or organic diluent.The monograph has suggestedtwo procedures for qualitativeanalysis:

Procedure A specifies a G43(Zebron ZB-624 or equivalent)phase and Procedure B specifiesa G16 (Zebron ZB-WAXplus orequivalent) phase. Procedure Cis for quantitative analysis. Pro-cedure A should be used first.

If a compound above the speci-fied concentration limit is deter-mined, then Procedure B shouldbe used to confirm its identity.Since there are known co-elu-tions on both phases, the orthog-onal selectivity ensures that co-elutions on one phase will beresolved on the other. Neither ofthe procedures is quantitative, soto determine the concentration,the monograph specifies Proce-dure C, utilizing whichever phasegives the fewest co-elutions.

Procedure A: G43 (6 %-cyano-propy -94 % dimethylpolysilox-ane)Procedure B: G16 (polyethyleneglycol)Procedure C: G43 or G16depending on the procedure withthe fewest co-elutions

Class 3 solvents may be deter-mined by <731> Loss on Dryingunless the level is expected to be > 5000 ppm or 50 mg. If the losson drying is > 0.5 %, then awater determination should beperformed using <921> WaterDetermination.

USP monograph <467> allowsthe use of alternate methodolo-gies as long as they have been �

significantly increases the requi-rements with which a pharmaceu-tical company must comply inorder to demonstrate that alldrug products (not just new) arecompliant with Chapter <467>limits. The change increases thenumber of solvents requiringtesting from seven to fiftynine.

While most companies have ex-tensive data on the solvents usedin the manufacturing of theirAPI, the information regardingsolvents present in the excipientsis usually much lower. The drugproduct manufacturer is respon-sible for control of the limit ofsolvents.

Testing is only required for thosesolvents used in the manufactur-ing or purification process ofdrug substances, excipients orproducts. This allows each com-pany to determine which solventsit uses in production and todevelop testing proceduresaddressing their individual needs.

Solvents and health risks

Solvents have been classified intothree main categories based ontheir potential health risks:Class 1: Solvents should not be

used due to unacceptabletoxicities or deleteriousenvironmental effects.

Class 2: Solvents should be limit-ed because of inherenttoxicities.

Class 3: Solvents may be regardedas less toxic and of lowerrisk to human health.

Method overview

The USP has provided a methodfor the identification, control andquantification of residual sol-vents. For solvents of Class 1 and

U.S. Pharmacopoeia – revised General Chapter

Monograph <467>

Figure 1:

GC-2014 and

HT200H

Drug TestingPharma Special

25

Drug Testing Pharma Special

26

• signal-to-noise ratio of eachpeak of Class 1 solvent shouldbe > 3

• resolution between acetonitrileand methylene chloride > 1.0

• At the concentration limitsspecified by the monograph,signal-to-noise ratio for 1,1,1-trichloroethane was 59.9; andall other compounds exceeded3. Resolution between acetoni-trile and methylene chloridewas 1.71 (Figure 2).

Conditions

Gas chromatograph: ShimadzuGC-2010Injection: Split 5 :1 @ 140 °C, 1 mLCarrier gas: Helium @ 35 cm/s(constant linear velocity)Oven program: 40 °C for 20 min to 240 °C @ 10 °C/minfor 20 minDetector: FID @ 250 °CHT200H headspace: 60 minuteconditioning at 80 °C, syringe at85 °C, 0.5 min on/off shaker, 1 min flushColumn: Zebron ZB-624, 30 m x0.32 mm x 1.8 μm

Procedure B – Confirmation(Class 2 and 3 Solvents)

• System suitability requirements:signal-to-noise ratio of benzene> 5

• signal-to-noise ratio of eachpeak of Class 1 solvent shouldbe > 3

• resolution between acetonitrileand trichlorethylene chloride > 1.0

• At the concentration limits spe-cified by the monograph, sig-nal-to-noise ratio for benzene

However, if a company changesany of the procedures specifiedby <467>, the new method willneed to be fully validated. Onlythose solvents used in the manu-facturing process must be testedin the final dosage form.

For the best solution, each com-pany must consider the numberof samples, analysis time, methodvalidation, accuracy, precisionand cost of equipment. Oncemethod performance has beenachieved, it is also important toconsider whether the method canbe transferred to other manufac-turing facilities. Do they have theknowledge and instrumentationto implement the method?

The changes to the <467> mono-graph have applied since July 1,2008, and it is essential to formu-late a strategy now for compli-ance. During the process, manyquestions and concerns willundoubtedly arise. To ensure theUSP addresses as many of theseconcerns as possible in the newmethod, an open dialog betweenindustry and the USP is criticial.

The combination of a ShimadzuGC-2010 with an HT200Hheadspace autosampler is a cost-effective solution to meet theneeds of static headspace injec-tion with GC analysis.

was 104.2; and all other com-pounds exceeded 3. Resolutionbetween acetonitrile and tri-chloroethylene chloride was1.52 (Figure 3).

Conditions

Gas chromatograph: ShimadzuGC-2010Injection: Split 5 : 1 @ 140 °C, 1 mLCarrier gas: Helium @ 35 cm/s(constant linear velocity)Oven program: 50 °C hold 20 min to 165 °C @ 6 °C/minhold 20 minDetector: FID @ 250 °CHT200H headspace: 60 minuteconditioning at 80 °C, syringe at85 °C, 0.5 min on/off shaker, 1 min flushColumn: Zebron ZB-WAXplus,30 m x 0.32 mm x 0.25 μm

Conditions

All pharmaceutical companiesneed to determine as soon as pos-sible how the changes to GeneralChapter <467> will impact theirtesting procedures. The moreexcipients and excipient vendorsa company uses, the more effortit will require to demonstratecompliance with the newmethodology.

The new USP regulations areaimed at improving patient safetyand will need to be implementedfor all products, whether existingor new. Although the USP hasprovided a testing method thatcan be used to identify and quan-titate most Class 1 and 2 solvents,the method can be improvedbased on each companies needs.

validated appropriately. However,only the results obtained by theprocedures given in the generalchapter are conclusive. So, theresults from the alternate methodwill have to be compared to themonograph before being accept-able to the Food and Drug Ad-ministration (FDA).

Instrumentation

All procedures were performedusing a Shimadzu GC-2010, anHT200H headspace autosampler(Figure 1) and Phenomenex GCcolumns.

The Shimadzu GC-2010 offershigh-end GC performance readyfor all analytical tasks includingfull fast GC functionality withfilter time constant and samplingfrequency of 4 ms and 250 Hzrespectively for all GC detectors.

The HT200H headspace auto-sampler has been specially de-signed to meet the needs of Gen-eral Chapter <467>. The sensitiv-ity and reproducibility have beenexhaustively evaluated at multiplelocations around the world toensure consistent method. Thesampler has also demonstratedperformance characteristics fordrug substances, excipients anddrug products. All show extreme-ly consistent data.

Procedure A – Identification(Class 1 and 2 Solvents)

• System suitability requirements:signal-to-noise ratio of 1,1,1-trichloroethane > 5

Figure 2: USP Method <467> Procedure A: Class 2 mix A for water

soluble compounds

Figure 3: USP Method <467> Procedure B: Class 2 mix A for water

soluble compounds

0.0

1.0

2.0

3.0

4.0

5.0

6.0

uV (x 100,000)

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5

0.0

1.0

2.0

3.0

4.0

5.0uV (x 10,000)

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

MinutesClass 2 mix AClass 2 mix A

Minutes

Extrusion testing made easy

Tablets, capsules, sera, oint-ments, aerosols – pharma-ceutical products must be

thoroughly and securely pack-aged in order to prevent damageduring the route from productionto patient. Depending on thedosage form, packagings are man-ufactured from various materials,equipped with specific function-alities and are available in variousforms.

The packagings are regularly tes-ted. One of these tests measuresthe press-through strength ofblister contents, which caninclude for instance tablets.

During the test, the sample ispushed through the packagingusing a constant force or speed.The packaging lies on a smooth,flat surface containing a holethrough which the sample can bepushed. Whether or not the pack-aging film ruptures evenly is test-ed. At the same time it can bechecked whether the blister pack-aging can withstand the variousstorage and transport conditionsundamaged. The test also deter-

cells and accessories, testingmachines in the EZ-Test modelseries are particularly versatileand convenient.

A distinct feature is the compre-hensive Trapezium-X softwarethat enables exceptionally flexibledata reporting and features amultitude of measuring modes.An intuitive user-friendly methodassistant supports the selection ofparameters. Versatile statisticalfunctions as well as control cardsystems complete the newTrapezium-X software.

mines whether the foil or thepackaging ruptures, in order toavoid possible cut injuries.

Two force values are determinedduring this test. The first maxi-mum value is the force needed torupture the packaging film. Thesecond, increasing value indicatesthe force needed to push thetablet out of the packaging. Thiscorresponds effectively with thetear propagation test on the film.

Shimadzu’s universal testingmachines are highly suitable forthese extrusion tests. Based ontheir compact dimensions andstraightforward exchange of load

Testing machine for blister packaging

EZ-Test universal testing machine.

Figure 1: Force-stroke diagram of an extrusion test of a blister packaging

Figure 2: Force-distance diagram of an extrusion test of a blister packaging

Figure 2: Trapezium-X software

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0

Stroke (mm)

Forc

e (N

)

0

2

4

6

8

10

12

14

16

18

20

22

24

25

Chemistry / Manufacturing / ControlPharma Special

27

Chemistry / Manufacturing / Control Pharma Special

28

Big potential with protein based drugsQuality control of recombinant proteins by N-terminal

Edman sequencing

The future of pharmacy willbe a special category oftherapeutics and diagnos-

tics referred to as protein-baseddrugs. To date, modern medicinehas relied heavily on syntheticallyor chemically produced drugs totreat and prevent ailments and dis-eases.

However, developments in mole-cular biology have led to an in-crease in knowledge of biologicalsystems and their interactions. Forexample, scientists now knowmore about the sources of manydiseases and how the human bodyfights them. They now focus onusing the body's own tools fordeveloping therapeutics that mimicthe actions of the body’s arsenal.Today, advances in biotechnologyhave led to increased use of livingorganisms and biological sub-stances in the production of pro-tein drugs for human health care.Specifically, the use of recombi-nant DNA (rDNA) technologyhas enabled the production oflarge quantities of protein drugs.

Today, several types of proteinbased-drugs are available, forexample:1) Cytokines

These drugs regulate the im-

of the protein or having a higherlevel of impurities in the sample.The method of choice is still theN-terminal sequencing method orso-called Edman Sequencing.

Edman degradation, developed byPehr Edman, is a method of se-quencing amino acids in a peptideor protein. In this method, theamino-terminal residue is labeledand cleaved from the peptide with-out disrupting other peptide bondsbetween other amino acid residues.Phenyl isothiocyanate is reactedwith uncharged terminal aminogroup under mildly alkaline condi-tions to form a phenylthiocar-bamoyl derivative. This derivativeof the terminal amino acid is thencleaved under acidic conditions asa thiazolinone derivative. The thiazolinone amino acid is thenselectively extracted into an organ-

mune system. They are proteinswhich activate the immune sys-tem cells to carry out differentimmune functions.

2) HormonesProtein drugs that regulatefunctions in the body. As drugs,these proteins can be used toelevate levels of specific hor-mones, such as estrogen duringmenopause or growth deficien-cy. They can also be used totreat certain diseases such asdiabetes, or conditions such asinfertility.

3) Clotting factorsProteins that regulate the clot-ting of blood. These drugs areused to treat blood clotting dis-orders such as hemophilia.

4) VaccinesProteins that stimulate theimmune system to produce specific antibodies used to prevent or treat diseases.

5) Monoclonal AntibodiesProteins that mark a specificforeign material (such as cancercells, disease-causing bacteria orviruses) for removal or destruc-tion by other components ofthe immune system. These arealso used as effective diagnostictools for many specific geneticdiseases and other conditionssuch as pregnancy.

The first substance of this kindapproved for therapeutic use wasbiosynthetic ‘human’ insulin madevia recombinant DNA technology.

Edman degradation forsequencing amino acids

Protein production and processoptimization needs good qualitycontrol in order to avoid condi-tions producing truncated versions

ic solvent and treated with acid toform the more stable phenylthio-hydantoin (PTH)-amino acidderivative that can be identifiedusing chromatography and com-parison with standards. This pro-cedure can be repeated to identifythe next amino acid.

Although the method was estab-lished more than 40 years ago it isstill used throughout differentstages of drug discovery or todemonstrate comparability andconsistency between batches forrelease during manufacturing. InEurope, different regulations ofthe European Medicines Agency(EMEA) need to be followed to characterize protein-basedproducts, e.g.• Guideline on Development, Pro-

duction, Characterization andSpecifications for Monoclonal �

Shimadzu has more than 20 years of

experience in the field of Edman sequen-

cing and has recently released the latest

product PPSQ 31/33A for the European

market

Table 1: Reproducibility of retention time of the different amino acids in comparison

to 1/7/15 days

Asp

Glu

Asn

Gln

Ser

Thr

His

Gly

Ala

Tyr

Arg

Met

Val

Pro

Trp

Phe

Lys

Ile

Leu

Day 1

2.45 min

3.14

3.84

4.06

4.25

4.61

4.90

5.26

6.65

7.04

7.66

11.44

11.79

12.22

14.43

16.49

17.03

17.58

18.99

Day 7

2.44 min

3.15

3.83

4.05

4.25

4.60

4.89

5.26

6.65

7.04

7.65

11.45

11.79

12.22

14.44

16.50

17.04

17.59

19.01

Day 15

2.44 min

3.15

3.83

4.05

4.25

4.60

4.90

5.26

6.66

7.05

7.65

11.47

11.82

12.25

14.49

16.55

17.11

17.64

19.06

PTH-amino acid Elution time

Antibodies and Related Products.(EMEA/CHMP/BWP/157653/2007)• Guideline on similar biological

medicinal products (EMEA/CHMP/437/04)

• Guideline on similar biologicalmedicinal products containingbiotechnology-derived proteinsas active substances: QualityIssues (EMEA/CHMP/BWP/49348/2005)

• Guideline on similar biologicalmedicinal products containingbiotechnology-derived proteinsas active substances: Non-clinicaland Clinical Issues (EMEA/CHMP/BMWP/42832/2005).

Analysis efficiency with single- and triple-reactor type sequencers

The robustness of the Edmansequencing method in routineenvironments such as quality con-trol, the possibility to correctly

determine isobaric amino acids(Leu / Ile) and acquiring of quanti-tative information from the prod-uct as well as from possible impu-rities makes it still the method ofchoice. Even where mass spectro-metry has replaced Edman degra-dation in the field of protein iden-tification, it cannot reliably answerall questions and is more difficultto interpret. Edman degradation istherefore also used for discoveryof de novo sequencing of newnovel proteins or peptides.

The PPSQ 31A/33A was releasedin Europe in 2009, based on thewell-established prominenceHPLC modules. The PPSQ 31 Ais a single-reactor type and thePPSQ 33A is a triple-reactor typepermitting sequential automatedanalysis of three samples to en-hance analysis efficiency.

PPSQ series protein sequencersachieve baseline stability and allow

high-sensitivity analysis of PTH-amino acids by separating themisocratically. Isocratic sequenceanalysis provides more stableretention times (Table 1). Peaksdetected in previous cycles canthen be cancelled using substationchromatogram processing, makingit easier for users to identifysequences. Performing PTH-amino acid analysis in isocraticmode makes it possible for labora-tories to reduce liquid waste andrunning costs. Reagents and sepa-ration column are available fromWako and enable further reduc-tions in operating costs(www.wako-chemicals.de).

New software significantlyimproves data analysis

In addition, the newly developedsequencer software providesimportant chromatogram repro-cessing for determination ofamino-acid sequences. This soft-

ware processes multiple chro-matograms aggregated by sample,thereby significantly improvingdata analysis functionality. Fur-thermore, by adopting the latestHPLC analysis system, noise lev-els are reduced, enabling high sen-sitivity detection of PTH-aminoacids.

PPSQ’s capability was demon-strated impressively in the analysisof an unknown protein sample,which was correctly sequenced upto 58 cycles starting with approx.200 pmol sample and up to 40cycles with approx. 40 pmol sam-ple material.

Figure 1: Chromatogram of standard amino acids

25 pmol PTH-amino acid standard analysis

Figure 2a: Cycle 2, amino acid Val

Figure 2b: Cycle 3, amino acid Gln

Figure 2c: Cycle 21, amino acid Leu

Figure 2a, 2b, 2c: Results of amino acids eluting in different cleavage cycles. The negative signal peaks result from the automatic substraction function, helping to identify rising

signals in the next cycle.

Chemistry / Manufacturing / ControlPharma Special

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Chemistry / Manufacturing / Control Pharma Special

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Today, the determination oftoxic inorganic analytes(such as metal residues in

pharmaceutical products) is atypical method for highly advan-ced instruments, such as induc-tively coupled plasma opticalemission spectrometers (ICP-OES). This technology enablesextremely accurate, quantifiableand reproducible analyses, and ahigh sample throughput.

Metal residues in pharmaceuticalsubstances or drug products mayoriginate from several sources,e.g. metal catalysts and metalreagents used during synthesis ofthe active pharmaceutical sub-stance and the excipients, manu-facturing equipment and piping,bulk packaging, the environment,cleaning solvents etc. Since metalresidues do not provide any ther-apeutic benefit to the patient, andproduct risk should commensu-rate with the level of productbenefit, the specification of apharmaceutical substance or thedrug product may need to inclu-de a limit and validated methodfor metal residues to guaranteeacceptable product quality.

Dedicated guidelines forresidues of metal catalysts

For this reason, the EuropeanMedicines Agency (EMEA), aLondon located decentralizedagency of the European Union,has been defining dedicatedguidelines, such as the guidelineon specification limits for resid-ues of metal catalysts or metalreagents in pharmaceuticals(EMEA/CHMP/SWP/4446/2000).The objective of this guideline is

to recommend maximum accept-able concentration limits of metalresidues arising from the use ofmetal catalysts or metal reagentsin the synthesis of drug substan-ces and excipients.

Since the use of these metals isrestricted to defined chemicalreactions, limitation of theirresidues in pharmaceutical sub-stances will normally be suffi-cient. Limitation of these metalresidues in the final drug productwill normally not be necessary.

The concentration limits in thisguideline are based on safety cri-teria and assure an adequate qual-ity of the pharmaceutical sub-stance and the drug product. It istherefore not considered appro-priate to expect that the pharma-ceutical industry tightens the

concentration limits in the regula-tory dossier based on GMP, pro-cess capabilities or any otherquality criteria. Since the originof metal residues is irrelevantregarding their potential toxiceffects, the concentration limits inthis guideline are in principle alsoapplicable to residues from sour-ces other than catalysts andreagents.

However, for these other sourcesadoption of a concentration limitand a validated method in thespecification is only necessary inthe very exceptional cases wherethese residues are known to beinsufficiently limited by GMP,GDP or any other relevant provi-sion. Pharmaceutical companiesare not expected to perform ex-tensive tests on metal residuefindings of unknown sources in

order to comply with this guide-line. They may rely on generalinformation from trustworthysuppliers.

The metals currently included inthis guideline are listed in table 1.The guideline may be updated toinclude other metal residues indue course. Any interested partycan submit relevant safety data.The classification and concentra-tion limits of the currently in-cluded metals may also changewhen new safety data becomesavailable.

EMEA guidelines

Table 1 shows the permissibleamounts and concentrations ac-cording to the guidelines issuedby the EMEA on February 21,2008. Based on risk to human

Analysis of residual catalysts in pharmaceuticalsICP-OES technology quantifies 14 elements

according to EMEA guidelines

Figure 1: ICPE-9000 simultaneous inductively coupled plasma optical emission spectrometer with CCD detector

health, these guidelines classified14 types of metals into threeclasses. Class 1 was further divid-ed into three sub-categories dueto concerns over potential car-cinogenic or other serious toxici-ty issues in humans. The permis-sible limits adressed the threeadministration pathways of oral,parenteral or inhalation exposurefor each class. Permissible levelswere based on the Guideline forResidual Solvents (ICH Q3C) 4.As the maximum permitted valuefor pharmaceuticals, the Permit-ted Daily Exposure (PDE, unitsμg/day) was used. The permissi-ble concentration values werebased on computing of the PDE,assuming a 10 g/day dosage ofthe pharmaceutical. Where dailydosage exceeds 10 g, the permissi-ble value was computed basedupon content of the active ingre-dients in the pharmaceutical.

Experimental work

The 14 elements of the EMEAguidelines were measured asresidual metal catalysts in thepharmaceutical substances.Reagents commercially availablefor research purposes were usedas the samples. For conveniencein these tests, an organic solventcontaining little contaminationwas selected for pretreatment ofthe test samples, rather thanresorting to acid digestion (thechoice in this case being DMSOdue to its high solubility).

0.5 g of each test sample wasweighed and dissolved in DMSO

spectrometry is an ideal methodfor analyzing residual catalysts inpharmaceuticals.

References:

Director of the Evaluation and Li-

censing Division, the Pharmaceutical

and Food Safety Bureau (Iyaku

Shokuhinkyoku Shinsa Kanrika), the

Ministry of Health, Labour and Wel-

fare: Revised Guidelines on Impuri-

ties in New Drug Substances, No.

1216001, issued by the Evaluation

and Licensing Division, the Pharma-

ceutical and Food Safety Bureau

(Iyaku Shokuhinkyoku Shinsa Kanri-

ka) December 16, 2002.

Committee for Medical Products for

Human Use (CHMP): Guideline on

the Specification Limits for Residues

of Metal Catalysts or Metal Reagent

(Doc. Ref. EMEA/CHMP/SWP/

4446/2000).

London (February 21, 2008)

USP Ad Hoc Advisory Panel on Inor-

ganic Impurities and Heavy Metals

and USP Staff: “General Chapter on

Inorganic Impurities: Heavy Metals,”

stimuli to the revision process,

Pharmacopeia Forum 34 (5), 1345 -

1348 (Sept.-Oct. 2008)

International Conference on Harmo-

nization of Technical Requirements

for Registration of Pharmaceuticals

for Human Use: ICH Harmonised

Tripartite Guideline, Impurities:

Guideline for Residual Solvents

Q3C(R3). Step 4 version

than 1 % for all elements. Fur-thermore, the addition and recov-ery samples to which the tosu-floxacin tosilate was added weresubjected to repeated testing overa three-day period in a repro-ducibility test. The results areshown in table 2.

In these reproducibility tests, acalibration curve was preparedeach time in performing thequantitative analysis. Very goodreproducibility was obtainedwith each element, with the 3-dayRSD being about 1 %.

Summary

In this paper, an overview of theICP optical emission spectrome-try was introduced and exampleswere presented using the ICPE-9000 to quantify 14 elements inpharmaceutical substances identi-fied under EMEA guidelines, as amethod for the analysis of residu-al catalysts in pharmaceuticals.The Japanese Pharmacopoeia(15th Revision) currently listsonly the colorimetric method, atest method for heavy metals, andatomic absorption spectrometryas analytical methods for metalsin pharmaceuticals. No othermetal analysis methods have beenadopted.

However, when reviewing thetesting for metals in pharmaceuti-cals that has been conducted on aglobal scale in recent years, it iscertain that the ICP analyticalmethod will be essential for theanalysis of pharmaceuticals. Asdemonstrated in this introduc-tion, the ICP optical emission

to bring the volume to 5 mL (a10-fold dilution). At this time,yttrium (Y) was added as aninternal standard element to pro-duce a Y concentration of 0.1 μg/mL, and a standard sample oftosufloxacin tosilate was added toa dilution solution to obtain atosufloxacin tosilate concentra-tion of 1 μg/mL. This was usedfor addition and for recoverytesting. The solutions used tomake the calibration curves werestandard single-element solutionsdiluted with DMSO solvent. Inpreparing these calibration solu-tions, Y was also added as aninternal standard element to pro-duce a Y concentration of 0.1 μg/mL.

1 μg/mL concentration solutionswere used to prepare calibrationcurves and changes over time andwere measured continuously overtwo hours. Good results wereobtained; the RSD (relative stan-dard deviation) over the twohours of measurement was less

Class 1A: Pt, Pd

Class 1B: Ir, Rh, Ru, Os

Class 1C: Mo, Ni, Cr, V

Metals of significant safety

concern

Class 2: Cu, Mn

Metals with low safety

concern

Class 3: Fe, Zn

Metals with minimal safety

concern

PDE (μg/day)

100

100

250

2,500

13,000

Conc. (ppm)

10

10

25

250

1,300

PDE (μg/day)

10

10

25

250

13,000

Conc. (ppm)

1

1

2.5

25

130

PDE (μg/day)

Pt: 70

Ni: 100, Cr (VI): 10

Classification Oral Exposure Parenteral Exposure Inhalation Exposure

Table 1: Class Exposure and Concentration Limits for Individual Metal Catalysts and Metal Reagents (PDE: permitted daily exposure)

Table 2: Reproducibility Results over 3 Days (units μg/mL)

Element 1st Day 2nd Day 3rd Day RSD (%)

Cr

CU

Fe

Lr

Mn

Mo

Ni

Os

Pd

Pt

Rh

Ru

V

Zn

1.00

0.99

1.42

0.98

0.99

1.01

0.99

1.00

0.98

1.00

0.98

0.98

1.00

0.99

1.00

1.00

1.43

0.98

0.99

1.01

0.99

1.00

0.98

1.00

0.97

0.98

1.00

0.98

1.02

0.99

1.43

0.97

1.00

1.01

0.99

1.01

0.99

1.02

0.97

1.00

1.00

0.97

1.18

0.46

0.19

0.60

0.89

0.26

0.24

0.66

0.85

0.96

0.55

1.35

0.23

0.87

Chemistry / Manufacturing / ControlPharma Special

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Chemistry / Manufacturing / Control Pharma Special

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Cleanliness is an importantissue in the productionof any type of drug, since

highest purity and careful han-dling of substances and activesubstances are essential in thepharmaceutical industry. The ba-sic request is the effective remo-val of residual products incurredduring production. A clean plantreduces contamination of themedicine. When using a batchprocess, subsequent cleaning isparticularly important due to theproduction of several drugs in thesame plant. Contamination bythe previous product must beprevented. Cleaning validationensures the efficiency of thecleaning process.

Cleaning validation is not a newissue. In 1963, the Good Manu-facturing Practice (GMP) alreadydeclared:

„Equipment shall be maintainedin a clean and orderly manner“(GMP Reg../FDA Part 133.4). A similar section on equipmentcleaning was included in the 1978CGMP regulations.

ly without dismounting the plant.In this case, the plant has to havea CIP-design (rinsing systemwith recycling possibility and nodead volumes) which usuallyrequires a high investment.

On the other hand, optimizing oftime, temperature, cleaning agentand solvents is possible, resultingin very effective cleaning. Theautomatic rinsing enables a stan-dardized and validated procedure.

2. COP (Cleaning Out of Place)With COP, the complete plantneeds to be dismantled and allcomponents cleaned individually.This procedure requires moretime and staff resources. Due tothe individual cleaning, standardi-zation and validation is more dif-ficult but low investment and theopportunity to inspect all compo-nents visually are among the ben-efits.

Sampling and analytics

Depending on the cleaning pro-cess, several facilities can be usedfor sampling to check the clean-ing efficiency. The easiest andfastest way is the analysis of thefinal rinse solution. Dependingon the solvent used, subsequentanalysis can be carried out withHPLC or TOC.

The swab method is applied totest the surface of the productioncontainer. In this way, areas canbe evaluated, which are difficultto clean but reasonably accessibleand the level of contamination ofresidues per given surface can bedetermined. In addition, residuesthat are “dried out” or insolublecan be sampled by physical re-moval. Traditionally, the swabsare subsequently extracted andthe resulting solution is furtheranalyzed. However, use of aTOC-solid module with glass-

The implication of cleaning vali-dation has lately been increasing-ly significant since the pharma-ceutical industry develops moreand more active substances whichare effective even in low concen-trations. Proof of contaminationneeds to be validated by analyticmethods which are sensitiveenough to define accepted limitsof residues. A typical criterion is10 mg/L or 1/1,000 of therapeuti-cal doses of the medication.

Based on the large variety ofpharmaceutical drugs, differentanalytical methods are applied inthe analysis of residues. In partic-ular, HPLC and TOC analysishave been proven to demonstratethe effectiveness of the rinsingprocess.

Cleaning process

Selection of the right cleaningprocess is the decisive factor ineffectively cleaning the plant.Two processes are normally used.

1. CIP (Clean In Place)The cleaning is done automatical-

Figure 1: Sampling with swabs

How cleaning validation p

Chemistry / Manufacturing / ControlPharma Special

33

fibre swabs free of any carbonenables direct analysis of theswabs.

TOC-Analysis

Total Organic Carbon measuresthe carbon content of organicmatter present in different matri-ces. The carbon content of thesample is oxidized to carbondioxide and detected with aNDIR-detector. Water samplescan be measured easily and veryquickly. (Typical measurementtime is four minutes).

In cleaning validation, TOC-values cover contamination of thepre-product and the remainingrinsing solution. If this result isbelow the defined limit, furtheranalysis is unnecessary. However,if the result exceeds the limit,HPLC-analysis is needed toidentify the source of contamina-tion.

Shimadzu’s modular TOC-Lseries simplifies TOC-analysis,

Figure 2: Calibration curve of TOC-solid module up to 50 μg

regardless of how samples of finalrinse or swab method are meas-ured. The TOC-L is based on theproven technologies of catalyticcombustion (680 °C) and NDIR-detection. In this way, it is possi-ble to detect low molecular massacids and invisible particles. Dueto its measuring range of up to30,000 mg/L (detection limit: 4 μg/L), the instrument is notonly suitable for the low rangebut also for analysis of highlycontaminated samples, for exam-ple during the cleaning validationprocess.

TNM (Total Nitrogen Measure-ment) is an analytical techniqueused for the determination oftotal water-borne nitrogen (TN)by means of oxidative combus-tion chemiluminescence. Bothcombustion tube and oxidationcatalyst are shared with TOC(Total Organic Carbon) analysis.The well-known TOC applica-tions used for Cleaning Valida-tion purposes are therefore com-plemented by the determination

Conclusion

Cleaning validation is an essentialpart of good manufacturing prac-tice, avoiding cross contamina-tion in the pharmaceutical pro-duction process. Fundamentalcomponents of cleaning valida-tion are the analytical methodsapplied. Regardless of the type ofcleaning procedure, Shimadzu’sinstrumentation and softwaresupports the examination ofcleaning validation samples withTOC. Specific software toolshelp to perform the partly com-plex analysis and support docu-mentation and archiving of theanalysis results.

of nitrogen in the related sam-ples. This enhances the analyticalinformation obtained from thecleaning validation samples, inparticular proteins and aminoacids since nitrogen is an essentialpart of these compounds.

Since the number of insolublecompounds in the pharmaceuticalindustry is increasing, directdetermination of swabs with theTOC-solid module has beengaining more significance. In thiscase, a carbon-free glass-fibreswab is wetted with purifiedwater and the defined surface isswiped according to a prescribedprocedure. The swab is then fold-ed and placed in a clean ceramicboat and inserted in the TOC-solid module. The system config-uration and calibration curve areselected depending on the expect-ed concentration or the definedlimiting value. The calculated car-bon content now correspondsdirectly to the area of theswabbed surface.

Figure 3: Calibration curve of TOC-solid module up to 250 μg/L

prevents contamination

Chemistry / Manufacturing / Control Pharma Special

34

Figure 1: Microsampling method on AA-7000F

Determination of sodium, potassium and calcium Analysis of Na, K and Ca with flame atomic

absorption spectrometry in microsampling mode

Determination of elementsin pharmaceutical prod-ucts such as crystalloid

solutions typically applies atomicabsorption spectrophotometerssuch as AA-7000. Concentrationsof sodium (Na), potassium (K)and calcium (Ca) are the same asin the body fluids/extracellularfluids and need to be monitoredcarefully.

Crystalloid solutions are rapidlyexcreted by the kidneys andappear virtually unchanged. Fur-thermore, the solutions are suit-able for use in dissolution andparenteral administration ofdrugs. Upon administration thecrystalloid solutions are rapidlyeliminated. They have therefore

The right choice: the flamemicro sampling method

When compared with the flamecontinuous method, flame microsampling has several advantages:the analysis is possible with asmall amount of sample, andwhen the autosampler is used,automatic dilution of the sampleand automatic addition of buffersolutions are possible in order tocompensate for interferences.Moreover, since only a smallamount of sample is introduced,the flame micro sampling methodis effective for analysis of highmatrix samples which may causeclogging of the burner in theflame continuous method.

The method is the right choicefor determination of alkaline andalkaline earth elements in crystal-loid solutions.

Sodium, potassium, and calciumbelong to the essential mineralsubstances in the human organ-ism. These elements are influen-tial in the generation of enzymesand hormones, control osmoticpressure in tissues and body flu-ids and are important for theexchange procedures in the cellmembranes.[1]

Particularly during surgery,severe loss of extracellular fluids(interstitial fluid and blood) iscritical and must be compensatedby crystalloid solutions having asimilar composition to the extra-cellular fluids. Blood losses mayalso be compensated by thesesolutions provided the quantityof the loss is not too critical.

proven to be the solutions ofchoice for administration ofdrugs. Table 1 shows typical ele-ment concentrations of crystal-loid solutions.

The AA-7000 combined with theASC-7000 sample preparationstation enables the automatedflame micro sampling method(Figure 1). In this method, flameatomic absorption analysis isconducted with small sample vol-umes (2 - 90 μL), while in theconventional flame method (here-after “flame continuous method”),the sample is aspirated continu-ously with a flow rate of approxi-mately 8 mL/min and larger sam-ple volumes are needed for aspi-ration.

Chemistry / Manufacturing / ControlPharma Special

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Table 1: Typical example of a full electrolyte solution/crystalloid solution

Reliable determination of elements in high matrixsamples

Control of Na, K, and Ca incrystalloid solutions according tothe European Pharmacopoeia hasbeen performed with the Shi-madzu atomic absorption spec-trophotometer AA-7000 in a ful-ly automatic multi elementsequence. The blank, standardsand the test samples have beenanalyzed in the direct calibrationmethod.

All solutions were placed in theautosampler ASC-7000 andmixed automatically with the

Electrolyte concentration in mmol / L

Sodium

147

Potassium

4

Calcium

2.3

Chloride

155.5

Instrument

Autosampler ASC-7000 incl. microsampling kit

AA-7000

Measurement element

Wavelength

Slit width

Lamp current

Backgroundcorrection

Flame type

Gas flow rate

Na

589.0 nm

0.2 nm

8/600 mA

SR

Air-C2-H2

1.8 l/min

K

766.5 nm

0.7 nm

8/600 mA

SR

Air-C2-H2

2.0 l/min

Ca

422.7 nm

0.7 nm

10 mA

D2

Air-C2-H2

2.0 l/min

Table 2: Instrument and Analytical Conditions

corresponding reagents necessaryto achieve reliable results. In thecase of sodium and potassium 40 μL of CsCl-solution (12.65 gCsCl + 50 mL HCl (d = 1.16)filled up to 500 mL volume withH2O) was added for a 400 μLmixing volume of standard andsample solution, homogenizedbefore injection to the flame. Inthe case of calcium, a La2O3-solution (5.875 g La2O3 + 50 mLHCl (d = 1.12) filled up to 250mL volume with H2O ) was used.

Instrumental parameters andmeasuring conditions were setelement-specific from the systemsoftware. These conditions are

automatically set for each ele-ment including optimized burnerheight and gas flow rates and arelisted in table 2.

Under these conditions a series ofcrystalloid solutions has beenanalyzed to demonstrate that theAA-7000 is an effective tool forthe reliable determination of ele-ments in high matrix samplesduring routine analysis.

[1] Mineralstoffe und Spurenele-

mente, Leitfaden für die ärztliche

Praxis, Bertelsmann, 1992

UV-1800

Quality control of special pharmaceutical organicsIdentification with UV spectroscopy

The European Pharmacopo-eia guideline for instru-ments details adjustment

parameters and promotes a com-mon understanding of their per-formance. Up to nine checks aredone to calibrate and validate aninstrument.

The most important requirementof scanning UV instruments istheir capability to perform 1 nmresolution. For the standardrequirements of the Pharmaco-poeia an instrument such as theShimadzu UV-1800 is a goodsolution. This double beam spec-trophotometer is equipped with aslit of 1 nm. The proof of per-formance is the signal resolutionof a mixture prepared from Hex-

ane and Toluene. The instrumentapplies all Shimadzu and Pharma-copoeia validation criteria. Itautomatically integrates valida-tion in one level and automatedtests with certified standards inanother level.

245.06

nm

Abs

.

246.00 247.00 248.00 249.00 249.040.173

0.180

0.200

0.220

0.241

Chemistry / Manufacturing / Control Pharma Special

36

The UV-1800 is a recommendedsystem because of its stability inbaseline and the accuracy of eachdata point over the completemeasurement range.

This system can be used for sim-ple quality control of pharmaceu-tical substances according to therequirements of EP, USP andFDA.

Some applications needing muchhigher resolution are also ofinterest. In these cases a high-per-forming instrument is required, inparticular when the analysis shallbe performed with a resolution oflower than 1nm.

An example of such a high-reso-lution request is the measurementof Gadodiamide in UV range incombination with a ShimadzuUV-2550 spectrophotometer.

The analysis

Gadodiamide (known under thebrand name of Omniscan, for

265

nm

270 2750

0.1

0.2

0.3

0.5

0.4

Figure 1: Graph-hexane/toluene resolution

Figure 3: Zoom into the region of interest. The dark green line has 0.5 nm resolution

and the red one 0.2 nm slit selection. The signal developed by the 0.2 nm slit is much

higher and sharper. Identification of the signal is much more accurate. The light green

spectrum has 1 nm resolution, showing the difficulty of detection.

Figure 2 and 2a: Spectra of Gadodiamide in the UV-range with automated peak detec-

tion; the spectra were measured with different resolution; the red spectrum was with

0.000

240.00

nm

Abs

.

260.00 280.00 300.00-0.028

0.200

0.400

0.593

Chemistry / Manufacturing / ControlPharma Special

37

example) is used as a magneticresonance (MR) contrast medi-um. Gadodiamide is used as anindicator for the neuro part orwhole body of a scan. From achemical point of view, this sub-stance is a chelate complex whichis a molecular structure of nega-tively charged groups surround-ing a metallic ion. In this case, itis the Gd (Gadolinium) ion. Thesubstance is also explained in thefollowing formula as Gd-DTPA-BMA. The characteristic is a lin-ear chelate, uncharged and withlow osmolarity.

For the identification high reso-lution is needed for the signalshape of position 247.3 and 253.1nm.

In figure 2 the spectrum with 0.2 nm is shown on the left side,and the same substance with0.5nm resolution is shown on theright side. Automatic determina-tion of peak position can verifyboth signals of interest only withhigher resolution out of the high

Instrument Properties

Instrument Type: UV-2550Measuring Mode: AbsorbanceSlit Width: 0.2 nmLight Source Change Wave-length: 360 nmS/R Exchange: Normal

*Sources:

1: http://chembank.med.

harvard.edu

2: Wikipedia

basic absorption at the area from260 to 240 nm. The signal at 247 nm disappears into a shoul-der when the measurement isdone with 1 nm resolution. Anautomated detection is impossibleand manual detection becomesdifficult. It will result in timeconsuming activities of spectrummanipulation.

The UV-2550 instrument wasapplied. The spectra generatedwere measured in the absorbancemode.

Following parameters were investigated under high resolu-tion measurement with the UV-2550:

Measurement Properties

Wavelength Range:240 to 300 nmScan Speed: Very SlowSampling Interval: 0.1 nmScan Mode: SinglePath Length: 10 mm; 1 cm standard quartz cell

Synonyms Omniscan

Molecular weight

Molecular formula

Classifications

576.68036

C16H29GdN5O8

known drug

FDA approved drug

bioactive

Figure 4: Chemical structure

of the Gadodiamide Molecule

0.2 nm resolution and the green spectrum with 0.5 nm

Table 1: First characteristics of Gadodiamide*

0.000

240.00

nm

Abs

.

260.00 280.00 300.00-0.028

0.200

0.400

0.593

Shimadzu Pharma Special

Customer Magazine of Shimadzu Europa

GmbH, Duisburg

Publisher:

Shimadzu Europa GmbH

Albert-Hahn-Str. 6 -10 · D - 47269 Duisburg

Phone: +49 - 203 - 76 87- 0

Telefax: +49 - 203 - 76 66 25

shimadzu@shimadzu.eu

www.shimadzu.eu

Editorial Team:

Uta Steeger · Phone: +49 - 203 - 76 87- 410

Ralf Weber, Tobias Ohme

Design and Production:

m/e brand communication GmbH GWA

Düsseldorf

Electronic version

©Copyright:

Shimadzu Europa GmbH, Duisburg, Germany

April 2011

Windows is a Trademark of Microsoft

Corporation

IMPRINT

Chemistry / Manufacturing / Control Pharma Special

38

Quality control of ultra pupharmaceutical manufact

Ultra pure water is one ofthe most widely usedreagents in industry and

its quality is therefore of utmostimportance in all industrial pro-cesses. Quality control has, formany years, been carried out anddocumented via conductivitymeasurements, which provide anassessment of the concentration ofall inorganic species present inwater. This detection method doesnot take organic pollutants intoaccount as they typically do notcontribute to conductivity. However, organic pollutants cantremendously influence furtherindustrial processes and it hasbecome increasingly more impor-tant to include quantitative deter-mination of all organic species inquality control of water samples.

The TOC value (Total OrganicCarbon) can be used as sum

Highly Purified Water is sterilewater for the production of drugsnot requiring ‘Water for Injec-tion.’ It is often used for the finalrinse during cleaning as well.Highly purified water is producedvia reversed osmosis. Its TOCcontent may not exceed 0.5 mg/L.However, the United States Phar-macopoeia does not apply thisclassification.

Purified Water is used for theproduction of drugs not requiringa separate standard. Organic con-tent is determined via TOC value(0.5 mg/L) or the permanganatetest for purified water in bulk.

TOC determination accordingto EP 2.2.44

The EP 2.2.44 guidelines do notprescribe any particular oxidationtechnique for TOC determination.But the TOC systems must differ-entiate between inorganic andorganic carbon. This can be uti-lized via removal of the inorganiccarbon species (NPOC method),or a separate determination (dif-ference method). The limit ofdetection for TOC should be atleast 0.05 mg carbon/L. Applica-bility of the method must bedetermined via a system suitabilitytest.

To carry out a system suitabilitytest, a standard solution consistingof sucrose with a carbon contentof 0.5 mg/L is prepared as well asa control solution of 1,4-benzo-quinone with the same carboncontent. The blank water referencesolution (ultra-pure water) usedfor this purpose may not exceed aTOC content of 0.1 mg/L. For the system suitability test, all solu-tions including the blank waterreference are subsequently meas-

parameter for organic compounds.Similar to conductivity signalsthat are composed of ionic com-pounds, the TOC value is a meas-ure of the contribution of manyorganic compounds present in awater sample.

Different areas of applicationtherefore require different gradesof pure water. The EuropeanPharmacopoeia (EP) defines sev-eral grades of quality including‘Purified Water’, ‘Highly PurifiedWater’ and ‘Water for Injection.’

Clarifying the Terminology

Water for Injection is ultra-purewater used in the preparation ofinjection solutions. Ultra-purewater is produced via distillation.Its TOC content may not exceed0.5 mg/L (water for injection inbulk).

Two TOC instruments offering different oxidation met

TOC-L mit ASI-L

100rss rw

rs rw

-

-

Chemistry / Manufacturing / ControlPharma Special

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closer examination of the particu-lar application.

TOC series

Shimadzu’s TOC series covers allwater quality analysis require-ments. Two systems are particu-larly suitable for ultra-pure waterTOC analysis. Both offer a widemeasuring range of 0.5 μg/L up to25,000 mg/L and support a broadrange of applications – from ultra-pure water up to highly pollutedwastewater (or cleaning valida-tion). Both systems are availableas a PC as well as a standaloneversion and are easily adjustableto any laboratory environment.

The two TOC instruments forultra-pure water analysis differ intheir methodology. The TOC-VWP/WS uses wet-chemical oxida-tion whereas the TOC-L uses the catalytic oxidation method at680 °C.

TOC-L: Oxidation via catalytic combustion

The TOC-L applies the provencatalytic oxidation at 680 °C. Theintegrated ISP sample preparationunit (an 8-position switching valvewith syringe and sparging gasconnection) considerably reducesanalysis time and complexity, asdilution, acidification and sparg-ing are fully automated by theinstrument. Automatic dilutionincreases the measuring range upto 25,000 ppm (detection limit: 4 μg/L).

In addition, the combustion tech-nique can be used in combinationwith the TNM-1 module, where-by a single injection is sufficientfor simultaneous TOC/TNb (totalbound nitrogen) determination.

This is carried out in accordancewith EN guidelines for TNbdetermination via chemilumines-cence detection.

In this case catalytic combustionhappens at 720 °C. SimultaneousTOC/TNb determination is high-ly suitable for cleaning validation,as this makes differential determi-nation between cleaning agent andproduct possible.

Wet-chemical oxidation usingthe TOC-VWP

The key technique of the TOC-VWP analyzer is the powerful oxi-dation via the combination ofsodium persulphate and UV-oxi-dation at 80 °C.

A persulphate solution is neededfor the determination. Therefore,it is important that this solutiondoes not contain any contami-nants affecting the measuring val-ue negatively. The TOC-VWPcontains an automatic reagentpreparation function that elimi-nates possible contamination ofthe persulphate solution in orderto assure that the average TOCvalue truly originates from the

ured and the resulting signals arerecorded.

Blank water reference: rwStandard solution (sucrose): rsControl solution (benzoquinone): rss

The signal of the blank water ref-erence is subtracted from theresponse signals of both standardsolutions. Then, the recovery ofthe benzoquinone standard is sub-sequently calculated with respectto the sucrose standard.

Recovery in %:

Results between 85 - 115 % areacceptable. The ultra-pure watersample corresponds to the guide-lines when its response signal (ru)does not exceed rs - rw.

TOC determination in ultra-pure water

Two oxidation methods are rou-tinely being used in TOC analy-sis: 1. Catalytic combustion where

carbon compounds are convert-ed into CO2 using a catalystunder high temperatures withsubsequent detection of theCO2 using a NDIR detector.

2. Wet chemical oxidation whichuses a combination of UV irra-diation and persulphate for oxi-dation.

Both methods can be applied forultra-pure water TOC determina-tion. Which of the two methodswill offer the best solution for dif-ferent applications can not beanswered in general, but requires

sample – and not from the reagentsolution used.

Combined with the large injectionvolume (up to 20.4 mL) and thehighly sensitive NDIR detector,this leads to an extremely lowdetection limit (0.5 μg/L) andexcellent reproducibility in thelower ppb range. The TOC-VWP/WS is therefore highly suit-able for TOC determination inthe ultra-trace range.

Conclusions

Both types of instruments withtheir different oxidation methodscan be used for TOC determina-tion according to EP 2.2.44 guide-lines. The advantage of the com-bustion method is its high oxida-tion potential, especially for sam-ples containing particulate matter.Moreover, simultaneous TOC/TNb measurements can be carriedout using this instrument.

The advantage of wet-chemicaloxidation is its high injection vol-ume, which leads to higher sensi-tivity and therefore enables highprecision measurements in thelower ppb range.

re water in uringthods

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How heavy metals can migrate into pharmaceuticals

Pharmaceutical substancescan be toxic and dangerousto the human health if not

manufactured with proper care.All substances from starting mate-rials to finished products aretherefore strictly regulated. Ingeneral, this covers proof of prod-uct stability, drug-release profilesand examination of the activepharmaceutical ingredients (API).Furthermore, product safety in-cluding sterility and absence ofnon-proprietary materials, e.g.heavy-metals is determined.

One of the analytical tests in thepharmaceutical industry is thequantification of heavy metals orinorganics in all substances andmaterials in a pharmaceuticalproduct. For example, toxic heavymetals such as cadmium (Cd),mercury (Hg) and lead (Pb) are

antimony content. The acid in thejuice seems to enhance the migra-tion process of antimony from thebottle into the liquid contained.PET bottles are used not only forjuice drinks, but also for pharma-ceutical products.

lyst. Residues of antimony remainin the bottle and can, under cer-tain conditions, migrate into thepharmaceutical product. It istherefore logical to control heavymetal content in the packagingmaterial.

Is there evidence that antimony migrates into the product?

Unfortunately, yes. Scientists atthe University of Copenhagen,Denmark, recently studied anti-mony levels in 42 juice drinks andfound concentrations above EUlimits for drinking water in eightof them.

Freshly produced juice drinksappear to be free from increased

checked normally. It is also com-mon practice to measure catalystssuch as palladium (Pd) and plat-inum (Pt). Throughout the manu-facturing processes, there aremany potential sources of con-tamination. In addition to testingthe raw materials it is thereforeessential to also measure all fin-ished products and in some casesintermediates to guarantee com-pliance with the appropriate regu-lations.

Contamination by the packaging

But even when the product is pro-duced without contamination, thefinal product can contain heavymetals. Contamination can becaused by the packaging material,for example by PET bottles con-taining antimony (Sb) as a cata-

EDX series detects elements from C to U – carbon and uranium

0.00.0

Standard Value (ppm)

Inte

nsit

y R

atio

50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0

0.2

0.4

0.6

0.8

1.0

1.2

Figure 1: Measurement of a PET bottle containing around 250 ppm of Sb

Figure 2: EDX measurements are a great tool for analyzing pharmaceutical products

and their packaging materials in a fast and cost-efficient way

0.000025.00

[KeV]

[cps

/uA

]

26.00 27.00 28.00

0.0050

0.0100

This discovery is of concern sinceantimony is a known toxic ele-ment. In small doses, it can causeheadaches, dizziness and depres-sion. Larger doses can lead to vio-lent and frequent vomiting, andeven to death within a few days.

Shimadzu’s EDX series of x-rayfluorescence spectrometers hasbeen designed for the analysis of awide variety of elements from car-bon (C) to uranium (U), in con-centrations from 100 % down to a few ppm. The instruments meas-ure the content of heavy metals in pharmaceuticals products andtheir packaging materials. Todemonstrate the performance ofthe EDX instrument a calibrationwas made using plastic standardsranging from 50 ppm to 400 ppm.