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7.0 and 8.0 ppm and an additional peak canbe found for the methyl group at around 2.3ppm (DMSO-d6). 4,4’-Difluorobenzophenone(4,4’-DFBP, cat.no. 07563) is solid and thetwo symmetrical fluorine atoms show amultiplet at around -106.5 ppm (DMSO-d6)in the 19F spectrum. Eight aromatic protonsgive signals between 7.0 and 8.0 ppm(DMSO-d6). All three compounds are solublein common organic NMR solvents. Molecularweights are 215 g/mol (2,4-DCBTF), 144.57g/mol (2Cl4FT) and 218.2 g/mol (4,4’-DFBP).Purity values, expanded measurementuncertainties, NMR solvent specific shiftsand relaxation times (T1) can be found inTable 1.Technical aspects of 19F qNMRA characteristic of 19F NMR is given by 13Cand 12C satellites that are present in the NMRspectrum. The interaction of 19F with 12C and13C leads to an isotopic effect and thereby tounsymmetrical satellites on the one handand to multiple satellites around the mainpeak in non-decoupled spectra on the otherhand.Additionally, peak shapes are differentdepending on the structure element. Ingeneral, CF3 peaks show singlet signalpattern and aromatic bound 19F atomsmultiplet signal pattern.As with 31P qNMR, inverse gated decouplingwas used during 19F qNMR data acquisition.Using this method instead of an e.g., powergated decoupler minimizes NOE (NuclearOverhauser Effect) build-up. With thisexperiment, decoupling is applied onlyduring data acquisition and thus allows thespin system to reach equilibrium betweendecoupling steps. By applying inverse gateddecoupling, only one satellite appears that ison only one side of the main peak (Figure 2).When performing pretests, a set ofdecoupled and coupled spectra wasrecorded to distinguish between satellitesand impurities. Integration of decoupledspectra (Figure 2) was done either includingboth satellites, only the 12C satellite, or ifpossible no satellite. No matter whichpossibility was chosen, integration wasperformed in the same way for the internalstandard and the sample compound withregard to the line width. Similar to 13C, the19F nucleus has a wide chemical shift range.To perform quantitative measurements,broadband excitation over the full spectralwidth is required. Due to insufficientavailable radiofrequency power for pulsedexcitation, signal intensities and thus signalintegration can be error-prone. The effectleads to relatively narrow ranges offrequencies (15 - 30 kHz, 600 MHz NMR, 90 pulse) where an accurate quantification canbe guaranteed.9

This requires sound pretesting, followed byaccurate adjusting of spectral width andtransmitter frequency offsets. Furthermore itis important to set the acquisition time asshort as possible to avoid NOE build up, butlong enough to avoid loss of spectral qualityby truncation of the Free Induction Decay(FID). That requires an additional analysis ofthe FID prior to quantitative measurements.All 19F NMR experiments were performed ona Bruker Avance III 600 MHz NMRinstrument equipped with a Prodigy TCIprobe head.Even though a standard probe (instead of adedicated 19F probe) was used, a goodspectralqualitycouldbeensured.Background distortions by probe head andsample tube materials, pulse breakthroughand ringing artifacts influence the spectralquality, especially the baseline (rollingbaseline), which is typical for 19F, 11B and 29Siand increases when measuring over largespectral width. This can be counteracted byeither applying additional processing steps(FID repair by cutting the first data pointsbefore data transformation) or by increasingthe pre-scan delay. For 19F qNMRexperiments during the development of ourCRMs, an increased pre-scan delay was usedand no FID cutting was done.T1 times were determined by inversionrecovery experiments. Typical T1 times for19F qNMR CRMs are between 1.2 and 4.8 sdepending on the concentration, of themixture and solvent. Multiplying T1 times bya factor of 7-10 gives D1 times between 20and 35s.CRM for 19F qNMR - traceability to the SIthrough primary CRMSimilar to the study published for 31P, atraceability scheme for 19F qNMR CRMs waselaborated to guarantee the traceability tothe SI unit and show the comparability of 1Hand 19F qNMR experiments and thus theindependency of the result of the measurednucleus (Figure 3, C). As primary referencematerial, 3,5-BTFMBA of the NationalMetrology Institute of Japan was selected.This reference is highly pure (99.96 %), has averysmallexpandedmeasurementuncertainty (0.06 %, k 2) and the twosymmetrical CF3 groups show a sharp 19Fsignal at -61.3 ppm (in DMSO-d6). The threearomatic protons give signals around 8.2 8.6 ppm (DMSO-d6), depending on thesolvent. 3,5-BTFMBA is soluble in allcommon organic solvents and is specified byNMIJ for 1H and 19F qNMR.10

The purity value of 2,4-DCBTF was certifiedby 19F and 1H qNMR using 3,5-BTFMBA. In asecond way, certification was done with 1HqNMRusing1,2,4,5-Tetrachloro-3nitrobenzene (TCNB, cat. no. 40384) withtraceability to the primary CRM BA (NISTSRM 350b). The three purity values andtheir expanded measurement uncertaintiesare in perfect accordance (SD 0.015,Figure 4). Values for uc(CRM) (k 2) are alsocomparablebetweenthedifferentexperiments (0.25 – 0.29 %). Due to differentsignal shapes and spectral regions of peaks,2Cl4FT and 4,4’-DFBP were certified byanother route. Traceability to the SI for2Cl4FT was achieved by determining a massfraction via 1H qNMR using 3,5-BTFMBA. In asecond way, Benzyl benzoate (BBO) was usedas internal standard. A third valueis assigned by 19F qNMR using 4,4’-DFBP asinternal standard. The purity values from heirexpandedmeasurement uncertainties and again showgood accordance (SD 0.053, Figure 4). Theuncertainty values uc(CRM) (k 2) are similarto that of 2,4-DCBTF (0.24 – 0.41 %).The purity value of 4,4’-DFBP was certifiedvia 1H qNMR using 3,5-BTFMBA, and in asecond way Maleic acid (MA, cat.no. 92816),as internal standard. 19F qNMR certificationwas performed using 2Cl4FT, showing againthe independency of the result of theobserved nucleus. All three values arecomparable and the SD of the three results issmall (SD 0.055, Figure 4). The values ofuc(CRM) are slightly higher compared withthe other two 19F qNMR CRMs (0.30 to 0.37%). The increased uncertainties (e.g., 0.41 %,2Cl4F2 and 0.37 % 4,4’-DFBP) do not resultfrom the measurement procedure but arecaused by a higher uncertainty contributionby the internal standard (4,4’- DFBP, MA) andhomogeneity of the material. In all other 19Fcertifications the overall repeatability of themeasurement represents the most significantuncertainty contribution.A last experiment was done to assign apurity value to the TraceCERT FlutamideCRM. It was possible to show, that via 19FqNMR and using 2,4-DCBTF as internalstandard, comparable results were achievedas by the common route via 1H qNMR usingan established CRM (BBO).11

Again,overallrepeatabilityofthemeasurement represents the most significantuncertainty contribution, which is in thesame order for certification via 1H and 19F.The purity values are overlapping within theirexpandedmeasurementuncertainties(Figure 4), which is again a clear indicatorthat 19F qNMR can be used routinelyas a stand-alone method to assign the purityof fluoroorganic substances.Reference1. Rigger R, Rück A, Hellriegel C, SauermoserR, Morf F, Breitruck K, Obkircher M (2017)Journal of AOAC International, Vol. 100, No.5, 1365-1375.ConclusionIn summary, qNMR using 1H, 31P, or 19FTraceCERT CRMs is a very valuablemethod. We outlined sensitive aspects thatare important for an accurate qNMRcertification and need particular awarenessby the operator. The presented set of 1H, 31P,and 19F qNMR CRMs is produced fulfillingthe requirements for a reference materialproducer under ISO 17034 ity of the material and short-termand long-term stability.For an overview on our qNMR products visitus at SigmaAldrich.com/qnmrThe full portfolio of organic TraceCERT certified reference materials (CRMs) can befound at SigmaAldrich.com/organiccrm12

- New Post -Read it now!Author: Travis Gregar, AnthonyBusche, Terry Downey, 3M Center,St Paul, MN 5514413

- The Impact of Weighing Accuracy andData Integrity for qNMR Applications AbstractOne of the major sources for measurementuncertainty in quantitative NMR applicationsis weighing of reference and analyte.Weighing has a strong bearing on the finalqNMR results. A balance must beconsistently accurate, which is achieved bycalibrating the device periodically and bydetermining the minimum weight and thesafe weighing range. Weighing sample sizesin the safe weighing range reduces themeasurement uncertainty of the weighingprocess below a predefined threshold.Further to accurate weighing, data integrityof plays a fundamental role in regulatedenvironments. With automated transfer ofweighing data and associated metadata thetraceability of the weighing process isestablished and operator errors can beavoided.Thesignificanceofmeasurementuncertainty and minimum weightWeighing is a critical step for qNMR analysis.It strongly and directly influences theaccuracy of the final result because theweight of the net sample and of thereference standard have a direct correlationon the determination of sample purity orcontent. To ensure that weighings areaccurate, laboratory managers often rely onquality management systems to define aweighing process. This includes properrecording criteria, calibration of theinstrumentanddeterminationofmeasurement uncertainty.To better understand minimum weight, it isimportant to recognize that the stand outprerequisite for traceable and accurateweighing is the effective calibration ofweighing instruments, which must include anestimation of measurement uncertainty.Historically, many laboratories have set uptheir own calibration procedures due to thelack of nationally or globally recognizedcalibration guidelines. Based on internationalcooperation from subject matter experts inthe field of metrology, efforts have beenmadetogloballyharmonizethemethodology of calibration of weighinginstruments1.The benefit of these harmonization activitiesis that the state-of-the-art calibrationconcepts not only stipulate how to estimatemeasurement uncertainty at the time ofcalibration, but provide guidance forestimation of uncertainty during the day-today usage of the instrument. This conceptleads to the calculation of the minimumsample weight, often referred to as theminimum weight. This is the smallestamount of net substance that must beweighed in order to achieve a specifieddegree of accuracy.All weighing instruments act in a similarmanner across the weighing range - as thesample size decreases, the , with a small enough mass, therelative weighing uncertainty can becomehigh enough that the weighing result is nolongeraccurate.Themeasurementuncertainty then becomes larger than thespecified threshold. This accuracy limit is theminimum weight (Figure 1).14

Based on the risk associated with theweighing process, it is also recommended toapply a safety factor to this value. This factorincreases the minimum amount that shouldbe weighed on a particular balance anddefines the starting point of the so-calledsafe weighing range. The safety factoraccounts for performance fluctuationscaused by environmental factors (air drafts,temperature, vibrations, and different usertechniques) that can affect the balanceduring normal use between calibrations.With the benefit of measurement uncertaintyand the resulting minimum weight defined,it is important to realize that ntuncertaintyvalues.AnAccuracy Calibration Certificate (ACC)containsbothcomponents,themeasurement uncertainty and the minimumweight for the required weighing tolerance.Therefore, it links the performance of theweighing instrument to the weighing processtolerances required by the user for theirspecific application.Figure 1: Typical behavior of measurement uncertainty across theweighing range of a balanceThe minimum weight is an extremelyimportant characteristic when performingquantitative NMR analysis because smallsample sizes are often used for the purposeof minimizing costs or limited valuableamount of samples.The associatedweighings of the samples and standardshave a direct impact on the analysis results.Therefore, weighing above the minimumweightunderconsiderationofanappropriate safety factor, i.e. weighing in thesafe weighing range of the instrument, isextremely critical.Based on the defined safety factor, the ACCallows the safe weighing range to bedetermined for each particular balance. Thislevel of detail from a calibration enablesbalance users to improve the quality of theirweighing, increase confidence in theweighing results and avoid weighing errors.15

Ultimately, understanding and implementinga quality system that adheres to weighingsufficiently more substance than theminimum weight and thus working in thesafe weighing range of the balance, ensuresinstrument accuracy and minimizes the riskof errors that could affect the correctness ofanalysis results.With respect to measuring instruments,many regulations and guidelines now requirecomplete data derived from all tests.2.This includes the raw data generatedthrough the course of an analysis and theassociated metadata.Metadata is thecontextualinformationrequiredto3understand data .Figure 2: Simple example of metadata in an everyday situationAvoiding incomplete data and achievingcomplianceTo help comply and meet the requirementson data integrity, especially in the , it is also important tounderstand the benefits of incorporating thecomponents of the weighing process in anintegrated data management system. Inrecent years, an increasing number ofassessments and FDA warning letters haverevealed incomplete data, the lack of audittrails, and falsification of results. Theproblems with data integrity could beeliminated by first focusing on the samplefile generated from the sample during thecourse of analysis. Many labs have turnedtoward LIMS systems with the idea ofreplacing the manual workflow. Thesesystems are designed primarily to aggregateresult data from an array of analytical tests,rather than to automate and documentbench top workflows or bind instrumentmetadata to the measurement.An example of the use of metadata in aneveryday situation is shown in Figure 2. If acar speeds through a traffic enforcementcamera and the only information captured isthe image, the speed of the automobile,and the associated unit of measure, thereisn’t enough information to link the car tothe speed. However, if the date, time, colorof the car, unique picture identifier, andlocation is included, the necessary contextualinformation is then available to link the carwith the speed.When the same principle is applied to theregulated laboratories , every critical weightmeasurement that is recorded should notonly include the weight and unit of measure,but the additional metadata necessary to beconsidered "complete data" (Figure 3).16

Figure 3: Examples of metadata available from tandardization of weighing workflowsMany labs have discovered that transferringmetadata from bench top analyticalinstruments is much more complex than onlythe transfer of a few parameters, such assample weight and unit of measure.Leveraging the potential of appropriatesoftware technology, such as LabX, enablesusers to transfer weighing results with all theassociated metadata directly to their LIMSsystems - thereby ensuring the data iscomplete and traceable.Additionally, the weighing workflow can beautomated and standardized to thespecifications of the unit or lab (Figure 4).This guarantees and proves that the sameweighing process is used for each sample,regardless of who performs the steps –ensuring consistency in every analysis. Forexample, the administrator can elect to havethe balances locked down every morninguntil an analyst logs in and performs anadjustment of the balance by means of thebuilt-in weights. Only once that has beencompleted can the balance user proceed to aguided weighing process on the terminal ofthe balance.Another example of a benefit the softwareprovides is the ability to capture not only thenet weight of the substance, but the weightof the tare vessel used in each weighingevent. This allows the analyst to providedocumentation during trial, confirming thetare vessel weight was not included in thenet weight of the substance in question.Automateddatatransferandstandardization of weighing workflowsMany labs have discovered that transferringmetadata from bench top analyticalinstruments is much more complex than onlythe transfer of a few parameters, such assample weight and unit of measure.Leveraging the potential of appropriatesoftware technology, such as LabX, enablesusers to transfer weighing results with all theassociated metadata directly to their LIMSsystems - thereby ensuring the data iscomplete and traceable.17Figure 4: Example of a standardized weighing method

ConclusionTo increase accuracy of qNMR analysis, it is crucial to minimize weighing and samplepreparation uncertainty. Error elimination, process simplification and data traceability are thekeys to succeed in qNMR application which can be supported by the following. Establish a harmonized approach to the calibration of balances Ensure all weighing is performed in the safe weighing range, well above the minimumweight Automate data capture and transfer of weighing data to ensure traceable data and toreduce operator errorAuthorsTucker RubinoMarket ManagerMettler-Toledo, LLC1900 Polaris ParkwayColumbus, OH 43240Tucker.Rubino@mt.com 1 (614) 438 4511Figure 5: XP205 Semi Micro BalanceKlaus Fritsch, Ph.DManager Compliance and Senior MetrologistMETTLERWeighingTOLEDOIm ensee,Direct dial 41-44-944 22 96E-mail: Klaus.Fritsch@mt.comMarket Development ManagerTOLEDOIm ensee,Direct dial 41-44-944 22 96E-mail: Nutsima.Schnell@mt.com1. Euramet, Guidelines on the Calibration ofNon-Automatic Weighing Instruments, No.18, Version 4.0, November 2015.2. EUROLAB Technical Report 1/2014, “Guideto NMR Method Development andValidation – Part 1: Identification andQuantification”, May nd%20Validation%20May%202014 final.pdfNutsima SchnellMETTLERWeighingReferences4. U.S. Food and Drug Administration, Codeof Federal Regulations, Title 21, Food andDrugs, Pt. 200-299, Revised 21 CFR 211.194(a)5. U.S. Food and Drug Standards (CGMP), Data Integrity andCompliance with CGMP, Guidance forIndustry, April 2016.18

- Proficiency Testing for yourQuantitative Nuclear MagneticResonance (qNMR) Analysis -Clickhere!1919

- Future validation guidance from USPand ICH By ValidNMR Committee,Lead author: Dan SorensenBoth the USP and the ICH have initiatedactivities to update their guidancedocuments to incorporate the application ofscientific knowledge and quality riskassessment to the development andvalidation of analytical procedures. Theseprinciples, that can be summarized anddescribed as Quality by Design (QbD), arefully embraced and endorsed by theValidNMR group. We are very excited aboutthese initiatives and encourage the widerNMR community

Bernie O'Hare “I began my career with Bruker BioSpin providing NMR installation, service, and applications support for a diverse customer base. Soon after joining Bruker, I became involved with their ssNMR-DNP product. I was eventually responsible for all DNP installations and service in the America’s and interacted directly with R&D