workshop 2 - festkörper nmr – ultraschnelle probenrotation, … · 2017-12-06 · • deployment...

Innovation with Integrity

RMN Quantitative

Dr. Fabrice Moriaud, Product Manager 31ème Réunion d’Utilisateurs RMN, 28 novembre 2017, Lyon, France


Quantification by NMR

Quantitation by NMR has many faces and can answer many questions

• Absolute concentration (mM) • Potency / Purity (%)

– … in full automation (but fully editable) or manual – … with internal or external standards – … high throughput or single samples

– Bruker provides solutions for all of these

[email protected]


Popular qNMR Applications

qNMR method: Internal standard (IS) External standard (ES) with PULCON Units: wt/wt% - potency mM Error (accuracy): < 1 % > 2% (typically 5%) Workflow: IS + analyte in 1 tube 1) calibrate with ES 2) measure analyte Sample preparation

(1) (2) 10-20 minutes: because of

manual and accurate weighing of two compounds < 1 minute and can be fully automatised

In automation with PotencyMR : typically 2 %


Potency: Round Robin Test

• 15 / 25 participants measured true potency with +- 1% • Using a standard operating procedure (SOP) • Deviation > 2% are traced back to problems in the sample preparation

NMR spectroscopy in pharmaceutical analysis, Editors U. Holzgrabe, I. Wawer, B. Diehl, 2008


that uses External Standard Method

Concentration Determination


Rapid and Automated Workflow

1H Experiment (with or without solvent suppression) : automatic processing

Result is the concentration:

• Submission in IconNMR

• Results: pdf

The external standard methodology is based on PULCON (Wider G, Dreier L. J. Am. Chem. Soc. 2006; 128: 2571.)


Today´s Bruker Automated Solution

SampleJet: 5 x 96 samples

SamplePro Tube Liquid handler


High Throughput Workflow

• Medchem compounds in NMR solvents (deuterated or not)

• E.g. for FBS ligands in well plates reference & QC

Samples

• Any Bruker sample changer SampleJet for high throughput

• Optimized parametersets

• Customization easy

Automation

• Fully automated, on-the-fly, analysis:

• Concentration, purity, ID, water

Analysis

• All information at a glance

• Easy manual cross check and editing

• Spread sheets, pdfs…

Results


Potency Determination

that uses Internal Standard Method

Sample 20 mg 18 mg API, 2 mg of anything else , detectable or not by NMR Potency = 90 %


Popularity of Potency Determination

Gaining popularity and adoption:

• Introduction Pharmacopeia

• Used in industry (Pharma, food, chem.)

• Deployment to open access (ease of use)

Planta Med 2013; 79 - SL79 DOI: 10.1055/s-0033-1351904


1.0208 a.u.

4.6817 a.u.

NMR: a Primary Ratio Method

8.56 mg 6.22 mg

1

1

3

Analyte Internal Standard

MW: 168.19 g/mol MW: 356.41 g/mol

I can quantify one compound with respect to another compound without a response factor!

2

𝑃𝑃𝑎𝑎 = 𝑃𝑃𝐼𝐼𝐼𝐼 ×𝐴𝐴𝑎𝑎𝐴𝐴𝐼𝐼𝐼𝐼

×𝑁𝑁𝐼𝐼𝐼𝐼𝑁𝑁𝑎𝑎

×𝑊𝑊𝑊𝑊𝐼𝐼𝐼𝐼𝑊𝑊𝑊𝑊𝑎𝑎

×𝑀𝑀𝑊𝑊𝑎𝑎𝑀𝑀𝑊𝑊IS

Sample prep. Known compounds NMR From supplier


NMR `One-Stop Shop´

potency determination purity assessment relative response factor calculation residual solvent moisture analysis identity testing

‘Potency determination by qNMR has been shown to be a single point replacement for routine development testing which previously involved several experiments and techniques.’*

1 single experiment -> qNMR

Efficiency, Economy Selectivity

* Webster G.F. and Kumar S., Anal. Chem., 86, 11474 (2014)


Fully Automated Potency Determination

Acquisition + Analysis + Reporting


Setup of Potency Determination in Icon


Results: PDF Report

Robustness and reproducibility Internal standard peaks ID and integration Analyte ID, check and quantification Potency calculation

Consistency check is done as an indicative statement: potency determination is independent of the consistency check.


Results in Excel

Quality Duplicates, triplicates … Error analysis – Intra and Inter sample Easy review

What if there would be an error in weighing ? inter sample RSD:

Triplicate measurement:

What if there would be an error in analyte integral (overlap with impurities, satellites) ? intra and inter sample RSD:


Reproducibility at 2 Magnetic Fields Sulindac API with 1,3,5-trimethoxybenzene as IS

500MHz 300MHz


Example of Drug Product

The potency of the formulated ibuprofen is: Potency = 400 𝑚𝑚𝑚𝑚

533 𝑚𝑚𝑚𝑚 = 75.0%

PotencyMR (300MHz) fully automated

The quantification is robust because ibuprofen aromatic integrals are separated from all excipients signals “Separation without physical separation“

Correct result in full automation:

The tablet weighs 533 mg Sample is a clear solution in dDMSO after centrifugation


Manual Interaction at any Time

Expert Analysis Interactively define integrals Use predefined regions

– Create and copy methods – Run methods in automation

• Make “methods” for re-occurring tasks • Set molecular mass • Define integral regions

• Use these in automation • Auto-report with your definitions


PotencyMR Software Summary

Problems it solves: streamlines potency/purity determination, which can now be run in automation by both NMR experts and non-experts.

• Easy sample submission through IconNMR, including replicate samples,

acquired and analysed in automation

• Excel report generated automatically after measurement. Describes how potency is computed and gives intra and inter sample RSD

• Easy-to-use GUI enables user interaction at any point

• Automatic analysis of the 1H-spectrum: analyte quantification, consistency analysis, overlap between reference and analyte determination.

• Flexible functionality allows to run a potency determination method with pre-defined analyte's integral regions

Software is available with TopSpin acquisition license (3.5.7 and 4.0.0)


Tips: FILCOR in automation, DE, local baseline correction in TS

• Optimize FILCOR is very important to get good automated phase correction with apk0

• When using a He

cryoprobe, make DE as short as possible (without ring down signal). Try back prediction on analog FID

• Integral definition with

local baseline correction in TopSpin : screenshots ->

Mephaquin Drug tablet


Thank you! …and please talk to us for demos and discussions

[email protected]

workshop 2 - festkörper nmr – ultraschnelle probenrotation, … · 2017-12-06 · • deployment...

Documents