la rmn quantitative appliquée aux petites molécules...• water karl fisher titration • residual...

December 9, 2016 1

La RMN quantitative appliquée aux petites molécules

Fabrice Moriaud - Applications Development - Fällanden

30ème Réunion d’Utilisateurs RMN Bruker

Quantification in liquids

External and Internal Reference

Concentration Determination (External Reference)

Potency Determination (Internal Reference)

Covered in this presentation

Demain matin 09:00

session3 RMN en milieu pharmaceutique

Quantification in Solids

Shortening Time to Market - Rapid Analysis of Drugs

Anna Codina

TD-NMR Workflow

The minispec Mq20

External and Internal Reference have very similar workflow and are available side by side

Workflow in IconNMR: analyte and reference (calibrant)

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Workflow in CMC-assist

External and Internal Reference have very similar workflow and are available side by side

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General Comments about External and Internal Calibration

• Spectroscopic methods are othogonal and, thus ideally suited to assess materials purified chromatographically.

• With NMR, the integral regions reflect the molar response of the protons.

• Absolute quantification is possible: no need of a reference sample of the analyte. A suitable (certified standard) can be chosen.

• Any mass related interpretation requires in addition the molecular weight, or the molecular Formula.

2

2/3

0

2/3T

T

NSBn

Noise

SignalDETEXC

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General Comments about External and Internal Calibration

Internal vs External Reference:

Internal calibration is a common method for absolute quantification

• It involves the addition of a known amount of a certified calibrant to the analyte. Accurate and precise.

• But signal overlaps challenge.

External calibration is also a common method for absolute quantification.

• Precious sample are not mixed with a calibrant.

• Higher throughput.

• But new sources of errors occur…

Accuracy and Precision

neither accurate nor precise

accurate but not precise

reproducibility error

not accurate but precise

systematic error

accurate and precise

Sources of Systematic Errors in quantitative NMR

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Effect of pulse miscalibration

… only important for external quantification

Effect of short recycle delay

… always important

Effect of low digital resolution


Effect of short acquisition time


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Effect of phase errors

… most important for external quantification

Effect of baseline offsets

… most important for external quantification

Effect of integral limits


Sample preparation


Signal to noise


Choice of integral regions for analyte


Sources of Reproducibility Errors in quantitative NMR

Analyte integrals automatic determination

• Integral regions and Proton content are determined in automation with CMC-assist algorithms.

• Selection of Integral region for quantification

• Tested/proven on Expert analysed Data and integrated in industry workflows.

• At the heart of several automation solutions: quantification within IconNMR, SmartDriveNMR, CMC-q

Analysing mixtures ! • One analyte • One internal standard • Known impurities in any amount • Unknown impurities: small integrals

compared to analyte integrals (0.3 Proton)

CMC-q: workflow package for library QC, Fragment Based Screening

• Medchem compounds in NMR solvents (deuterated or not)

• well plates and SDF information

Samples

• Any Bruker sample changer

• SampleJet for high throughput

• CMC-q 2.0: optimized experiments

• In H-DMSO or H2O buffer

NMR Automation

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

• Verification, concentration, purity, water

Analysis

• All information at a glance

• Easy manual cross check and editing

• Spread sheets, pdfs…

Results

CMC-q launches Batch experiments in IconNMR

•Batch Setup from SDF

•Automatic structure file extraction

•Single click setting of global parameters

•Different levels of confidence

•Customizable

•Automatic acquisition, processing and analysis

Batch results

Inspect batch results

• Interactive well-plate

view

• Export to external formats

CMC-q concentration test results

• Software Statements:

• green: high confidence

• red (with concentration): lower confidence

• light blue: no concentration

• Light background: in concentration

CMC-q automatically delivers: integrity, concentration, purity and water content (for H-DMSO)

QA results for DMSO stock solutions

Compound integrity Compound concentration

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Library QC on-the-fly in buffer Tailor libraries & get ligand reference data

We prepared a library from a supplier, in aqueous solvent : ca. 30% bad samples!

• 20% “no compound” no compound in stock solution, or not soluble in buffer

• 10% decayed or wrong compound

• 50% concentration off by more than +/- 30%

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Howto: External reference definition

External Reference definition in TopSpin & CMC-assist: separate reference spectrum (PULCON, ERETIC-II)

Define integral region

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External reference definition with CMC-assist or TopSpin

Select integral region

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External reference definition with TopSpin or CMC-assist

Define as Eretic reference

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Define Reference concentration and number of protons


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All done. The dataset becomes a reference dataset (with a file called eretic)


Potency of Drugs Definition

mpoundsInactiveCoDrugDrugP )(

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

Inactive Compounds:

• Degradation substances LC-UV

• Process impurities LC-UV

• Water Karl Fisher titration

• Residual Solvents GC

• Inorganic material residue in ignition

Needed before administrating the drug to determine the correct dose based on the amount of

active drug in the preparation.

Typically measured by HPLC. Characterised reference standard of the drug itself is needed.

Otherwise is determined by difference:

NMR ‘One-Stop Shop’

potency determination

purity assessment

chromatographic 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

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

Efficiency, Economy

Selectivity

qNMR Internal Standard

IS

IS

IS

IS

Mw

Mw

Wt

Wt

I

IPP

a

a

aa

Accuracy

Eliminates errors introduced by inherent sample

differences

Pa, PIs = potency of analyte/standard

Ia, IIs = integral area of analyte/standard from NMR spectrum normalized by number of nuclei

Mwa, MwIs = molecular weight of analyte/standard

Wta, WtIs = weight of analyte/standard.

e.g. Potency = 90 % means that out of 100mg, only 90 mg are from analyte. Remaining 10% can be anything, moisture, solvent, impurities: the method captures what frequently escapes detection

Rapid and Flexible Workflow

Sample Submission

Maleic acid, 0.99, # 05427ES

Results in PDF and Excel

Robustness and reproducibility

Internal standard peaks ID and integration

Analyte ID, check and quantification

Potency calculation

Results in PDF and Excel

Area** IS Area** analyte

Prep. Wt a. [mg] Wt IS [mg] CH Region 1 Region 2 Region 3 Averaged Area a. SD Area a. Potency [%] RSD Potency [%]

1 10.30 5.10 1.03 1.00 0.99 0.98 0.99 0.01 99.11

2 13.10 5.60 0.88 1.00 0.98 0.97 0.98 0.01 99.19

3 12.40 17.80 2.93 1.00 0.98 0.82 0.94 0.08 95.42

Average 97.91 1.80

Quality

Duplicate, triplicates …

Error analysis – Intra and inter sample

Easy review

Flexibility: User Intervention Possible at any Time

Calculated potency 99.1 %

• License

• TopSpin 3.5pl7: acquisition license allows to use quantitative NMR, External and Internal Reference (No need of CMC-assist license at the spectrometer)

• External Reference: several possibilities

• IconNMR

• CMC-q

• TopSpin, CMC-assist

• Internal Reference qNMR: IconNMR, CMC-assist. Next release TopSpin 3.5pl7

• Rapid and cost effective solution to determine the amount of active drug in a formulation

• Integrated in IconNMR and CMC-assist, side by side with external reference quantification

December 9, 2016 36

la rmn quantitative appliquée aux petites molécules...• water karl fisher titration • residual...

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