La RMN quantitative appliquée aux petites molécules

La RMN quantitative appliquée aux petites molécules Fabrice Moriaud - Applications Development - Fällanden 30ème Réunion d Utilisateurs RMN Bruker December 9, 2016 1

Covered in this presentation Quantification in liquids External and Internal Reference Concentration Determination (External Reference) Potency Determination (Internal Reference)

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

La RMN quantitative appliquée aux petites molécules Quantification in liquids External and Internal Reference Concentration Determination (External Reference) Potency Determination (Internal Reference)

External and Internal Reference have very similar workflow and are available side by side Workflow in IconNMR: analyte and reference (calibrant)

External and Internal Reference have very similar workflow and are available side by side Workflow in CMC-assist December 9, 2016 7

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. Signal n EXC DET 3/ 2 B 3/ 2 NS T Noise 0 T 2 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. December 9, 2016 8

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 December 9, 2016 9

Accuracy and Precision neither accurate nor precise accurate but not precise not accurate but precise accurate and precise reproducibility error systematic error

Sources of Systematic Errors in quantitative NMR Effect of pulse miscalibration only important for external quantification Effect of short recycle delay always important Effect of low digital resolution always important Effect of short acquisition time always important December 9, 2016 11

Sources of Reproducibility Errors in quantitative NMR Effect of phase errors most important for external quantification Effect of baseline offsets most important for external quantification Effect of integral limits always important Sample preparation always important Signal to noise always important Choice of integral regions for analyte always important December 9, 2016 12

Analyte integrals automatic determination Integral regions and Proton content are determined in automation with CMC-assist algorithms. Selection of Integral region for quantification Analysing mixtures! One analyte Tested/proven on Expert analysed Data and integrated in industry workflows. One internal standard Known impurities in any amount Unknown impurities: small integrals At the heart of several automation solutions: quantification within IconNMR, SmartDriveNMR, compared to analyte integrals (0.3 Proton) CMC-q

CMC-q: workflow package for library QC, Fragment Based Screening Samples Medchem compounds in NMR solvents (deuterated or not) well plates and SDF information NMR Automation Any Bruker sample changer SampleJet for high throughput CMC-q 2.0: optimized experiments In H-DMSO or H 2 O buffer Analysis Fully automated, on-the-fly, analysis: Verification, concentration, purity, water Results All information at a glance Easy manual cross check and editing Spread sheets, pdfs

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

QA results for DMSO stock solutions CMC-q automatically delivers: integrity, concentration, purity and water content (for H-DMSO) Compound integrity Compound concentration

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% 20 December 9, 2016

Howto: External reference definition 21

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

External reference definition with CMC-assist or TopSpin Select integral region December 9, 2016 23

External reference definition with TopSpin or CMC-assist Define as Eretic reference December 9, 2016 24

External reference definition with TopSpin or CMC-assist Define Reference concentration and number of protons December 9, 2016 25

External reference definition with TopSpin or CMC-assist All done. The dataset becomes a reference dataset (with a file called eretic) December 9, 2016 26

Webster G.F. and Kumar S., Anal Chem, 86, 11474 (2014) Potency of Drugs Definition 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: P ( Drug) Drug InactiveCompounds Inactive Compounds: Degradation substances LC-UV Process impurities LC-UV Water Karl Fisher titration Residual Solvents GC Inorganic material residue in ignition

* Webster G.F. and Kumar S., Anal Chem, 86, 11474 (2014) NMR One-Stop Shop Efficiency, Economy Selectivity potency determination purity assessment chromatographic relative response factor calculation 1 single experiment -> qnmr 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. *

qnmr Internal Standard Accuracy Eliminates errors introduced by inherent sample differences P a P IS I a Wt IS Mw a I IS Wt a Mw IS 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 Quality Duplicate, triplicates Error analysis Intra and inter sample Easy review 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

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