The Need for Proficiency Testing in the Scope of NMR Method Validation

A Role for the ValidNMR Organization?

Presented by
John C. Edwards, Ph.D.
Process NMR Associates, LLC, Poughkeepsie NY
Validation Workshop, PANIC 2020

Abstract: In the scope of analytical method validation it is generally accepted, in the arena outside of NMR spectroscopy, that
validation should include inter-laboratory comparison (ILC) studies or proficiency testing coordinated by a relevant third-party
organization such as AOAC, AOCS, ASTM, ISO, USP, etc. Validated NMR methods that are in general use by the industrial
community under the auspices of official organizations are very limited. The reasons for the lack of official NMR methods is often
stated to be due to the high cost of NMR instrumentation and the fact that it resides in research centers rather than in
manufacturing plant laboratories. Over the years, chromatography has been preferred in these environments even though it
requires constant calibration with expensive standards. As a primary method NMR does not require continued calibration with
standards and appropriate utilization of superconducting and benchtop systems to provide ID and qNMR in purity, potency, and
complex mixture analysis could greatly reduce the burden on chromatography approaches. A short review of the limited number
of NMR-based inter-laboratory comparisons will be presented to provide a background to this topic. Further discussion will
elaborate on the role that the ValidNMR organization could play as an interface between standard method development in
industry and the requirement to determine intra-lab and inter-lab error and bias through ILC studies or third-party coordinated
proficiency testing.
 Section 7.7 Ensuring Validity of Results
• Record data in such a way that trends and statistically quantifiable variations can be observed in the measured results

• Monitoring should be planned and reviewed.

• Execution of monitoring can be done by utilization of:

❖ Reference materials
❖ Quality control materials
❖ Alternative instrumentation (Cross Check)
❖ Control charts maintained for working standards
❖ Replicate testing
❖ Re-testing retained samples
❖ Interlaboratory comparisons
❖ Proficiency Testing

Section 7.7.2: Monitor performance by comparison of results from other laboratories where available and appropriate
by participation in Proficiency Testing (PT) and Inter-Laboratory Comparison (ILC)

This monitoring is to be planned and reviewed.

 Proficiency Test (PT) – Method to Validate a qNMR or an NMR-Based Non-Targeted Measurement Process
A Proficiency Testing Plan, undertaken as part of ISO 17025 accreditation, validates a laboratories measurement processes, personnel
technical training, traceability of standards and CRM, quality of calibrations, and determination of uncertainty budgets.
In a qualified PT a sample with an unknown reference value is provided for analysis by an accredited organization (ISO 17043 + ISO
17034) under conditions where the reference value was determined by a competent laboratory that has demonstrated competency
through interlaboratory comparisons or PT appropriate to validate their measurement capability.

Accreditation Company Example:

Chemical - General - Sub-Disciplines

Summary of Requirements Chromatography: GC, GC-MS, HPLC, IC, TLC
(PT1) Laboratories shall conduct proficiency tests in accordance with their normal Combustion: LECO
testing/calibration and reporting procedures, unless otherwise specified in the instructions from Spectroscopy: AA, CVAA/GFAA,
the proficiency test provider. Fluorescence, ICP, IR/FTIR, MS (MSD,
(PT2) Laboratories shall also ensure that proficiency testing samples are equally distributed among
trained and qualified personnel and satellite locations (where applicable) for the relevant tests.
MS/MS, etc.), OE, UV/Vis, XRF, NIR
(PT3) Unless otherwise specified within this document or in A2LA R103a – Annex – Proficiency Wet Chemistry
Testing for ISO/IEC 17025 Laboratories, at minimum, laboratories shall participate in at least two
proficiency-testing activities per year, every year. Laboratories with 4 or less sub-disciplines on their
scope are required to participate in at least one proficiency testing activity per year, every year.
(PT4) In fields without specific requirements, and in addition to meeting PT3, all accredited
laboratories must participate in relevant and commercially available proficiency testing at a
frequency sufficient to ensure that all sub-disciplines and materials/matrices/product types (as
defined in each section of R103a – Annex – Proficiency Testing for ISO/IEC 17025 Laboratories
document) on the scope(s) of accreditation are covered over a four-year period.
(PT5-PT11) Etc.
NMR Official Standard Test Methods
ASTM NMR Test Methods
E2977: Standard Practice for Measuring and Reporting Performance of Fourier Transform Nuclear Magnetic Resonance (FT-NMR) Spectrometers
for Liquid Samples
D7171-05: Standard Test Method - Hydrogen Content of Middle Distillate Petroleum Products by Low-Resolution Pulsed NMR Spectroscopy
D4273-05: Standard Test Methods - Polyurethane Raw Materials Determination of Primary Hydroxyl Content of Polyether Polyols
D4875-05: Standard Test Method - Polyurethane Raw Materials: Determination of the Polymerized Ethylene Oxide Content of Polyether Polyols
D5017-96: Standard Test Method - Determination of Linear Low-Density Polyethylene (LLDPE) Composition by 13C NMR
D5292-99: Standard Test Method - Aromatic Carbon Contents of Hydrocarbon Oils by High Resolution NMR Spectroscopy
F2259-03: Standard Test Method - Determining the Chemical Composition and Sequence in Alginate by 1H NMR Spectroscopy
F2260-03: Standard Test Method - Determining Degree of Deacetylation in Chitosan Salts by 1H NMR Spectroscopy
USP<2251> Adulteration of Dietary Supplements with Drugs and Drug Analogs
USP <761>: General Chapter – Nuclear Magnetic Resonance Spectroscopy
Krill Oil Monograph – 1H and 31P NMR, Beta Glucan Monograph – 1H NMR,
Heparin Sodium Monograph – 1H NMR Poloxamer Monograph – 1H NMR
European Pharmacopoeia
Salmon Oil, Farmed 01/2005:1910 13C NMR for distribution of fatty acids.
ISO
10565:1998 Oil and Moisture in Seeds by Low Field NMR Spectroscopy
8292 Animal and vegetable fats and oils -- Determination of solid fat content -- Pulsed NMR method
21461:2006 Rubber - Determination of the aromaticity of oil in vulcanized rubber compounds
21561:2005 Styrene-butadiene rubber (SBR) - Determination of the microstructure of solution-polymerized SBR
11543:2000 Modified starch -- Determination of hydroxypropyl content -- Method using proton NMR spectrometry
10632:2000 Oilseed residues -- Simultaneous determination of oil and water contents -- method using pulsed NMR spectroscopy
 AOCS
Cd 16b-93 : (revised in 2000): Direct Method - Determination of Solid Fat Content -- Pulsed NMR Method
Cd 16-81 : (revised in 2000): Indirect Method - Determination of Solid Fat Content -- Pulsed NMR Method
Ak5-01 (01) : Oil Content of Oilseed Residues by NMR
Ak3-94 (00) : Oil Content in Rapeseed and other Oilseeds by NMR
Ak4-95 (99): Simultaneous Determination of Oil and Moisture Contents of Oilseeds using Pulsed NMR Spectroscopy
American Herbal Pharmacopoeia
Aloe Vera - 1H NMR Method for Quality Inspection and Identification
Canadian Crude Quality Technical Association
Maxxam V1.04 11/05 : Determination of Olefin Content of Crude Oils, Condensates and Diluents by 1H NMR
AOAC
2008.06: Moisture and Fat in Meats Microwave and Nuclear Magnetic Resonance Analysis

PT Problem (even for Official Standard Methods)

1) Few Standard Samples Available (except for some TD-NMR Standards for AOCS Methods and CRM from USP)

2) No Mechanism to Create Standard Samples particularly for NMR

3) No Mechanism to Develop a Proficiency Test or Inter-Laboratory Comparison particularly for NMR

4) Business Model and Funding Requirements for PT Development Not Discussed within NMR Community
or the general testing community with respect to NMR testing.
The qNMR Enabler
See Also PANIC Poster #12
Presenting Author: Alexander Rueck, Sigma-Aldrich Production GmbH
Title: Development of a novel Proficiency Testing (PT) material for quantitative NMR (qNMR)
 Millipore
Proficiency Testing Sigma
Concept

Slide provided by Alexander Rueck - MilliporeSigma

 Millipore
Proficiency Testing Sigma
qNMR Scheme

 Mass fraction of main component Dimethyl sulfone shall be determined by 1H qNMR experiment (≥90% content of the analyte)

 Product is set up as a quick-turn study material and can be ordered

any time

 The PT material is produced under ISO/IEC 17025 and ISO 17034

accreditations and available worldwide

 Reports generated according to ISO/IEC 17043- MilliporeSigma holds

the accreditation as PT Provider

Slide provided by Alexander Rueck - MilliporeSigma

Courtesy of Freddy Thomas - Eurofins PRO-PTS: Schedule and order form for 2020

Round Date Deadline sample Matrix Parameters

(sample (reporting of code Price
dispatch) results) (EURO)

1 February 20 March 20 20/1/A Red wine Country, Region, Variety, quantitative parameters 120
20/1/B White wine Country, Region, Variety, quantitative parameters 120
20/1/C Apple Juice Country, Type of Product, quantitative parameters 120
20/1/D Orange Juice Country, Type of Product, quantitative parameters 120
20/1/E Honey 1 Addition of sugar, botanical and geo origin, quantitative parameters 120
20/1/F Honey 2 (Manuka) Addition of sugar, botanical and geo origin, quantitative parameters 120
20/1/G Spices Adulteration, conformity vs label, quantitative parameters 80
20/1/H Coffee Variety (Arabica/Robusta), country, adulteration, quantitative parameters 80
20/1/I Oil Adulteration, conformity vs label, quantitative parameters 80
20/1/J Agave syrup Adulteration,quantitative parameters 80
2 June 20 July 20 20/2/A Red wine Country, Region, Variety, quantitative parameters 120
20/2/B Rosé wine Country, Region, Variety, quantitative parameters 120
20/2/C Grape Juice Country, Type of Product, quantitative parameters 120
20/2/D Pineapple Juice Country, Type of Product, quantitative parameters 120
20/2/E Honey 1 Addition of sugar, botanical and geo origin, quantitative parameters 120
20/2/F Honey 2 Addition of sugar, botanical and geo origin, quantitative parameters 120
20/2/G Spices Adulteration, conformity vs label, quantitative parameters 80
20/2/H Coffee Variety (Arabica/Robusta), country, adulteration, quantitative parameters 80
20/2/I Oil Adulteration, conformity vs label, quantitative parameters 80
20/2/J Agave syrup Adulteration, quantitative parameters 80
3 October 20 November 20 20/3/A Red wine Country, Region, Variety, quantitative parameters 120
20/3/B White wine Country, Region, Variety, quantitative parameters 120
20/3/C Mango Juice Country, Type of Product, quantitative parameters 120
20/3/D Lemon Concentrate Country, Type of Product, quantitative parameters 120
20/3/E Honey 1 Addition of sugar, botanical and geo origin, quantitative parameters 120
20/3/F Honey 2 Addition of sugar, botanical and geo origin, quantitative parameters 120
20/3/G Spices Adulteration, conformity vs label, quantitative parameters 80
20/3/H Coffee Variety (Arabica/Robusta), country, adulteration, quantitative parameters 80
20/3/I Oil Adulteration, conformity vs label, quantitative parameters 80
20/3/J Agave syrup Adulteration, quantitative parameters 80
 PRO-PTS: Results Table Round 2 2020
Courtesy of Freddy Thomas - Eurofins Deadline : 31/07/2020

20/2/A 20/2/B 20/2/C 20/2/D 20/2/E 20/2/F 20/2/G 20/2/H 20/2/I 20/2/J
Red wine Rosé wine Grape Juice Pineapple Juice Honey 1 Honey 2 Spices Coffee Oil Agave syrup
in accordance with database of authentic samples ?
Y/N
if yes, deviations observed
Adulterated ? Y/N galacturonic acid
If yes, type of adulteration succinic acid
Conformity with the indicated geographical origin gallic acid
(country)? Y/N shikimic acid
If no indication, state the origin found.
proline
Conformity with the indicated geographical origin
trigonelline
(region)? Y/N
If no indication, state the origin found.
turanose
Conformity with the indicated variety? Y/N maltose
If no indication, state the variety found. mannose
Conformity with the indicated vintage? Y/N methylglyoxal
If no indication, state the vintage found. dihydroxyacetone
Quantitative parameters : Arabica%
alcoholic grade % vol Robusta%
ethanol cafeine
glycerol trigonelline
fructose
cafestol
glucose
16-O-methylcafestol
sucrose
tartaric acid kahweol
malic acid HMF
lactic acid furfuryl alcohol
citric acid chlorogenic acids
acetic acid palmitic acid
fumaric acid palmitoleic acid
gluconic acid oleic acid
quinic acid linoleic acid
methanol linolenic acid
acetaldehyde
stearic acid
ethylacetate
sum omega3
2,3-butanediol
3-methyl-butanol sum omega6
benzoic acid sum saturated fatty acids
sorbic acid sum monounsaturated fatty acids
HMF sum poylunsaturated fatty acids
Innovative Solutions, S.r.l
http://www.innovative-solutions.it/
Innovative Solutions, S.r.l
http://www.innovative-solutions.it/
Innovative Solutions, S.r.l
http://www.innovative-solutions.it/
 Another Approach –
Utilize Pre-Existing Programs Designed for Proficiency Testing of Chromatography

34 public and 33 private labs

Results are submitted in triplicate
on the analytes and methods used PNA HPLC
in each laboratory.
•Δ9-THC using HPLC
•Δ9-THCA using HPLC
•Total Δ9-THC using HPLC or GC
•CBD using HPLC PNA NMR
•CBDA using HPLC
•CBN using HPLC or GC
•Total CBD using HPLC or GC

1H NMR: CDCl3 Extract, 10 Minutes

HPLC: Methanol Extract, 10 Minutes

My Own Questions
• How do I find out about up-coming Inter-Laboratory Comparison (ILC) studies before it’s too late?

• What happens to the valuable samples used in these ILC studies?

• Should ILC samples be manufactured in enough volume that they can be used as a PT for laboratories
working in similar areas? For a purchasing fee, of course!

• What happens to all the data in these studies? Is it openly available or restricted to those that
participated? If available, how do we use it for the good of the NMR community?

• What organizations are interested in curating ILC data and/or selling standards created by the ILC as
validated reference standards or as blinded PT samples?

• Is there enough of a business driver for NMR reference standards and PT samples to be produced by ISO
17034 Accredited CRM Manufacturers who are also qualified to coordinate PT under ISO 17043 ?
Eurofins,USP, Sigma-Aldrich, Etc. ?????
 Role for ValidNMR Organization – My Personal View / Hope
• Provide a forum for development of PTs requested by NMR community
• Provide a centralized clearing house for “Call for Participants” in NMR Inter-Laboratory
Comparisons
• Provide a forum for development of industrial, governmental or organizational funding for
PT and ILC in identified analysis areas
• Provide a forum for development of a business model for companies to coordinate PT, ILC
and CRM production and to curate NMR data generated by such activities (thinking non-
targeted NMR databases).
• Provide a forum for the development of a central data repository for the development of
standard methods based on qNMR and non-targeted methods.

The development of NMR based standard methods, mechanisms to obtain ISO 17025
accreditation as NMR testing laboratories, and an acceptance of NMR as a routine testing
procedure are important for the future development of the NMR technique if not it’s very
existence.