High-Performance Quantitative 1H-NMR (HP-qNMR®) yields organic certified

reference materials (CRM) with traceability and low measurement uncertainty

A. Rueck, C. Hellriegel, R. Sauermoser, J. Wuethrich, M. Weber – Sigma Aldrich GmbH, Industriestrasse 25, CH-9471 Buchs Switzerland A. Nichols – Sigma Aldrich, 595 North Harrison Road, Bellefonte, PA 16823 USA

References [1] Weber M, Hellriegel C (2010) GIT Labor-Fachzeitschrift, 07, 527 [2] Malz F, Jancke H (2005) J Pharm Biomed Anal, 38(5):813- 23 [3] Eurachem/CITAC Guide, 1-37, 1st Ed. (2003) “Traceability in chemical measurement” [4] Eurachem/CITAC Guide, 1-120, 2nd Ed. (2000), “Quantifying uncertainty in analytical measurement” [5] DeBièvre P, Dybkaer R, Fajgelj A, Hibbert B (2011) Pure Appl. Chem.Vol 83 (10): 1873-1935 [6] Weber M, Hellriegel C, Rück A, Sauermoser R, Wüthrich J (2013) Accred Qual Assur, online

Summary

Introduction Validation experiments: 6 Different traceability chains for Maleic acid

Quantitative NMR spectroscopy (qNMR) has become an invaluable instrument for the exact content assignment and quantitative determination of impurities [1]. A main property of 1H-qNMR is, that it is a relative primary method, as the signal intensity is directly proportional to the number of protons contributing to the resonance [2]. It is therefore possible to directly compare the signal intensities of a substance to quantify and a reference standard, and in doing so, the structures of the substances are fully irrelevant. Thus, the measurement results are directly traceable to an internationally recognized primary reference standard, e.g. NIST SRM, and therefore traceability to SI units is obtained [3]. Chromatographic techniques such as HPLC or GC usually do not provide this traceability, because they require a highly pure standard of the substance of interest, which is often not available. Thus, chromatographic purities are often given in area %, whereas the content determination by qNMR gives a result in weight %. In this work, we demonstrate the power of the qNMR technique by optimizing the combination of qNMR with metrological weighing. It is possible to certify the purity of organic reference materials (expressed as mass fraction) with relative expanded uncertainties of < 0.1 % for a 95 % confidence interval (k=2). Following well-defined selection criteria, a set of 13 different chemical compounds is evaluated and certified to serve as internal references for 1H-qNMR measurements. Comparison measurements is made amongst a sub-set of the selected compounds. The purity of maleic acid is determined by 6 different 1H-qNMR measurement series and all results show full consistency. In 2 more measurement series, four different nuclei are analysed within the same sample against one calibrator. Even with non-optimised signal intensity ratios and varying signal pattern a high consistency was obtained. Therefore, the validity and robustness of 1H-qNMR measurement results are demonstrated. All experiments are performed under ISO/IEC 17025 and ISO Guide 34 accreditation [4].

This poster shows that 1H-qNMR, combined with metrological weighing, can be optimized to obtain results to certify the purity of organic reference materials (expressed as mass fraction) with less than 0.1 % relative expanded uncertainty for a 95 % confidence interval (k=2). For this reason, the authors decided to call this approach “High-performance qNMR” (HP-qNMR®) [6]. The validity, accuracy and robustness of the technique was proven by a series of experiments. The purity of maleic acid was determined by six different 1H-qNMR measurement series and all results showed full consistency. All the six mean values are covered within the range of +/- 0.05 %. 1H-qNMR measurement results are directly traceable to a variety of internationally accepted primary reference materials, and therefore traceability to SI units is obtained.

www.sigma-aldrich.com/organiccrm

Fig. 2+3 Validation of qNMR measurement results for the certification of maleic acid purity through 6 different traceability chains. Upper values refer to the assigned purity expressed as mass fraction in %, lower values refer to the corresponding expanded absolute uncertainty of the mass fraction value in % (k=2). The 6 different values assigning a purity value for MA are given in bold. The comparability of these 6 measurement results is further illustrated in Fig. 3 (below).

According to De Bièvre et al. [5], a metrological traceability scheme can be drawn: An internationally accepted primary reference material serves as the primary calibrator and ensures SI traceability. A set of well selected substances serves as 1H-qNMR references, whereby the certified mass fraction content can be assigned to each of these calibrators. Sigma-Aldrich provides up to now a set of 13 of these calibrators (see figure 4). This set of 1H-qNMR calibrators is then used to assign certified values to a wide range of organic substances (samples), i.e. pesticides, pollutants, drugs, metabolites and many others. Sometimes the sample purity (expressed as a mass fraction) can also be assigned by direct measurement against a primary calibrator. Influence of signal intensity and pattern (example: Ethyl 4-(dimethylamino)benzoate)

Fig.5: Some molecules offer more than 1 signal for purity determination and in most cases also different signal patterns. Are these 2 effects of significant relevance? Using maleic acid as internal calibrator, the purity of Ethyl 4-(dimethylamino)benzoate (EDAB) was assigned evaluating four different protons. Two experiments were realized with different optimized ratios of signal intensities between EDAB and calibrator. The optimal ratio of 1:1 signal intensity for each experiment is indicated by a black dot.

Metrological traceability scheme (example: Caffeine)

Fig. 4 Set of 1H-NMR spectra of 13 CRM to serve as qNMR standards covering a wide range of chemical shifts and different solubilities.

Fig. 1 shows caffeine as an example. Caffeine is certified using 1H-qNMR with respect to maleic acid as the (secondary) calibrator, and maleic acid is certified by 1H-qNMR against the primary calibrator KHP from NIST. Therefore the traceability to the SI unit ampere is obtained by coulometry as the primary measurement technique.

• 6 different mass fraction values for the same MA were observed • 3 of the results came from a direct comparison measurement to a primary calibrator (Exp. A to C) • 2 of the results were generated by a three-step comparison measurement (Exp. D and E) • 1 result was observed from a four-step comparison measurement (Exp. F) • all 6 results show full consistency in terms of overlapping of the uncertainty budgets with all the other mean values (Fig. 3) • the relative standard deviation of the 6 mean values from Exp. A to F is 0.04 % and all the 6 measurement results are covered

within a range of +/- 0.05 % • the direct measurements (Exp. A to C) show the lowest uncertainties (all below 0.09 % relative) and due to uncertainty

propagation, the uncertainty values are higher for Exp. D to F • Exp. F comprises 4 comparison measurements and therefore shows the highest uncertainty. Nevertheless, Exp. F shows the

enormous potential of the 1H-qNMR method: the content of MA was confirmed within an expanded relative standard uncertainty of < 0.2 % although this value was generated over a series of 4 comparison measurements using 3 intermediate references

BA Benzoic acid KHP Potassium hydrogen phthalate MA Maleic acid DNB Dinitro benzoic acid DMSO2 Dimethyl sulfone

Suitable substances as qNMR reference materials

• availability in very high purity • non-hygroscopic and non-volatile • low chemical reactivity and toxicity • simple signal pattern (few signals only) • chemical shift of signals covering different

areas • low ratio of isochronic protons to molecular

mass • solubility in multiple deuterated solvents • high chemical stability • purity under double accreditation ISO/IEC

17025 and ISO Guide 34 • all these CRMs are certified with relative

expanded standard uncertainty values of 0.08 % to 0.17 %

Exp.1: the mass ratio of sample and calibrator was optimized to obtain a 1:1 peak ratio of MA and the 6 methyl protons of the amino group of EDAB Exp.2: the mass ratio of sample and calibrator was optimized for a 1:1 peak ratio of MA and the ethyl group of EDAB (2 isochronic nuclei) Results: • comparability of experiments with optimized signal intensity ratio is extremely high: almost identical values of the 3 results

shown with black dots • all 8 values show a good consistency in the range of +/- 0.07 % relative standard deviation of the mean values • the signal pattern (singlet vs. multiplet) seems not to be of highest relevance in terms of accuracy (see 3 black dots)

15639 3,5 Dinitrobenzoic acid

40384 1,2,4,5-Tetrachloro3-nitrobenzene

03826 Calcium formate

06185 Benzoic acid

14659 Potassiumphthalate monobasic

92816 Maleic acid

07038 Dimethyl terephthalate

41867 Dimethyl sulfone

74658 Durene

06856 Duroquinone

89151 Dimethylmalonic acid

2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5 ppm

55177 Benzyl benzoate

42582 Ethyl 4-(dimethlamino) benzoate

QUANTITY UNCERTAINTY ( k =2)

CALIBRATOR or SAMPLE ACTION MEASURING

SYSTEM MEASURING PROCEDURE

U rel ( w KHP )

= 0.0076 %

U rel ( w MA )

= 0.08 %

U rel ( w Caffeine )

= 0.2 %

balance NMR instr.

HP - qNMR procedure

mass fraction of MA in calibrator w MA = 99.78 %

calibrator MA

sample of Caffeine

mass fraction of Caffeine in sample w Caffeine = 99.9 %

HP - qNMR procedure

balance NMR instr.

Primary measurement procedure coulometry

primary meas. system coulom. titrator mass fraction of

total acid exp. as KHP in primary calibrator w KHP = 99.9934 %

primary calibrator KHP

SI

CR

M p

rod

uce

r N

MI

[mas

s fr

acti

on

, %]

99.5

99.6

99.7

99.8

99.9

100.0

NIST - BA

350b

MA

Exp. A

MA

BA

DNB

Exp. D

MA

DMS

DNB

Exp. E

MA

BA DMSO2

Exp. F

NIST - BA

39j

MA

Exp. B

NIST - PPM

84l

MA

Exp. C

Pu

rity

of

Mal

eic

Aci

d (

MA

) 99.5

99.6

99.7

99.8

99.9

100.0

NIST - BA

350b

MA

Exp. A

NIST - BA

350b

MA

Exp. A

MA

BA

DNB

Exp. D

MA

BA

DNB

Exp. D

MA

DMS

DNB MA

DMSO2

DNB DNB

Exp. F Exp. F

NIST - BA

39j

MA

Exp. B

NIST - BA

39j

MA

Exp. B

NIST - PPM

84l

MA

Exp. C

NIST - KHP

84k

MA

Exp. C

T413049