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Pharmaceutical and Biomedical Analysis

Manuscript Draft

Manuscript Number: JPBA-D-13-00596R1

Title: Reference and normalization methods: essential tools for the

intercomparison of NMR spectra

Article Type: Special issue: NMR Spectroscopy

Keywords: qNMR; Quantitative NMR Spectroscopy; NMR Spectrometry; Chemical

Reference; Electronic Reference.

Corresponding Author: Prof. Serge Akoka, Ph.D.

Corresponding Author's Institution: Université de Nantes

First Author: Patrick Giraudeau, Ph.D.

Order of Authors: Patrick Giraudeau, Ph.D.; Illa Tea, Ph.D.; Gerald

Remaud, Ph.D.; Serge Akoka, Ph.D.

Abstract: This review aims at providing an analysis of the various

available reference methods in Nuclear Magnetic Resonance, based on

recent literature. This paper will provide a tool to help users in

choosing the appropriate method depending on the NMR experiment, sample

and targeted application. For each method, the crucial parameters that

should be particularly considered to reach the accuracy objectives of

quantitative NMR are described. After defining the main concepts, the

principle, the analytical performance and the main recent applications of

each method are described, and a critical discussion is finally provided.

Suggested Reviewers:

Professor Serge Akoka

(33) 2 51 12 57 07

serge.akoka@univ-nantes.fr

July 15, 2013

Jun Haginaka, Ph.D.

Editor

Journal of Pharmaceutical and Biomedical Analysis

Dear Prof. Haginaka,

Thank you very much for being interested in our work. Please

find enclosed the revised version of the manuscript entitled:

“Reference and normalization methods: essential tools for the

intercomparison of NMR spectra”

for submission as an invited Review in the special issue “NMR

Spectroscopy in Pharmaceutical and Biomedical Analysis” of the Journal

of Pharmaceutical and Biomedical Analysis.

We have carefully modified the manuscript, taking into account

your comments and suggestions and those of the 3 reviewers, as

described in the list of changes below. A highlighted version of the

manuscript is provided, where all the changes have been marked.

We trust that you will find this revised manuscript acceptable for

publication in Journal of Pharmaceutical and Biomedical Analysis, and

thank you by advance for your attention regarding this matter.

Sincerely,

Professor Serge Akoka

Response to Reviewers

List of changes

In response to referee #1: The manuscript seems to be written by different people which is allright but there is a variation in the language. It would be preferred that the same language and quality of facts are found throughout the manuscript. The electronic reference section have a higher quality to facts, some errors were found in the Chemical references section, see below. The conclusion needs some refinement.

Response: the manuscript was carefully checked by a native English speaker. The conclusion was modified (see response to reviewer 2). Abstract: Rewording is neaded, the word "critical" is used three times and makes the abstract too repetitive.

Response: the abstract was modified in order to avoid repetition. Error: Page 17 It is the side bands (i.e. the 13C satellites) of DMSO-d5 that are used, DMSO-d6 have no signal at all in a proton spectrum!

Response: it is right! The correction made in the revised manuscript. Spelling: Page 13, line 15 acetylpyrazine. Corrected. Page 16, line 5: TSP. Corrected. Page 31, line 11: stable Corrected. Page 17 and Figure 5: I don't agree or don't understand section two page 17 about the SI (line 8 to 19) and therefore see no point in Figure 5. I would for example use another method to verify an NMR-standard. There are other ways.

Response: the reviewer is right. There are other ways to determine the purity level. But the method should be primary -which is not as common as one could think- and since we are discussing on quantitative NMR we have proposed to use NMR. We added “for example” in the text to offer to the reader other possibilities as well as in the caption of figure 5. Page 20, line 3: Even though 1% is good related to accuracy however generally you which to acheive a high accuracy.

Response: the 1% accuracy was reported in the context of metabolic studies; in this case, this value can be considered as a high accuracy, as it is much lower than the concentration variations due to biological differences (typically several tens of %). The sentence was modified to indicate that this accuracy value was reported in a metabolic context. References: Some references don't have the correct format, 29, 48, 108, 112, 115?,118, 16?,

Response: the reference formats were checked and corrected. Indeed, ref 115 and 16 were correct (Plos one has a non-conventional page numbering, and eMagRes is the new version of “Encyclopedia of Magnetic Resonance” and does not have page numbers). Note that the reference numbers are modified in the new version due to the introduction of new references.

In response to referee #2: This paper reviews reference and normalization methods essential for the quantitative NMR spectroscopic analysis including metabonomics. It seems that recent literature is fully surveyed and well-ordered. This is an interesting review in the area of quantitative NMR, and the information presented will be a great help to NMR users. However, a number of points need clarifying and certain statements require more explanation. (General points) 1) 13C NMR spectroscopy : Many researchers also use quantitative 13C NMR spectroscopy, frequently coupled with 13C labeling. 13C NMR is quite different from 1H NMR in that the NMR sensitivity differs depending on the observed 13C nuclei under the usual NMR conditions, due to variable nuclear Overhauser enhancements and spin-lattice relaxation times. Although 13C NMR has been mentioned on page 6, line 11, it has not been addressed in other parts at all. Some considerations and statements are required for the lack of the description about 13C NMR.

Response: the referee is right; 13C NMR is quite different from 1H NMR. However, these differences concern the conditions needed to get quantitative spectra while the aim of this review is restricted to the reference and normalization methods which are identical whatever the nucleus. We cannot be exhaustive in a limited volume, we have therefore chosen to focus on 1H applications. A sentence has been added at the end of the introduction to explain this point. 2) quantitative NMR conditions: It is of course desirable to use so-called "quantitative NMR conditions" (page 11, line 10) for quantitative 1H NMR spectroscopy. However, this point is ambiguous in this paper, and the quantitative NMR conditions are not fully explained.

Response: we added a short sentence with appropriate references. 3) page 5 (1. Introduction) and page 31 (7. Conclusion): The conclusion is unclear and seems to be still insufficient. The aim presented in Introduction (page 5, lines 12-16) is only repeated in Conclusion (page 31, lines 2-7). Such duplication should be avoided. Some answers for the aim or concluding remarks are essentially required in the conclusion. Thus, an idea is to move some sentences (page 5, lines 10-16) in Introduction to Conclusion so that the duplication is removed and the conclusion made clearer. Additionally, the sentences (page 31, lines 11-14) should be reconsidered, as shown below.

Response: the introduction and the conclusion have been modified in order to take into account the remarks of referees. 4) English was checked by a native speaker? There are many grammatical errors and inappropriate expressions. The examples were shown below:

Page 7, line 14: The word "a" should be corrected to "an" Corrected

Page 11, line 20: The expression "The reparation of the NMR tube" is OK? Corrected: “preparation” Page 11, line 22: The words "section 3.4" should be corrected to "section 4.3. Corrected Page 12, lines 8-9: The expression ", i.e. the reference and the compound do not exist as ..(see Fig. 3)" is OK?

Corrected as “the reference and the compound are not mixed as” Page 12, line 14: The word "capillary" should be corrected to "solution" ?

The sentence has been rewritten in the text to avoid confusion Page 13, lines 17-18: There is no verb in the sentence.

Response: we do not understand the reviewer’s remark, as this sentence already contains two verbs.

Page 14, line 22: What is "the accuracy" of the gravimetrically prepared external standards ?

Response: the accuracy on the standard concentration is approximately 0.5%, as reported by Burton et al. This is now indicated in the revised version.

Page 15, line 10: The word "an" should be corrected to "a". Corrected

Page 15, line 14: The word "as" should be corrected to "to be" or eliminated. Corrected

Page 15, line 16: The word "to" should be corrected to "with". Corrected Page 15, line 18: The phrase "non-reactive with each other" should be changed to a noun phrase?

Done Page 15, line 23: What is "this product"?

It is suppressed in the text. Page 16, line 22: What does the word "which" indicate? The sentence "which is dependent enrichment" thus does not make sense.

The sentence has been rewritten to be clearer. Page 17, line 9: The word "Unit" should be corrected to "Units". Corrected Page 17, line 12: What is "uncertainty budget"?

Response: These are the words used in all analytical protocols and guides for estimating the uncertainty. It is the determination of the sources of uncertainty and the quantification of the individual uncertainties in order to get the combined uncertainty leading to the expanded uncertainty. Page 19, line 12: The phrase "the one of the sample" is OK?

Response: the initial sentence is correct, “the one” stands for “the NMR response” and does not need to be repeated twice in the same sentence. Page 20, line 7: It is unclear what the word "it" indicates.

Response: “it” was replaced by “this sample” to make it clearer. Page 21, line 14: The word "a" should be corrected to "an". Page 25, line 19: The words "the TSP one" are OK? Page 29, line 15: The words "depending of" are OK?

Response: these three points have been modified according to the referee's remarks. (Other points) 1) The character "k" of the equation S = kN (page 4, line 14) is italic, whereas the character "k" of the equation S = k[C] (page 11, line 13) is not italic. The character "k" on page 12, line 23 is also italic. In addition, the area is indicated by both of "S" and "S" in these equations. Which is right? Variables should be essentially indicated by italic characters. These descriptions are confusing although the meaning of the character "k" is different from that of "k". The authors mean that S = kN and N=k'[C], and then S=kk'[C]=K[C] ? Additionally, the character "k" is also used in the equation 4 (page 8, line14). The authors should arrange how to use these characters.

Response: In order to avoid any confusion, k is no more in italic, it is replaced by k' page 11 since it is effectively another constant and it is replaced by j in equation 4 and the following paragraph. We have added a paragraph in the definition section (section 2), page 7 to further describe k and k’. 2) page 7, line 16 Another external reference method without a capillary should be also included.

Response: Done 3) page 11, line 14: The words "Q factor" should be briefly explained.

Response: we have added a paragraph in the definition section (section 2), page 7. 4) page 11, lines 15-20: It is hard to understand the explanation. For example, it is probably effective to change the order of explanation. The description "It is assumed that kc=kR" seems to a mistake because k depends on the number of 1H nuclei which resonate at the same chemical shifts. For example, the values of k are different among methyl, methylene, and methine.

Response: as described in the answer above we have distinguished k and k’. It should now read k’. 5) page 11, lines 22-23: This description does not hold for the case of (ii) on page 12, lines 2-3.

Response: we have modified the sentences to be clearer. 6) page 12, line 7: The differences of the salinity and pH between the solutions in the outer and inner tubes also affects the quantitation? Such factors should be also mentioned here.

Response: we agree with the reviewer we have added this sentence. 7) page 12, line 11(equation 5): The significance of the equation [5] in this part is obscure, and the equation is hard to understand. The style of the equation is strange. PR is unnecessary in this equation? The word "100%" should be "x 100 (%)"? What is the material for d? Why does the issue of purity suddenly appear here? The connection from the sentence in lines 6-9 to the sentence in line 9 is not understandable. I suppose that the authors only want to explain the correction of SC/SR by multiplying VR/VC. The equation 5 is necessary? If the equation is presented, more explanation is required. The description after the equation 5 (lines 17-20) is also hard to understand in this context.

Response: this equation is directly extracted from the paper of Henderson, we have therefore chosen to keep it as it was written. We have added a sentence to be clearer and to precise that the purity of the reference compound (PR) is assumed to be 100%. 8) page 12, line 16: The VR/VC ratio seems to be basically constant irrespective of spectrometers. It only depends on the sizes of the outer and inner tubes?

Response: no, since it is the effective volume of matter detected by the coil. 9) page 12, line 24: What is the meaning of the word "NMR titration"? This word is generally used?

Response: changed to “quantitation”. 10) page 13, lines 5-8 I think that the external reference method has also an advantage that the same compound as the targeted one can be used as a reference. This point should be added here?

Response: we agree with the reviewer and we have added this sentence.

11) page 13, line-19-20: The phrase "This can be partially......sealed glass tubes," seems to be meaningless.

Response: this part of the sentence was removed. 12) page 13, lines 21-25, page 14, lines 1-8: The content is hard to understand because the significance of PULCON is not clearly explained. I understand that the significance of PULCON is in that the difference of NMR sensitivity between samples can be corrected using pulse widths.

Response: we are glad that the reviewer understood correctly the principle of the PULCON method. His explanation was inserted in the revised version. 13) page 13, line 24: The word "proportional" should be corrected to "inversely proportional".

Response: corrected as suggested. 14) page 14, lines 7-8: The sentence is errorneous. The meaning of factor f is differently explained in the literature.

Response: as described in Ref 44, “the factor f accounts for any signal loss that occurs between the excitation and the acquisition of the signal, for a possible use of different receiver gains and for incomplete relaxation in the case of short interscan delays.” Therefore, our sentence was not erroneous but only incomplete. We modified it to give a more complete description of the factor f. 15) page 16, line 25: The expression "An extension of this concept is the use of the solvent signal.." seems to be erroneous because the use of DMSO-d5 in DMSO-d6 is also "use of solvent signal".

Response: already mentioned by reviewer 1 and corrected. 16) page 17, lines 21-22: I think the expression is not reasonable because the selection of external references is much easier than that of inernal references.

Response: the word “external” was removed from the sentence to account for the reviewer’s remark. 17) page 17, lines 23-25: The content is hard to understand. I recognize the use of targeted compound itself as an external reference is the merit of the external reference method, and "the external calibration curve" (from page 18, line 3 - page 19, line 12) is an extension of the external reference method. In addition, both of the internal and the external reference methods are also a kind of calibration method. Thus, it seems to be unnatural to discriminate the calibration strategies from those reference methods. It seems to be beneficial that "the external calibration curve" is moved to the section 4.1. or after that. The external reference method (including "the external calibration curve") is similar to the absolute calibration methods used in spectrophotometric and chromatographic methods. On the other hand, the standard addition methods should be discriminated from the internal reference method.

Response: we agree with the reviewer in that the “external calibration” shares some common features with the “external reference”, and is similar to calibration methods used in other types of spectroscopy. In the same way, the standard addition procedure can be considered as a calibration approach relying on an internal reference. The external calibration and the standard addition methods have in common that they rely on a series of

sample to plot a calibration curve; this is why they are described in the same sub-section. In order to make this notion clearer, the first paragraph of section 4.4 was modified. 18) page 18, line 21: I can not understand the expression "the use of classical internal and external standards is not possible". Even if the NMR response is analyte-dependent, calibration is possible for each analyte.

Response: the reviewer is right; we should rather say that the use of classical reference methods such as those described in previous paragraphs is not possible. The sentence was modified. 19) page 19, lines 18-19: There are also the cases that S represents 1D peak area.

Response: the sentence was modified to take this remark into account. 20) page 19, line 20: The character "[C]i" should be eliminated for the clear explanation and because of no definition.

Response: removed as suggested. 21) page 22, line 1 (equation [8]): When in vivo NMR and solid-state NMR are used, how do we estimate [ERETIC]?

Response: a sentence has been added page 22 which to precise that the calibration must be previously performed using a sample of known concentration and with dielectric characteristics as close as possible to that of the measure sample. 22) page 25, line 20: The meaning of the values are obscure.

Response: the sentence was modified to be clearer. 23) page 31, line 8: What is the "ring test"?

Response: the sentence was removed. 24) page 31, line 10: What does the word "This" indicate?

Response: the sentence was modified to be clearer. 25) page 31, lines 11-14: The sentence (lines 11-12) seems to mean that the precision of few percent is acquired when the same sample is measured in separate NMR tubes under the same NMR conditions. However, this sentence is misunderstood. It seems as if no reference or calibration had been required, even though different samples with various salinity and pH are measured. The unclear meaning of the words "in this case" (line 13) also leads to misunderstanding. The main point of the sentences is considerably important, especially in relation to the external reference methods including "the external calibration curve". This main point should be stated in more suitable parts (for example, section 4.1. External reference).

Response: this part of the conclusion has been rewritten to be clearer.

26) page 46, line 7: The title is missing. The word "Biochem" should be corrected to "Biochim".

Response: Corrected as suggested. 27) page 50 (Figure 4): The solvent, sample composition, and NMR conditions are all missing. The panels a and b are both necessary?

Response: the experimental conditions are described in the cited reference there is no need to repeat them in the paper. Yes we think that is useful because as any analytical method using a standard one should assess the instrumental response (panel a) and then the calibration curve linear or not (panel b).

In response to referee #3: This review is generally well-written, comprehensive and for the most part fulfills the objectives stated in the abstract. It should be accepted with minor revision. Some suggestions for changes follow. 1. Introduction There are some areas in the Introduction (and elsewhere) where the English is wordy or slightly awkward and would benefit from editing and some condensation, e.g. P4 L6, P4 L14-25, P5 L2-7 (e.g. "to eliminate" instead of "in order to get rid of").

Response: a few words were removed or modified to make this part clearer. The manuscript was checked by a native English speaker. At P4 L18, "the absolute number of spins cannot be known" is debatable - in certain cases it could be measured at least as a concentration e.g. by dissolving a certified crystalline reference material or by use of a pure liquid as a calibrant.

Response: the sentence was modified to “the absolute number of spins is not easily accessible”. 2. Definitions P6 L2-15 The discussion advocating the use of "NMR spectrometry" is rambling and it is not clear what is actually being proposed for general use. At least, "qNMR" is short.

Response: as mentioned in the text our aim is to suggest a common language that could be useful for non NMR specialists which use quantitative NMR among other tools. As such abbreviations or short names could describe at once the type of experiments used.

P6 L17 Reference needed for the definition of a primary method. Done. P6 L20-24 Editing needed

Response: The sentence has been rewritten.

P7 L3 Point out here that Fig.1 contains equations 1 to 3 Done. P7 L16 Add within (ii): "or placed in an identical NMR tube to the one containing the compound under study, and recorded separately with precautions to compensate for changes in probe Q loading

(tuning and matching, 90o pulse measurement)." Done. 3. Normalization Some areas could be edited to condensed\ and clarify.

Response: this part has been condensed and clarified by removing some details such as the explanations on vector length and on GAN normalization (p9 line 12-17 and p10 line 15-21). A clarification has been made in equation (4) p8 as suggested by another referee. 4. Chemical references

P12 L5 add ref [17] after "reached". Done

P13 L5 Eliminate "The" at start of sentence. Done

P13 L8 Add "or separate identical tube of standard" after "insert". Done

P13 L11-15 needs editing. Note incorrect spelling of "acetylpyrazine". Done

P13 L21-25 Ref [17] was the first (2005) to introduce the idea of compensation for Q changes by means of 360o pulse measurement when using external standards in identical tubes, although the technique was not given a name or acronym at that time. PULCON came later (ref [42], 2006) but the authors were apparently unaware that the same method was fully explored and explained in [17] (and its supplementary information).

Response: we fully agree with the reviewer in that PULCON was initially proposed by Burton et al. This part was modified to fully acknowledge their contribution. Add ref [17] after "signal strength" in L24.

Response: corrected. 5. Electronic references The discussion of these techniques is balanced and makes clear the limitations of variants of ERETIC which do not allow for probe tuning effects or introduce the reference signal after the probe.

Response: the reviewer is right. 6. Discussion Generally a fair summary. Some other uses of quantitative NMR could be mentioned in the text, such as international CCQM comparisons of organic substances for chemical purity, in which 1H-qNMR has been directly (and favourably) compared with results of other techniques e.g. Westwood et al Metrologia 49, 08009, 2012. An excellent review of many qNMR applications by Pauli et al. J. Nat. Prod 2012, 75, 834-851 should be incorporated among the references.

Response: a paragraph has been added at the end of the discussion. 7. Conclusion

P31 L11 and L12 delete "s" from "stables", "percents" Corrected. References P34 L10 Accredit . Qual. Assur., 9 (2004) 55-63 (not 2009). P36 L20 Authors' names missing (R. S. Harrison, G. Ruiz-Gomez..) P43 L14 Authors' names missing (Heinzer-Schweitzer etc...)

Response: corrected. Note that the reference numbers are modified in the new version due to the introduction of new references.

In response to editor: 1. We changed our reference style: please add the reference title.

Response: the reference titles were already included in the initial version of the manuscript.

1

Critical analysis of the various available reference methods in Nuclear Magnetic

Resonance

To help users in choosing the appropriate method

The analytical performance and the main recent applications of each method

*Highlights (for review)

1

Reference and normalization methods:

essential tools for the intercomparison of NMR spectra

Patrick Giraudeau, Illa Tea, Gérald S. Remaud, Serge Akoka*

Abstract

This review aims at providing a critical analysis of the various available reference methods in

Nuclear Magnetic Resonance, based on recent literature. This paper will provide a tool to help

users in choosing the appropriate method depending on the NMR experiment, sample and

targeted application. For each method, the critical parameters that should be particularly

considered to reach the accuracy objectives of quantitative NMR are described. After defining

the main concepts, the principle, the analytical performance and the main recent applications

of each method are described, and a critical discussion is finally provided.

*Abstract

1

1

Reference and normalization methods: 2

essential tools for the intercomparison of NMR spectra 3

4

5

Patrick Giraudeau, Illa Tea, Gérald S. Remaud, Serge Akoka* 6

7

EBSI Team, 8

Chimie et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), 9

Université de Nantes, CNRS, UMR 6230, 10

LUNAM Université, 11

B.P. 92208, 12

2 rue de la Houssinière, 13

F-44322 Nantes Cedex 03, France 14

15

16

*Corresponding author: 17

Serge AKOKA, Chimie et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), 18

UMR 6230, Faculté des Sciences, BP 92208, 2 rue de la Houssinière, F-44322 Nantes 19

Cedex 03, France. 20

Tel. +33(0)2 51 12 57 07 21

E-mail: serge.akoka@univ-nantes.fr 22

*Revised ManuscriptClick here to view linked References

2

Abstract 1

This review aims at providing an analysis of the various available reference methods in 2

Nuclear Magnetic Resonance, based on recent literature. This paper will provide a tool to help 3

users in choosing the appropriate method depending on the NMR experiment, sample and 4

targeted application. For each method, the crucial parameters that should be particularly 5

considered to reach the accuracy objectives of quantitative NMR are described. After defining 6

the main concepts, the principle, the analytical performance and the main recent applications 7

of each method are described, and a critical discussion is finally provided. 8

9

10

Key words 11

qNMR; Quantitative NMR Spectroscopy; NMR Spectrometry; Chemical Reference; 12

Electronic Reference. 13

14

15

3

Table of contents 1

1. Introduction ........................................................................................................................ 4 2

2. Definitions .......................................................................................................................... 6 3

3. Normalization ..................................................................................................................... 9 4

4. Chemical references ......................................................................................................... 12 5

4.1. External reference ..................................................................................................... 12 6

4.2. PULCON ................................................................................................................... 14 7

4.3. Internal reference ....................................................................................................... 16 8

4.4. Calibration strategies ................................................................................................. 19 9

5. Electronic references ........................................................................................................ 22 10

5.1. The ERETICTM

method ............................................................................................. 22 11

5.2. The ARTSI method ................................................................................................... 27 12

5.3. Computer generated reference signal ........................................................................ 28 13

6. Discussion ........................................................................................................................ 30 14

7. Conclusion ........................................................................................................................ 33 15

References ............................................................................................................................ 34 16

17

4

1. Introduction 1

Nuclear Magnetic Resonance (NMR) is a highly versatile spectroscopic technique with 2

applications in a wide range of disciplines, from physics and chemistry to biology and 3

medicine. While its potential to provide structural information is highly recognized in all 4

chemistry and biochemistry laboratories, it also presents a considerable potential for 5

quantitative analysis. Quantitativity has been associated with NMR spectroscopy since its 6

very early days, starting with the structural elucidation of organic compounds for determining 7

the number of protons on each site of a molecule by the measurement of integrals. [1, 2] In 8

the field of analytical chemistry, the first quantitative analysis of a mixture by 1H NMR was 9

reported in 1963 by Hollis. [3] Since then, the application of quantitative NMR has been 10

reported in numerous fields, including but not limited to pharmaceutical analysis, [4, 5] 11

metabolic studies [6-8] or the authentication of natural products. [9, 10] 12

The most general equation describing the quantitativity of the NMR signal is S = k·N. In this 13

expression, S is the signal intensity (area or volume measured by integrating the target 14

frequency region), N is the number of spins responsible for this signal (proportional to the 15

concentration), and k is a proportionality constant. The latter depends on a great number of 16

factors such as the spectrometer, the probe, the NMR pulse sequence, the temperature and the 17

sample itself (relaxation times, J couplings, etc.). [11] As a consequence, determining the 18

value of k is an utopia, but quantitative results can still be obtained provided that k is stable 19

during a given experiment (or series of thereof). Another consequence is that the absolute 20

number of spins is not easily accessible, and only signal ratios can be determined. A first 21

solution is to perform relative measurements within the same spectrum (this is how the 22

integrals values are determined in structural NMR), but this does not allow the 23

intercomparison of NMR spectra. In order to be able to compare different samples, to 24

5

determine absolute concentrations and/or to determine the purity of samples, either a 1

normalization or a reference method is needed in order to eliminate the unknown k constant. 2

Numerous methods have been proposed to this end, which can be mainly divided into three 3

groups. The first one performs the normalization of a series of spectra, the second one relies 4

on the use of an internal or external chemical reference, while the third one involves an 5

electronic reference. The detailed definitions of the different reference methods are given in 6

the ―definitions‖ part below. Each group of reference methods includes a number of variants; 7

all of them have been widely described in the literature and often praised as being the ideal 8

method. Of course, each method presents its own advantages and drawbacks, and each of 9

them is generally associated with one or several application domains. 10

The present review aims at providing an analysis of the various available reference methods, 11

based on recent literature in the field. Although it is non-exhaustive, we hope that this paper 12

will provide a tool to help users in choosing the appropriate method depending on the NMR 13

experiment, sample and targeted application. We also aim at describing, for each method, the 14

critical parameters that should be particularly considered to reach the accuracy objectives of 15

quantitative NMR. After defining the main concepts, the principle, the analytical performance 16

and the main recent pharmaceutical and biomedical applications of each method are 17

described, and a critical discussion is finally provided. In this field, NMR studies have been 18

performed using different nuclei (1H,

13C,

15N,

19F, …). The experimental constraints depend 19

on the nucleus used (as the relaxation times or the nuclear Overhauser effect for example). 20

However, these differences concern the conditions needed to get quantitative spectra while the 21

aim of this review is restricted to the reference and normalization methods which are identical 22

whatever the nucleus. We have therefore chosen to focus on 1H applications. 23

24

6

2. Definitions 1

The quantitative property of NMR has been formulated in different ways in the literature. The 2

present review may be the place to harmonize the expressions. The following definition is 3

proposed: ―Quantitative NMR spectroscopy‖ could be advantageously replaced by ―NMR 4

spectrometry‖. These two terms have been used indifferently so often that there is no longer 5

any clear difference between them and therefore either may be used. Though, it is now 6

commonly accepted that ―spectroscopy‖ means the general study of spectra, and 7

―spectrometry‖ means the use of spectral information to make a quantitative measurement, as 8

in analysis; and this one has been used in one of the very first quantitative NMR experiments 9

[3]. Similarly the expression ―qNMR‖, usually given for 1H NMR, could be the abbreviation 10

for any quantitative NMR experiments whatever the nucleus studied. The type of nucleus 11

would just be added in front, e.g. 1H-qNMR or

13C-qNMR. Then the nature of the analysis 12

could be simply described, for example: (i) ―isotopic NMR spectrometry‖ or ―isotopic 13

qNMR‖ for the determination of isotope ratios or intra-molecular isotope composition; (ii) 14

―profiling qNMR‖ for the percentage of the relative composition (impurities profile, ―omics‖, 15

…). 16

According to the definition of a primary method by the CCQM (Comité Consultatif pour la 17

Quantité de Matière) [12, 13], NMR spectroscopy could fulfil the criteria for such a label 18

because there are no empirical factors in the description of the physical phenomena. However 19

it is not an absolute method as coulometry, gravimetry and titrimetry; a standard is necessary. 20

As such, qNMR is a relative method which measures the relative ratio between analytes. The 21

CCQM definitions suggest that a primary method could be either ―primary direct‖ (defined as 22

a method that measures the value of an unknown without reference to a standard of the same 23

quantity); or ―primary ratio‖ (defined as a method that measures the value of a ratio of an 24

unknown to a standard of the same quantity) [12, 13], NMR being in the second case. 25

7

Practically, there are three different quantitative NMR approaches [14]: (i) ratio 1

measurements, (ii) content measurements and (iii) purity determination. The first one is often 2

qualified as relative approach, while the two other are considered as absolute methods. Figure 3

1 depicts the position of NMR within a general analytical scheme and the equations (1 to 3) 4

describe each approach with the symbols used in the present review. 5

As any analytical method, qNMR has to be validated before routine applications on qualified 6

instruments. The analytical methods committee proposed an evaluation of general use NMR 7

spectrometers [15]. In the present paper, the word accuracy is defined as the contribution of 8

trueness + precision, as recommended in Refs. [16-18]. Finally, an uncertainty budget should 9

be established in order to define the expanded uncertainty associated to the experimental 10

result (see Ref. [19] as an example). 11

Several approaches for comparing spectra are currently used in qNMR: (i) the data 12

normalization which consists in the correction of the NMR spectra to compensate the 13

differences in dilution, extraction yield, volume transfer, water content and the receiver gain 14

encountered in the analysis of biological samples; (ii) the chemical reference which uses an 15

NMR signal from a chemical substance either mixed with the compound under study (internal 16

reference) or placed in a capillary in the NMR tube (external reference) or placed in an NMR 17

tube identical to the one containing the compound under study, and recorded separately with 18

precautions to compensate for changes in the probe Q loading (tuning and matching, 90° 19

pulse measurement); and (iii) the electronic reference where the NMR signal used for the 20

reference is created from the hardware or the software of the spectrometer as an artificial peak 21

in the spectrum. 22

As mentioned in the introduction, the basic concept of quantitative NMR depends on the 23

relation between the signal intensity S and the number of spins N giving this signal: S = k.N. 24

k is a characteristic of the spectrometer, particularly through the quality factor of the receiver 25

8

coil Q. N is related to the number of moles of the compound associated to the concentration 1

[C] and to the number of equivalent spins e leading to the equation: S = k’[C]. In the present 2

paper k’ encompasses Q, N and e. 3

4

9

3. Normalization 1

Normalization is a crucial step for the NMR quantitation of biological samples. While the 2

NMR peak areas are assumed to be proportional to the concentrations of molecules, they are 3

affected by both biological and technical variations. For example, these variations could be 4

the result of dilution phenomena, especially for urine samples. Extraction efficiency, pipetting 5

errors and differences in water content can lead to concentration changes. If spectra are 6

recorded using different numbers of scans or different instruments, the comparison between 7

the spectra is impossible without prior normalization. Therefore, a normalization step allows 8

the data from all the spectra to be directly comparable. 9

All normalization procedures scale the complete spectra so that these spectra represent the 10

same overall concentration [20]. The scaling factor of each spectrum corresponds to the 11

dilution factor of the corresponding sample. Most normalization methods depend on the 12

general equation: 13

j

j

j

n

n

old

uj

lj

dx.)x(S

)i(S)i(S

1 [4] 14

Where Sold

(i) and S(i) are the intensities of the variable i (spectral feature, bin, chemical shift) 15

before and after normalization, respectively, j is an index of the spectral regions used for 16

normalization, jjl and jj

u are the lower and upper borders of the spectral region j, for which the 17

power n of the intensities S(x) is integrated [20]. 18

The most commonly used normalization methods include integral normalization where the 19

power n is to set to 1. Integral normalization assumes that the integrals of the spectra are 20

mainly a function of the overall sample concentrations. A linear concentration series of 21

10

biological samples should result in a linear series of integrals of the corresponding spectra. 1

The spectra are normalized by dividing each signal or bin (following binning or bucketing 2

operation) of a spectrum by the total peak areas. This method is called constant sum 3

normalization (CSN) [21, 22]. It assumes that the total peak area of a spectrum remains 4

constant across the samples. However, it is not always the case for biological samples. If the 5

integral of a spectrum is dominated not only by the overall concentration but also by specific 6

changes of molecules, the CSN does not scale the corresponding spectrum correctly. Thus, the 7

robustness which is crucial for analysing highly varying biological samples is the main 8

weakness of the CSN method. 9

To compensate for this limitation, other normalization techniques have been developed such 10

as normalization to the creatine concentration [23] or to an internal quantitative reference [24] 11

(e.g., DSS-d6 or other suitable small molecules) by which the intensity of the spectrum is 12

scaled. 13

The approach of probabilistic quotient normalization (PQN) overcomes some limitations of 14

the method described above. Indeed, PQN assumes that a majority of molecule concentrations 15

remain unchanged across the samples. In the PQN method, individual spectra are compared to 16

a reference spectrum (normally a control spectrum or an average of several spectra) by taking 17

the quotient of the bins [20]. In the PQN method, the median is used as an estimation of the 18

most probable quotient rather than a single sum as the standard of normalization. The PQN 19

method was shown to be robust in a variety of metabolomics studies [25, 26]. However the 20

PQN method is insufficient for data sets in which the concentrations of a large number of 21

molecules change simultaneously. 22

Recently, a new approach of normalization called group aggregating normalization (GAN) 23

has been proposed [27]. In this method, the information about the group classification is 24

incorporated into the normalization procedure. GAN normalizes the data so that they 25

11

aggregate to the center of groups in a subspace of principal component analysis (PCA). This 1

is in contrast with CSN and PQN which rely on a constant reference for all samples. Dong et 2

al. demonstrated that GAN produces a more robust model than CSN and PQN methods [27]. 3

Figure 2 shows, as an example, a section of differential spectra (2.46-4.10 ppm) between the 4

centre of a diabetic group (i.e., mean spectrum) and a control group, following different 5

normalization procedures. The differential spectrum obtained following the GAN method is 6

similar to the differential spectrum of the original data, as shown in figures 2c and 2d. This 7

means that the GAN method is less susceptible to variations in group mean, noise and inter-8

individual difference. 9

10

12

4. Chemical references 1

The normalization process allows the comparison of spectra but it is not possible to indicate a 2

quantity of matter, such as a concentration determination or a purity determination. To 3

achieve such goals a reference should be used. As a result, a quantity of chemical substance is 4

used to provide the reference signal, the integral of which is proportional to a known amount 5

of the material. The addition of this standard can be made in the homogenous solution 6

containing the unknown compound (internal reference, calibrations) or separately as an 7

external reference. 8

4.1. External reference 9

When the quantitative conditions (for the definition of these conditions see [5, 28-31]) are 10

met, the NMR signal areas S are directly proportional to the total number of their respective 11

nuclei N, resonating at the observed frequency. N is proportional to the number of moles of 12

the molecule under study and therefore to the concentration [C]: S = k'·[C]. The constant k’ 13

(see definitions, section 2.) cannot be known with sufficient accuracy, since it depends on 14

measurement conditions, i.e. the spectrometer (especially the probe Q factor), the sample and 15

the temperatures of the probe, of the sample and of the spectrometer. For the determination of 16

the concentration of a compound ([C]), a reference substance with a known concentration 17

([R]) should be used. [C] can be calculated from equation 2 (Fig. 1) when the measurement 18

conditions are strictly identical for SC and SR. 19

It is assumed that k’C = k’R. This equality is achieved when the compound is analyzed in the 20

same time as the reference. The preparation of the NMR tube, by mixing a known amount of 21

the compound and reference, ensures that these materials occupy the same effective volume in 22

the probe: this is the internal reference approach (see section 4.3). The external referencing 23

approach does not always fulfil this condition. 24

13

Two methods can be labelled as external standards: (i) the use of coaxial inserts containing 1

the reference and (ii) the analysis of separate but identical tubes for the compound and for the 2

reference. In both cases k’C can be different to k’R, and it is then generally admitted that the 3

accuracy of such measurements cannot be better than 5% for most cases [32]. Different 4

sources can affect k’ as the differences in salinity and pH between the solutions in the outer 5

and inner tubes. Though, when precautions are taken, a precision of 1% or lower can be 6

reached [28]. A detailed work was published independently for each method. Henderson [33] 7

showed that, in the case of a capillary, the difference in the k’ values originates mainly from 8

the effective volume detected by the receiver coil of the probe, VC and VR, respectively, i.e. 9

the reference and the compound are not mixed as homogeneous liquid (see Fig 3). Therefore, 10

Henderson proposed a new relation (equation 5) which can be derived from equation 2 in Fig 11

1: 12

C

C

C

R

R

C

R

RC

d

M

V

V

S

S

M

dP [5] 13

where P is the purity of compound C (the purity of the reference compound is assumed to be 14

100%); MC and MR are the molecular weight of compound C and of reference R, respectively; 15

d is the density; S is the area or the volume of the NMR signal and VR and VC are the 16

effective volumes of the capillary tube containing R and the NMR tube containing C. 17

Thus the VR/VC ratio is a characteristic of the spectrometer. The determination of this insert 18

volume calibration should be performed on the same molecule. As a result, conditions should 19

be found for obtaining a different chemical shift for the inner (capillary) and for the outer 20

(tube) solutions, in order to avoid overlapping. As shown by Henderson [33], the chemical 21

shift phenomenon related to the pH values could be very suitable for peak separations. Burton 22

et al. [28] have addressed the issue concerning quantitative NMR measurements on separate 23

experiments for the tube containing the compound and for the tube containing the reference. It 24

is clear that k differs for each experiment, mainly due to the variation in the probe Q factor. In 25

14

that work the authors have shown that the success of the NMR quantitation depends on the 1

following factors: (i) probe tuning and matching is essential, (ii) the quality factor Q is 2

damped according to the nature of the sample (especially salt solutions). Based on the 3

principle of reciprocity, they have shown that a direct measurement of the 90° pulse length 4

may be used to correct intensity changes arising from the Q-variations. This is in fact the 5

protocol for the PULCON experiment (see section 4.2). 6

External referencing has the advantage that there is no contact between the compound to be 7

tested and the standard substance. It also has the advantage that the same compound as the 8

targeted one can be used as a reference, when the reference is in a different tube or when a 9

shift reagent or different pH are used between the coaxial insert and the main tube. Therefore, 10

the chemical compatibility (solubility, reactivity, polarity, homogeneity) between the two 11

molecules is not an issue, unlike the internal referencing (see section 4.3). Using an insert or 12

separating the standard in an identical tube has a further advantage in the preparation step: 13

once the appropriate solution of the reference is made in deuterated solvent or not (for 14

providing the lock signal), the capillary may be sealed and kept as such as long as its stability 15

allows it. Several examples are found in the literature (non exhaustive list). Just to mention a 16

few products used as reference: 1,1,2,2-tetrachloroethane [34]; 3-(trimethylsilyl)propionic-17

2,2,3,3-d4 acid sodium salt –TSP-d4- [35] it should be noticed that TSP cannot be primary 18

standard since it absorbs to glass surface; trimethylphosphate for 31

P NMR [36]; 19

acetylpyrazine [37]. 20

4.2. PULCON 21

As described above, using an external quantification standard placed in a different tube is not 22

without drawbacks, as the proportionality factor between the NMR signal and the 23

concentration may be significantly sample-dependent. In particular, it does not account for 24

probe Q-factor variations due, for instance, to a different sample salinity [28]. The PULCON 25

15

(pulse length based concentration determination) correction was introduced to deal with this 1

limitation [28, 38]. It is based on the principle of reciprocity, which basically states that the 2

90° and 360° pulse lengths are inversely proportional to the NMR signal strength [28, 39]. As 3

a consequence, the difference of NMR sensitivity between samples can be corrected using 4

pulse widths. Starting from this concept, the PULCON method was first developed by Burton 5

et al., then extended by Wider et al. to determine the concentration of protein samples [38]. It 6

consists in measuring, after tuning and matching the probe, the 360° pulse length on a sample 7

C of unknown concentration [C] and on a reference sample R of concentration [R]. The target 8

concentration can therefore be calculated by the relation: 9

ns.PW.T.S

ns.PW.T.S.R.fC

CR

RR

RC

CC

360

360 [6] 10

where PWC360 and PW

R360 stand for the 360° pulse width measured on the unknown and 11

reference samples, respectively, while T is the temperature in Kelvin and ns the number of 12

transients [40]. The factor f accounts for possible signal losses occurring between excitation 13

and acquisition, for a possible use of different receiver gains and for incomplete relaxation in 14

the case of short interscan delays. This method was further improved by Mo et al. who 15

introduced a ―receiving efficiency‖ factor to characterize how efficiently a unit magnetization 16

can be detected under the best probe tuning and matching conditions [41]. 17

The reported applications of PULCON have been mainly restricted to the area of protein 18

NMR [42-45]. Its precision for the determination of protein concentrations was assessed, and 19

a precision of 3 to 8% was reported, depending on the acquisition method used. The use of 20

PULCON for small molecule quantitative analysis is much more limited [46, 47]. It was 21

applied by Garcia-Manteiga and co-workers in a metabolomic study of lymphocyte cell 22

cultures, but the potential improvement brought by the PULCON correction was not 23

mentioned [47]. In 2005, Burton et al. showed that the precise measurement of 360° pulses 24

16

could provide direct and reliable corrections for the variations in probe Q-factors between 1

different solvents in the context of quantitative NMR of natural products [28]. The same 2

authors evaluated the factors affecting the accuracy and precision of quantitative experiments, 3

and they showed that the estimated contribution of the Q-factor variation to the overall error 4

was less than 0.2%, while the main source of error arose from the accuracy and purity of the 5

gravimetrically prepared external standards (ca. 0.5%). They also noticed that since the signal 6

amplitude is proportional to the cosine of the pulse width, a missetting of the 90° pulse by 7

several percent would be necessary to observe a loss of 0.1% of the peak amplitude. As a 8

consequence, the correction brought by the PULCON procedure seems relatively negligible 9

out of the protein NMR context where samples with very different salinities are compared. It 10

should be added that this correction requires a very precise determination of the 360° pulse 11

length for each sample, which is a rather lengthy procedure. However, a method has been 12

recently described which dramatically shortens the time necessary to determine the 360° pulse 13

length. This approach could potentially help in lightening the PULCON procedure [48]. 14

4.3. Internal reference 15

The principle of the internal referencing consists in mixing the compound and the reference as 16

a homogeneous solution. Thus the analytical protocol is: weighing of the substance used as a 17

reference, weighing of a quantity of matrix, integrating a selected NMR signal of the 18

compound and of the reference from the resulting spectrum. Equations 2 and 3 in figure 1 19

express the quantitative equation for content measurements and for purity determination, 20

respectively. 21

This approach appears as generic whatever the investigated nucleus, the type of NMR (one 22

pulse 1D or multi-pulse 1D or multi-dimensional) or the research area. But behind this 23

versatile use, constraints are associated with this methodology. Once the instrumental 24

parameters are properly set, the compound and the reference substance should fulfil the 25

17

followings: no chemical reaction between each other and/or with the solvent, complete 1

solubility, no overlapping signal (at least one peak of each should be well resolved), no slow 2

exchange (labile hydrogens, conformation, isomerisation…). It is not always easy to meet all 3

these conditions, and there is no official substance for quantitative NMR. For a given problem 4

a chemist may develop a specific reference by choosing a molecule from a catalogue or a 5

homemade one, but with no universal status. A comparative study of some of the main 6

molecules used as an internal reference has been recently published for quantification by 1H 7

NMR, covering the 0-10 ppm range [49]. Among them, dimethylsulfone was proposed as a 8

universal standard for 1H NMR [50] even for

17O NMR applied to the determination of 9

oxygenated additives in gasoline [51] and it is very useful for quantifying samples soluble 10

in water or polar solvents [52]. The silyl-derivatives are very common substances used for 1H 11

NMR, most probably because of their chemical shifts close to 0 ppm. Thus TSP-d4 [53, 54], 12

hexamethyldisilane [55], sodium 3-(trimethylsilyl)-1-propane sulfonate -DSS- [56, 57] have 13

been used. The 1H spectrum of 1,3,5-trimethoxy benzene shows two singlets (3.7 ppm and 6.1 14

ppm for the methoxy and aromatic protons respectively) and therefore it has been proposed 15

for several studies due the possible choice between two resonances for the peak integration 16

[58-60]. For polar solutions, tert-butanol found some applications [61, 62]. Others substances 17

are used, and without the ambition to be exhaustive it is worthwhile to mention some of them: 18

leucine [63]; trifluoroacetic acid for 19

F NMR [64, 65]; N,N-dimethylformamide [66]; 4-19

fluorobenzoic acid for 1H and

19F NMR [67]; hexamethylphosphoroamide for

31P NMR [68]; 20

3,4-dimethoxybenzaldehyde [69]; dioxane [70]; and tetrachloronitrobenzene [71]. Figure 4 21

illustrates the type of results which can be obtained using an internal standard, for example a 22

good linearity is achieved using tert-butanol [62]. 23

A simplification of this approach has been proposed by setting the residual proton signal from 24

the deuterated solvent as the reference. For example, the protonated portion of DMSO-d6, i.e. 25

18

DMSO-d5, is assumed to be constant in dilute solution and therefore it can be set arbitrarily to 1

100. Thus the spectra are normalized and sample concentrations can be compared [72]. 2

However, the samples under investigation have to be prepared using the same lot of DMSO-3

d6, since each batch is dependent on the level of 2H enrichment. When the concentration of 4

DMSO-d5 is known (NMR determination by the internal referencing, for example), the 5

solvent residual peak acts as a real internal substance, leading to absolute concentration 6

measurements [73]. An extension of this concept is the use of the solvent signal as an NMR 7

concentration reference. Mo and Raftery [74] showed that the water signal in 1H NMR can 8

lead to accurate (better than 2%) concentrations within a broad range (100 M to 75 M). The 9

radiation damping is often the main difficulty of these approaches when the analyte signal is 10

small compared to the solvent signal. Letot et al. [75] proposed to integrate the 13

C satellites 11

of DMSO-d5 rather than the main signal. Acetanilide was used to calibrate the 13

C satellites 12

since, as mentioned above, they vary considerably according to the DMSO-d6/DMSO-d5 ratio. 13

The ultimate step of the absolute quantitative analysis is the traceability to the International 14

System of Units (SI). Very often the purity grade of the chemical used as an internal reference 15

is the value indicated by the manufacturer. Since it is considered as a common chemical 16

(reagent, solvent…), the purity level is only indicative because there is no link to the SI. It 17

means that no complete uncertainty budget can be performed on the final result, even if the 18

purity value is not false. The last link of the quality chain is ensured by the determination of 19

the working standard purity from a certified reference material (CRM), equivalent to a 20

primary standard, by performing, for example, quantitative NMR experiments (Figure 5). In 21

that case the primary standard is the internal reference substance and the working standard 22

(molecules described above) is the compound to titrate. Several substances are available as 23

CRM and can be used as primary standards: benzoic acid [76-80]; diethylphtalate [78, 79, 81, 24

82]; bisphenol A [83], calcium formate, maleic acid and 3,5-dinitrobenzoic acid [78]. 25

19

4.4. Calibration strategies 1

As discussed above, finding the ideal internal reference compound is not always 2

straightforward. An alternative approach consists in using, as a concentration reference, the 3

targeted compound itself. This is made possible, as for many other analytical techniques, by 4

relying on calibration curves. The latter can be obtained by relying on external standards 5

containing the targeted analyte(s), which actually consists in an extension of the external 6

reference method. This is similar to the absolute calibration methods used in 7

spectrophotometry and chromatography. An alternative approach, closer to the internal 8

referencing, is to rely on a standard addition procedure. Both approaches are particularly 9

essential when the NMR response depends on the molecule, such as in multipulse or multi-10

dimensional spectroscopy. 11

External calibration curve 12

In this approach, calibration curves are first obtained by recording NMR spectra on a series of 13

samples in different concentrations, and by plotting the NMR signal (peak area or volume) of 14

the targeted compound versus its concentration determined by another method –generally by 15

gravimetry. Then, a spectrum is recorded on the sample containing the targeted analyte in 16

unknown concentration, and this concentration is determined through the equation of the 17

regression curve. This approach was successfully applied in 1D NMR [53, 61], and the 18

excellent linearity of the NMR response was highlighted. An accuracy of a few percent was 19

reported. In some cases, however, pure standards are not always commercially available. In 20

the case of biologically produced metabolites, Espina et al. demonstrated that metabolite 21

concentrations could be obtained from a calibration curve constructed with a parent 22

compound [72, 84]. They reported a precision of 5% and a trueness of 15 % for their method. 23

As discussed above, the use of an external calibration curve is particularly essential in 24

quantitative 2D NMR. Two-dimensional spectroscopy offers an invaluable alternative to 1D 25

20

methods in the case of complex samples where peak overlaps prevent the precise and accurate 1

quantification for 1D spectra, such as biological samples. However, in the case of 2D NMR, 2

2D peak volumes are influenced by factors such as relaxation times, pulse sequence delays 3

and coupling constants [85]. As a consequence, the NMR response is analyte-dependent and 4

the use of classical reference methods such as those described in previous paragraphs is not 5

possible. Therefore, the use of a calibration curve offers an invaluable solution to the problem 6

of accuracy, provided that the 2D NMR response is linear, which was demonstrated in several 7

recent studies [86, 87]. Moreover, as the aim of these quantitative 2D experiments is generally 8

to quantify a high number of analytes simultaneously, a single calibration procedure can be 9

sufficient, if the calibration samples contain all the targeted molecules. This approach was 10

successfully applied to quantify the most abundant metabolites in plant tissue extracts [88] or 11

in blood plasma [89] by heteronuclear 1H-

13C NMR. A similar strategy was used for 12

quantifying organic compounds in whole milk [34]. An accuracy of a few percent was 13

generally reported. 14

While this calibration strategy gives relatively precise results and is well adapted to the 15

simultaneous quantification of multiple compounds, it suffers from the differences that 16

inevitably occur between the model samples used for calibration and the complex sample 17

where the quantification is performed. This is the case, for example, when the sample studied 18

is a complex biological sample characterized by a high diversity of molecules. The external 19

standards used for calibration cannot fully reproduce these conditions, even though they are 20

trying to mimic them, by the use of buffers for instance. As a consequence, the NMR 21

response of the external standards may differ from the one of the sample of interest. 22

Standard additions 23

The limitations discussed above can be circumvented by relying on a standard addition 24

procedure [30, 90], which combines the advantage of an internal standard with those of the 25

21

calibration procedure. Known amounts of the target analyte (or mixture of analytes) are 1

gradually spiked to the sample analyzed. For each analyte to be quantified, a standard addition 2

curve is fitted by the linear regression equation: S = a·[C] + b, where S represents the 1D peak 3

area or the 2D peak volume and [C] the concentration of the analyte in the sample. The initial 4

concentration of the analyte in the sample is calculated by the b/a ratio, where a is the slope 5

and b the y-intercept of the linear regression curve. 6

This approach was employed with success in quantitative 1D NMR to determine the 7

concentration of impurities in heparin [91] or to quantify formic acid in fruit juices [92]. But 8

as for the external calibration strategy, it is particularly useful in quantitative 2D NMR. In two 9

recent studies, we highlighted the excellent analytical performance of the standard addition 10

procedure [93, 94]. Associated with fast homonuclear 2D NMR acquisition procedures, it was 11

applied to determine the concentration of major metabolites in breast cancer cell extracts 12

(Figure 6). An accuracy as low as 1% was reported which can be considered as very good in 13

the metabolic context making it possible to observe significant differences between breast 14

cancer cell lines. The accuracy was calculated via the recovery factor [94]. 15

The standard addition procedure is obviously a heavy and time-consuming approach, as it 16

requires taking the sample in and out of the spectrometer several times, spiking this sample 17

and recording several NMR spectra. It is therefore ill-suited for high-throughput studies 18

involving a large number of samples. However, it has two major advantages: first, it is 19

potentially extremely accurate, because the target analytes are acting as their own reference, 20

and because the whole procedure is carried out within the same NMR tube, thus avoiding the 21

inter-sample variation drawback of the external calibration approach. Secondly, the standard 22

addition procedure, via the calculation of the recovery factor, is the only method that allows 23

for accuracy determination when the true value is not known. 24

25

22

5. Electronic references 1

All the previously mentioned reference methods use a real NMR signal produced by a 2

chemical compound. Alternative strategies have been proposed using an electronically 3

produced reference signal. The ERETICTM

method (Electronic REference To access In vivo 4

Concentrations) was initially proposed as a way to avoid the addition of a reference 5

compound in in vivo NMR [95, 96]. Other methods, such as ARTSI [97] or QUANTAS, [98] 6

have been more recently proposed to try to reduce the constraints of implementing the 7

ERETICTM

method while keeping its benefits. 8

5.1. The ERETICTM

method 9

Principles 10

The basic concept of the ERETICTM

method, close to the "calibrator" proposed by P. Mahon 11

[99], is to use as a reference, not a real NMR signal but rather a pseudo-FID, synthesized by 12

an electronic device and transmitted inside the NMR probe during the reception of the NMR 13

signal. If this electronic signal has the same shape and the same frequency as an FID, it gives 14

rise, after Fourier transform, to an additional peak in the spectrum. 15

After calibration of the ERETICTM

peak, the concentration of any compound can be simply 16

determined by: 17

S

S.ERETIC.kC

ERETIC

C [7] 18

where: k takes into account the number of protons per chemical group 19

Sc = area of the peak to be quantified 20

SERETIC = area of the ERETICTM

peak 21

[C] = concentration of analyte 22

[ERETIC] = Equivalent concentration of the ERETICTM

line determined after a 23

calibration acquisition by the expression: 24

23

S

S.R.kERETIC

R

ERETIC [8] 1

Where: [R] = concentration of a calibration solution 2

SR = area of the calibration peak 3

This calibration must be previously performed using a sample of known concentration and 4

with dielectric characteristics as close as possible to that of the measured sample. The 5

reference signal is received, amplified and digitalized in exactly the same way as the NMR 6

signal; it therefore takes into account the receiver gain, the number of scans and all the 7

potential variations of the receiver chain. The ERETIC-to-NMR signal ratio is then 8

independent of these parameters. With this method there is no need to add anything inside the 9

sample and most of the drawbacks of the chemical reference methods are therefore 10

eliminated. Since the first application [99], different hardware configurations have been 11

proposed and the precision and robustness of the measurements are highly dependent on these 12

choices. 13

From practical considerations, the ERETICTM

signal is provided by one of the RF channels of 14

the NMR spectrometer and it is derived just before the amplification stage since the amplitude 15

needed is that of the regular NMR signal. The frequency, the amplitude and the phase of this 16

signal can therefore be freely chosen by the operator as NMR parameters. One of the main 17

features of the method is that the pseudo-FID has to be transmitted by an antenna which is not 18

tuned at the operating frequency in order to avoid any variation in its quality factor. When this 19

condition is fulfilled, the ratio between the NMR signal and the ERETICTM

signal is 20

independent of the NMR coil loading variation [99]. This result can be explained by the fact 21

that during the emission, the quantification can only be affected by the modification of the RF 22

flip angle, however nobody would plan to perform an accurate measurement by NMR without 23

a previous RF calibration. And during the reception, both ERETICTM

and NMR signals are 24

24

received within the same coil and a variation in the coil loading reduces both signals by the 1

same factor, keeping the ratio unchanged. 2

3

Injection of the ERETICTM

signal 4

This aspect of the method is crucial; the accuracy highly depends on the way used to inject 5

the pseudo-FID in the NMR coil. The first method used consisted in transmitting the pseudo-6

FID with an additional broadband antenna placed close to the NMR coil (Figure 7a). This 7

approach was used for in vivo applications [99, 100] and was adapted to high-resolution NMR 8

by incorporating an untuned broadband coil inside the NMR probe [101, 102]. However, there 9

are several drawbacks associated to this injection mode: (i) it requires a modification of the 10

probe, which is often difficult and can even be impossible in the case of a cryoprobe; (ii) 11

signal variations can be induced in case of a change in the mutual coupling between the 12

broadband antenna and the NMR coil; (iii) part of the RF pulses can be picked up by the 13

broadband antenna, which can damage the RF channel used for the ERETICTM

generation. 14

For in vivo applications, Marro et al. [103] proposed to introduce the pseudo-FID via 15

induction rather than radiation. The drawbacks previously mentioned are avoided, but this 16

implies cables inside the magnet bore, which is a source of inaccuracy because of parasitic 17

coupling. This problem was solved by using an optical signal transmission [104]. The method 18

was tested in clinical situation showing a good stability with a mean error of 2.83%. 19

For high resolution applications, another way to inject the pseudo-FID is to use one coil of the 20

probe which is not tuned at the operating frequency (i.e. the 1H coil in the case of a

13C-NMR 21

experiment). In order to allow the regular use of this probe, the ERETICTM

signal is then 22

introduced via a RF-coupler (Figure 7b) [105-107]. In this case, no probe modification is 23

needed and that is probably the reason why this method was the most used in the last decade. 24

However, with this configuration the method is very sensitive to the mutual coupling between 25

25

RF channels. The amplitude of the ERETICTM

signal highly depends on the Q factor of the 1

probe and it must be calibrated against a sample very similar to the analyzed one. In 2

particular, any significant variation in the ionic strength of aqueous solutions has to be 3

avoided. 4

Ziarelli et al. [108] proposed a third way to inject the ERETICTM

signal in the NMR probe 5

(Figure 7c). In this case, called PIG for ―Pulse Into the Gradient‖, the reference signal is 6

transmitted inside the probe by the gradient coil. The ERETICTM

signal is injected in the cable 7

connecting the gradient amplifier to the gradient coil of the NMR via a capacitive coupler in 8

order to electrically isolate the low voltage ERETICTM

signal source from the gradient 9

channel. The PIG method is undoubtedly the best method for the injection of the ERETICTM

10

signal. It is quite simple, it only requires a probe equipped with gradients, which is very 11

common today, and it avoids the drawbacks of the other approaches. With the PIG method, 12

Ziarelli et al. showed that the use of the ERETICTM

signal compensates for loading factor 13

variations caused by the change in ionic strength. This compensation is explained by the 14

concomitant decrease in the efficiency of both the NMR and ERETICTM

signal reception. The 15

variation of the accuracy with the ionic strength was less than 5% for NaCl solutions whose 16

concentrations ranged from 0 to 2 M [108]. 17

18

Pharmaceutical and biomedical applications 19

The applications of the ERETICTM

method in the field of biomedical NMR are numerous. 20

Two papers reported applications in imaging [109, 110]. In reference [109], the ERETICTM

21

was used to normalize cortical and medular kidney signal intensity evolution after contrast 22

agent injection. Marro et al. [110] demonstrated that the method they had previously 23

developed for MRS applications [103] could be adapted to 19

F imaging and therefore be used 24

to map quantitatively the 19

F content. The method was also used for in vivo spectroscopy. The 25

26

ERETICTM

signal was used to calculate the Pi, PCr, and ATP concentrations from resting 1

human tibialis anterior muscles [111]. The authors mentioned that this protocol could be 2

readily translated for use in patients with mitochondrial disease, where a sensitive assessment 3

of the metabolite content could improve diagnosis and treatment. Recently, the method 4

proposed in reference [104] was applied to in vivo 13

C-NMR of human skeletal muscle [112]. 5

In this study, the ERETICTM

method was cross-validated against internal and external 6

reference standards and measurements were in excellent agreement (less than 2%) for 7

glycogen and unsaturated fatty acid concentrations. 8

In the case of solid-state NMR, the usefulness of the ERETICTM

method was demonstrated 9

for both single and double resonance experiments [107, 113]. The comparison of the results 10

obtained with and without ERETICTM

indicates that the use of ERETICTM

systematically 11

reduces the standard deviation, whatever the sample or the rotor used [113]. This approach 12

was used for the precise quantification of active principles in pharmaceutical formulations 13

[114]. The Meprobamate was dosed in its commercial solid formulation (Equanil®) containing 14

other excipients (such as starch, talc, magnesium stearate, etc.) with a precision of 1%. 15

In the last years, the domain where ERETICTM

was the most applied is certainly high-16

resolution magic angle spectroscopy (HR-MAS), especially in the determination of tissue 17

metabolite levels for diagnosis and clinical prognosis of patients [74, 114-119]. As an 18

example, Albers et al. [117] evaluated the accuracy, precision, and stability of the ERETICTM

19

method as a quantitative reference in solution and human prostate tissue samples. For 20

comparison, the reliability of TSP as a quantitation reference was also evaluated. The 21

reproducibility obtained with the ERETICTM

signal as reference was superior to that obtained 22

with TSP, in both prostate surgical and biopsy samples (4.53% vs. 21.2% and 3.34% vs. 23

31.8%, respectively). 24

27

In high resolution NMR of liquids, the biomedical applications of the ERETICTM

method 1

concern the quantitative analysis of complex mixtures [120, 121]. The method was recently 2

used to simultaneously determine the concentrations of five active alkaloids in Rhizoma 3

Coptidis, a traditional Chinese medicine [122]. The relative accuracy error was less than 3% 4

for a concentration range of 0.1 to 20 mM and less than 0.9% for a concentration range of 1.0 5

to 20 mM. Lokeren et al. [121, 123] combined the ERETICTM

and DOSY methods in order to 6

determine the power and limitations of different processing algorithms in discriminating 7

diffusion coefficients in multi-diffusion regime. 8

5.2. The ARTSI method 9

As previously mentioned, with certain hardware configurations, the ERETICTM

signal does 10

not accurately track the changes in probe efficiency. Other electronic reference methods have 11

therefore been proposed in order to circumvent this drawback and the first one was the 12

ARTSI method (Amplitude-corrected Referencing Through Signal Injection [97]. As for the 13

ERETICTM

signal, the ARTSI signal is provided by one of the RF channels of the NMR 14

spectrometer and it is derived just before the amplification stage. However, in the case of the 15

ARTSI method, the reference signal is not sent through the NMR probe; it is directly sent to 16

the receive path of the spectrometer, using a directional coupler between the probe and the 17

preamplifier (Figure 8b). Thereby, the ARTSI signal follows any variation of the receiver 18

chain efficiency but it is not naturally modulated by the effective probe sensitivity changes 19

induced by the differences in sample properties. The 90° pulse width is therefore used to 20

measure the quality factor change, accordingly to the reciprocity principle [39], and the power 21

of the ARTSI signal is then scaled as follows [97]: 22

PW

PW.ERPWRERPWR

C

R

RC

360

360 [8] 23

28

where ERPWRC is the ARTSI signal power used, ERPWRR is the ARTSI signal power used 1

on a reference sample for which the 360° pulse width was PWR360

, and PWC360

is the 360° 2

pulse width measured for the sample. 3

In order to show how this strategy can compensate for loading factor variations, the 4

concentration of 2 mM sucrose samples with increasing NaCl concentrations (0 to 250 mM) 5

was measured using the ARTSI method and the ERETICTM

method (with injection via one of 6

the probe coils) [97]. The accuracy of the ARTSI method was found better than that of the 7

ERETICTM

method using injection via a RF coil of the probe (about 10 % for NaCl 8

concentrations ranging from 0 to 250 mM) but is below the performance of the ERETICTM

9

method using injection via the gradient coil [108]. 10

5.3. Computer generated reference signal 11

A few years ago, Farrant et al. proposed a method where the artificial reference signal is no 12

longer injected into the spectrometer but only mathematically generated and merged with the 13

spectrum during the data processing, to be used as a quantitative reference [98]. 14

The artificial signal is first scaled against a reference sample of known concentration, then a 15

nominal concentration is attributed to it. This signal is then added to the spectra of analyzed 16

samples, with a chemical shift which can be freely chosen. The intensity of the artificial 17

signal is modulated in order to take into account differences between acquisitions performed 18

on the analyzed sample and on the reference sample respectively: receiver gain, number of 19

scans and eventually nature of the solvent. As mentioned by the authors themselves: 20

"Naturally, similar scaling methods operating directly upon the absolute intensity of the 21

integrals also work, without the need of adding the artificial signal". The authors called this 22

method QUANTAS for QUANTification by Artificial Signal. As for the ARTSI method, the 23

reference signal is not naturally modulated by the effective probe sensitivity change induced 24

by the differences in sample properties. In the case of samples significantly different from the 25

29

reference tube, the loading changes are corrected using the PULCON strategy. However the 1

artificial signal does not test the receiver path and requires the accurate consideration of a 2

wider range of parameters than required for ARTSI or ERETICTM

. 3

In order to show how this strategy can compensate for loading factor variations, the 4

concentration of 50 mM sucrose samples with increasing NaCl concentrations (0 to 700 mM) 5

was measured with PULCON correction. The results are very similar to those obtained with 6

the ARTSI method [97]. This method is now included in the Bruker software as ―ERETIC 2‖, 7

even though it is very far from the original ERETIC method. A very similar method was also 8

implemented by Walker et al. in the context of drug metabolism studies [124]. This method is 9

named aSICCO (artificial Signal Insertion for Calculation of Concentration Observed). The 10

method was evaluated against the internal reference method and produced similar results, i.e. 11

accuracy within ±5%. In the two papers [98, 124], a good week-long stability was observed 12

(±5%) and the authors attributed it to the high degree of reproducibility of the spectrometers 13

they used. 14

The QUANTAS method was recently applied to the determination of the absolute 15

concentration of metabolites (glucose, lactate, alanine, glutamate, GABA, glutamine and 16

aspartate) in brain extracts [125], and a similar approach was used for the analysis of fruit 17

juices [126]. 18

19

20

30

6. Discussion 1

As shown above, numerous NMR methods have been proposed in order to be able to compare 2

different samples, to determine absolute concentrations and/or to determine the purity of 3

samples. They have been presented in the previous sections of this paper and are divided into 4

two groups. The first one relies on the use of an internal or external chemical reference, while 5

the second one involves an electronic reference. 6

Each of these methods presents its own advantages and drawbacks, and each of them is 7

generally associated with one or several application domains. The review of the literature 8

shows that good accuracy and precision (around 1%) can be achieved with any of these 9

approaches but it can be very tedious and time-consuming for some of them. 10

For each method, one or several aspects are especially critical and must be carefully chosen or 11

optimized. When a chemical reference is used, the choice of the reference compound is of 12

particular importance. As an example, Rundlöf et al. showed that significantly different 13

results can be obtained in the determination of the active principal concentration of 14

paracetamol depending on the reference compound used [49]. Concerning the applications of 15

the ERETICTM

method, the way used to inject the pseudo-FID in the NMR coil is crucial and 16

the accuracy obtained highly depends on it. Finally, the external chemical reference method 17

and some of the electronic reference methods do not account for probe Q-factor variations 18

due, for instance, to a different sample salinity [28]. Therefore, the PULCON correction must 19

be applied in order to deal with this limitation [38], although the impact of this correction 20

procedure is not always obvious out of the protein NMR context [28]. Therefore, none of 21

these methods can be seen as universal, and it is essential to choose between them taking into 22

account: (i) the target precision, (ii) the application domain and (iii) the experimental 23

constraints. 24

31

Certain application domains are not compatible with a given referencing method. Thereby, 1

internal reference and standard additions cannot be used for in vivo or ex vivo studies. And 2

from a general point of view, it is often hazardous to use these methods in complex matrices. 3

The concept of 1H NMR invisibility was reported [127]; it corresponds to the immobilization 4

by non-specific binding of the interacting compound and therefore it can significantly affect 5

the accuracy. Furthermore, the internal reference method is not straightforward to implement 6

in solid-state NMR because of the difficulty to prepare a homogeneous mixture of the sample 7

and of the reference. This is probably the reason why the electronic reference has been 8

extensively applied during the last years to solid and HR-MAS studies (see section 5). 9

Experimental constraints are very important criteria for the choice of the referencing method. 10

The hardware configuration has to be considered and can be an obstacle to the 11

implementation of methods like ERETICTM

. However, this difficulty can generally be 12

circumvented with the help of the spectrometer manufacturer. 13

The time needed for the complete experimental procedure is also particularly relevant, 14

especially when numerous samples are analysed. Standard additions could appear as very 15

efficient as they cumulate the advantages of the internal reference and of the calibration curve. 16

Furthermore, this method is not affected by different response coefficients for different peaks 17

induced by multipulse sequences or partial saturation. However, this method implies a very 18

long time because of the multiple additions and also requires having pure standards available 19

for all targeted compounds. 20

Finally, the experimental time is governed by the longest longitudinal relaxation time (T1). In 21

the case of a chemical reference, it is therefore essential to include the T1 of the reference 22

compound in the criteria of choice and to keep in mind that it depends on the temperature, the 23

solvent and the concentration. 24

32

Although we tried to survey the recent literature in the domain, it is obvious that there are still 1

some uses of quantitative NMR that are not evocated in the text. We can mention an 2

international CCQM comparison of organic substances for chemical purity, in which 1H-3

qNMR has been directly compared with results of other techniques [128]. All the participants 4

of this comparison who used quantitative NMR as a major or contributing technique and 5

included it as part of, combined it or confirmed it with a conventional 'mass balance' data 6

estimate, obtained very good results. Furthermore, an overview of literature on quantitative 7

1H-NMR methodology and its applications in the analysis of natural products can be found in 8

the review of Pauli et al. [29]. 9

10

11

33

7. Conclusion 1

In this paper we have presented the various available reference methods and their main 2

applications in the field of pharmaceutical and biomedical analysis. There is actually no 3

universal method, and it is often difficult for NMR users to determine which method would be 4

best suited for their particular sample and application. Reference methods are often well 5

described in review articles on quantitative NMR [5, 30, 129-131]. However, their critical 6

analysis is much more difficult to find in the literature, and often restricted to the comparison 7

of a limited number of specific methods, such as the comparison of different electronic 8

references [132], or the assessment of different chemical standards [28, 58]. 9

In the context of hardware improvements making NMR spectrometers more and more stable, 10

it is probable that a precision and accuracy of a few percent will be reached without any need 11

of calibration between consecutive spectra when the same sample is measured in separate 12

NMR tubes under the same NMR conditions. However, even in this case, the use of a 13

reference method should be useful in order to compensate for any drift of the spectrometer 14

due to aging or failure and to ensure traceability. 15

16

Acknowledgements 17

The authors are grateful to Michel Giraudeau for linguistic assistance. 18

19

34

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24

25

26

49

Figures 1

2

Figure 1: Schematic description of qNMR as a primary analytical technique. 3

n: mole number 4

N: number of equivalent spins under the NMR peak 5

m: mass 6

M: molecular weight 7

P: purity 8

S: area or volume measured by integrating the target frequency region 9

x, y, C, R stand for compound x, compound y, compound to be studied, reference, 10

respectively 11

nx/ny = molar ratio between x and y 12

nC/m = number of mol of compound C in a weight m of a given matrix. m can be changed in 13

V: volume of a given matrix 14

15

50

1

2

Figure 2: Comparison of different normalization methods on a metabolomics study. A 3

section of differential spectrum between the mean spectra of the diabetic group and the 4

contral group; (a) Data normalized by the constant sum normalization (CSN) method; (b) 5

Data normalized by the probabilistic quotient normalization (PQN) method; (c) Data 6

normalized by the group aggregating normalization (GAN) method; (d) Original data. From 7

Dong et al. [27], with permission from the editor. 8

9

51

1

Figure 3: Schematic illustration of the internal and the external referencing (b) and (c) 2

respectively. The illustration (a) shows that the volume of sample which is detected by the 3

coil, Vd, is smaller than the volume occupied by the sample in the NMR tube, Vsample. In the 4

external standard method (c) the volume of the reference, VR, is different from the volume of 5

the compound to be studied, VC. 6

7

52

1

Figure 4: Titration of arginine using tert-butanol as an internal reference. (a) instrumental 2

NMR response (Integral) with respect to the concentration of arginine; (b) linear calibration 3

curve relationship between the actual arginine concentration (theoretical) and the arginine 4

concentration measured by NMR (experimental); (c) repeatability of the method during a 5

short period. From Watanabe et al. [62], with permission from the editor. 6

7

53

1

Figure 5: Illustration of the relationship between the results of a qNMR experiment: purity or 2

concentration [C] determination, using a chemical as reference and the primary standard. 3

Each step (arrows) may be achieved by qNMR: these links ensure the traceability and thus 4

allow the result to be expressed in SI unit. It should be noticed that any other primary methods 5

may be used to ensure these links. For a definition of each standard see ref [133]. Each 6

standard may be of different chemical nature. 7

8

54

1

Figure 6: (a) Concentration of major metabolites in three different breast cancer cell extracts, 2

determined from spatially-encoded 2D COSY NMR spectra via a standard addition 3

procedure. The concentrations are normalized to a 100 mg mass of lyophilized cells. (b) 2D 4

COSY NMR spectrum of a SKBR3 breast cancer cell extract, recorded in 20 minutes at 293 5

K on a 500 MHz Bruker NMR spectrometer equipped with a 1H/

13C dual cryoprobe. 6

Reprinted with permission from Ref. [93]. Copyright (2012) American Chemical Society. 7

8

55

1

Figure 7: Three main methods used for the injection of the ERETICTM

signal using a 2

spectrometer equipped with three RF channels, one gradient output and a dual probe. M 3

stands for the coil used for transmitting and receiving the NMR signal, S stands for the 4

secondary channel (eventually used for decoupling), and G stands for the gradient coil. In (a) 5

the ERETIC signal is transmitted through an additional antenna placed inside the probe, in (b) 6

the ERETIC is transmitted by the secondary channel thanks to a directional coupler and in (c) 7

the ERETIC signal is transmitted by the gradient coil thanks to a capacitive coupling. 8

9

56

1

Figure 8: Three approaches to use an electronic signal as a reference for quantitative NMR. 2

In the ERETICTM

method, the reference signal is generated by one of the RF channels of the 3

spectrometer and transmitted inside the probe to be received as a regular FID (a). In the 4

ARTSI method, the reference signal is also produced by one of the RF channels, but it is 5

injected using a directional coupler between the probe and the preamplifier (b). In other 6

methods (QUANTAS, aSICCO), the reference signal is only mathematically generated and it 7

is inserted into the spectrum during data processing (c). 8

Primary method Direct method

Ratio method

Ratio

measurements

Content

measurements

Purity

determination

x

y

y

x

y

x

N

N

S

S

n

n

Equation 1 Equation 2

R

C

R

R

C

C

R

R

CC P

m

m

M

M

N

N

S

SP

Equation 3

RR

RC

R

R

CC Pm

m

M

1

N

N

S

S

m

n

Figure-1

Figure-2

Vsample Vd

VR

VC

VC+R

(a)

(b)

(c)

Figure-3

(c)

(b)

(a) Figure-4

Primary

standard

(CRM)

Traceability versus SI unit

Working

standard

Purity

or [C]

Primary

direct

methods Primary ratio method: qNMR

Secondary

standard

Figure-5

0

1

2

3

4

5

6C

on

ce

ntr

ati

on

(m

M) MCF-7

SKBR3

Cal 51

0

5

10

15

20

25(a) (b) Figure-6

Duplexer P

rob

e

G S M

RF3

Gradient

RF1

Receiver

RF2

Additional

antenna

(a)

Duplexer

G S M

RF3

Gradient

RF1

Receiver

RF2

Directional

coupler

(b)

Pro

be

Duplexer

G S M

RF3

Gradient

RF1

Receiver

RF2

Capacitive

coupling

(c)

Pro

be

Figure-7

Duplexer

RF2

RF1

Receiver

Directional

coupler

(b) Probe

Computer

Duplexer

RF2

RF1

Receiver

(a)

Probe

Computer

Additional

antenna

Duplexer RF1

Receiver

(c)

Probe

Computer Artificial

signal

Figure-8