Industrial Crops and Products 50 (2013) 904908

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Using SNIF-NMR method to identify the adulteration of molasses

spirit vinegar by synthetic acetic acid in rice vinegar
Chang-Wei Hsieh a, , Po-Hsien Li a , Ju-Yun Cheng b , Jung-Tsun Ma c
a
Department of Medicinal Botanical and Health Care, Da-Yeh University, No. 168, University Road, Dacun, Changhua 51591, Taiwan, ROC
b
Department of BioIndustry Technology, Da-Yeh University, No. 168, University Road, Dacun, Changhua 51591, Taiwan, ROC
c
Taiwan Miles Biotechnology, 13, Lane 185, Sec 3 Chungsan Road, Changhua City 50075, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: Vinegar is one of the most commonly used sauces in Asia. However, cheap synthetic acetic acid or molasses
Received 26 March 2013 spirit vinegar is widely used to adulterate pure fermented products. The aim of this study was to identify
Received in revised form 30 July 2013 and evaluate pure rice vinegar by three model of rice vinegar authentication test. According to the analysis
Accepted 1 August 2013
of SNIF-NMR method, the (D/H)CH3 values for rice vinegar, molasses spirit vinegar, and synthetic vinegar
are 98.50, 108.46 and 131.58 ppm. The results of (D/H)CH3 showed that there are signicant differences
Keywords:
for different materials (p < 0.05). In the rice vinegar authentication model test, the results showed that
SNIF-NMR
(D/H)CH3 increases with the addition of molasses spirit vinegar or synthetic vinegar, and that there is a
Adulteration
Rice vinegar
good linear correlation (R2 > 0.97). According to these results, we consider the SNIF-NMR method to be
an effective way of determining the degree of adulteration in fermented rice vinegar.
2013 Elsevier B.V. All rights reserved.

1. Introduction degeneracy of the nuclear energy levels to be lifted. The elec-

Vinegar is one of the most widespread and common products
in the world (Mazza and Murooka, 2009). It is usually used as a
food additive to improve the taste, odor, and color of food prod-
ucts. In conventional industrial manufacturing of vinegar, there are
two steps for fermentation: alcoholic fermentation and the trans-
formation of alcohol to acetic acid. The quality and authenticity of
vinegar are of particular importance because of the large quanti-
ties of vinegar consumed in daily diets. The main adulteration is
the adding of cheap synthetic acetic acid to vinegar. However, the
content of synthetic acetic acid must be declared on the packaging
to inform consumers for safety reasons.
Many researchers have indicated that stable isotope ration anal-
ysis (SIRA) is a reliable method for identifying adulteration in
food (Rossmann, 2001). The main reason is that each plant has
its unique natural stable isotope, including 13 C, 2 H and 18 O, and
the distribution of those stable isotopes is affected by certain
physical and biochemical processes (Haines and Montague, 1979).
SNIF-NMR (site-specic natural isotopic fractionation by nuclear
magnetic resonance) is an accurate and effective analytical method
for detecting the isotope ratio on a specic position of a molecule
(Martin and Martin, 1990; Martin et al., 1983, 1986).
Deuterium nucleus with a spin 1 has a quadrupole moment
which will interact with an electric eld gradient causing the
Corresponding author. Tel.: +886 4 8511888x6230; fax: +886 4 8511326.
E-mail address: welson@mail.dyu.edu.tw (C.-W. Hsieh).
tric eld gradient is provided by an asymmetric distribution of
electron density around the nucleus. The trace of the electric
eld gradient tensor is nearly zero in solution, so the quadrupole
interaction is averaged to zero. Deuterium NMR has the same
chemical shift range as for proton NMR. Deuteriumdeuterium cou-
plings are about 40 times smaller than protonproton couplings
and are therefore not observed. However, in partially deuterated
molecules small protondeuterium couplings can be observed.
Using deuterium NMR spectroscopy, this technique is able to mea-
sure non-statistical distribution of deuterium in different sites of
a given molecule. For a natural substance, SNIF-NMR provides
the information about the chemical pathway of biosynthesis and,
in some cases, the geographic region of the sample (Martin and
Martin, 1990).
It is hard to identify the source of acetic acid in vinegar by
common chemical analysis. Remaud et al. (1992) were the rst to
use the SNIF-NMR method to examine adulteration in vinegar. The
method was based on the ratio of deuterium to hydrogen (D/H) at
a specic position (methyl group) of acetic acid obtained by fer-
mentation through different biosynthetic mechanisms that result
in different isotopic ratios.
In Asian countries, there is still no proper way to identify adul-
teration in vinegar. The developing of such a detection method for
adulteration in vinegar is urgently needed, as well as the estab-
lishing of a databank. In previous studies, it was found that the
SNIR-NMR method could be applied for the identication of rice
wine made with different Taiwan rice species (Hsieh et al., 2005).
A convenient sample preparation method for SNIF-NMR analysis

0926-6690/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.08.014
 C.-W. Hsieh et al. / Industrial Crops and Products 50 (2013) 904908
which reduces the work of sample preparation has also been devel-
oped (Ko et al., 2013). Therefore, the goal of this study was to
identify and evaluate the degree of adulteration in rice vinegar by
the SNIF-NMR method in order to have the best quality control for
consumers.
2. Materials and methods
2.1. Microorganisms and chemicals
Three varieties of rice, Taikeng 9 (TK-9), Taichung sen 10
(TCS-10) and Tainan 11 (TN-11), were obtained from the District
Agricultural Research and Extension Station, Taichung, Taiwan.
The 95% edible alcohol (a molasses spirit; MS) produced from
cane molasses was purchased from the Taiwan Sugar Corpora-
tion, Taiwan. The microorganism species Rhizopus formosaensis
(BCRC 31150), Saccharomyces sake (BCRC 20262) and Acetobac-
ter aceti (BCRC 12325) were obtained from the Food Industry
Research and Development Institute (FIRDI) in Taiwan. Acetic acid
(99.99%) and N,N-tetramethylurea (TMU, 99%) were obtained from
SigmaAldrich (St. Louis, MO, USA). All solvents and other chemi-
cals, including diethyl ether, mannitol, yeast extract and peptone,
were analytical grade and purchased from Katayama Chemical Co
(Osaka, Japan).
2.2. Fermentation of rice into rice wine
Rice wine was made with a 1:1 ratio of rice grains (10 kg) and
water (10 kg). The rice/water mixture was simmered at 125 C
for about 60 min and cooled to room temperature (25 C). R. for-
mosaensis (BCRC 31150) was then inoculated (3 107 cells/mL)
into the rice to produce amyloglucosidase, -amylase and -
glucuronidase. These enzymes hydrolyzed the starch polymers into
smaller starch oligocarbohydrate polymers and monosaccharides,
and also liqueed the rice for further and complete fermentation.
When the temperature reached 25 C after approximately 60 min,
at 2.8 107 cells/mL S. sake (BCRC 20262) was inoculated into the
rice, 30 kg water was added and then the temperature was main-
tained at 25 C and was fermented for 12 days.
2.3. Fermentation of wines into vinegars
The three rice wines and MS were diluted with water to 7%
(v/v) in ethanol concentration. The strain A. aceti (BCRC 12325) was
inoculated into mannitol broth (25 g/L mannitol, 5 g/L yeast extract,
3 g/L peptone) and incubated at 30 C for 3 days. The culture was
then inoculated into the diluted wines: (1) three rice wines; (2)
MS; and (3) rice wines mixed with MS (0%, 25%, 50%, 75%, 100%) in
a 28 L jar at the ratio of 10% (v/v) and incubated at 30 C. After fer-
mentation for 30 days, new corresponding vinegar products were
obtained and then diluted with water to 4% (v/v) in acetic acid
concentration.
2.4. Rice vinegar authentication model test
Authentication model tests of vinegars for SNIF-NMR are
divided into 3 models. Model I: 3 kinds of rice vinegar were mixed
with 0%, 25%, 50%, 75%, and 100% synthetic vinegar, which was
diluted from synthetic acetic acid and adjusted to 4% (v/v) in acetic
acid concentration. Model II: 3 kinds of rice vinegar were mixed
with 0%, 25%, 50%, 75%, and 100% of MS vinegar. Model III: 3 kinds
of rice wine were mixed with 0%, 25%, 50%, 75%, and 100% of MS
and then processed by fermentation from wine into vinegar.
905
2.5. NMR measurements
For the SNIF-NMR measurements, tetramethylurea (TMU) with
a known isotopic content was used as an internal standard. The
TMU solution (1.3 mL) was placed in a previously weighed bottle
and weighed to the nearest 0.1 mg (mst). Then, 3.2 mL of the sam-
ple was put into the bottle and again weighed to the nearest 0.1 mg
(mA), and then homogenized by shaking (Martin et al., 1996). The
2 H NMR spectra were recorded on an NMR spectrometer (Bruker
DMX 600, Bruker Biospin GmbH, Rheinstetten, Germany) equipped
with a 10 mm probe, operating at 1 H frequency of 299.9 MHz and
at 92.0 MHz for deuterium. Measurements were taken according to
the AOAC protocol (Martin et al., 1996). Transients were accumu-
lated with 16,320 data points over a 1200 Hz spectral bandwidth
with a 30 s (90 ) rf pulse. The acquisition time was 1.5 s. This
set of accumulations was repeated 2000 times for each sample.
The D/H ratios were measured by recording the intensities of the
deuterium signals corresponding to the methyl sites of acetic acid
and tetramethylurea. Means and standard deviations of the isotopic
ratios were calculated (Freddy and Eric, 2009; Ogrinc et al., 2001).
The D/H ratios of (D/H)I were expressed in ppm. The average pre-
cision values of the measurement of ratios were below 0.5 ppm for
(D/H)CH3 .
The equation for calculation was as follows (Freddy and Eric,
2009):
P st Maa mst Saa
(D/H)CH3 = (D/H)st
P aa Mst maa Sst
where aa is acetic acid; st, the internal standard TMU; P, the num-
ber of equivalent deuterium positions for the considered molecular
site; M, molecular weight; m, weighted mass; S, NMR signal area;
and (D/H)st (ppm), certied deuterium content of TMU provided by
the supplier.
2.6. Statistical analysis
All of the experiments were performed on triplicate samples.
Data were analyzed by ANOVA with SAS for Windows v8 (SAS Inst.,
Cary, NC, USA). Signicant differences were analyzed by Duncans
multiple range test (p < 0.05).
3. Results and discussion
3.1. (D/H)CH3 value in SNIF-NMR index of various vinegars
The 2 H-NMR spectra of TK-9 rice vinegar are shown in Fig. 1.
Peaks A and D were residual ester after the extraction process, peak
B was acetic acid, peak C was N,N-tetramethylurea (TMU, internal
standard) in the sample. The deuterium of TK-9 will rapid exchange
with the proton of H2 O in solution. The peak E was the signal of
H2 O. The other 2 H-NMR spectra of TCS-10, TN-11, MS and synthetic
vinegar were similar to those of TK-9. The (D/H)CH3 value was cal-
culated based on Freddy and Eric (2009), and the results are shown
in Table 1. The (D/H)CH3 values in TK-9, TCS-10, TN-11, MS and syn-
thetic vinegar were 98.62 ppm, 98.45 ppm, 98.62 ppm, 108.46 ppm
and 131.58 ppm, respectively. Statistically, there was no signicant
difference between TK-9, TCS-10 and TN-11; however, the MS and
synthetic vinegar samples were signicantly different from the 3
kinds of rice vinegar. The (D/H)CH3 value of MS in our study was
close to the results of Vallet et al. (1998) ((D/H)CH3 = 107.3 ppm).
The (D/H)CH3 value of synthetic acetic acid was close to that of
Ko et al. (2013), Remaud et al. (1992) and Vignali et al. (2007) at
130.37132.5 ppm. In Hsieh et al. (2005) study, the (D/H)CH3 val-
ues of TCS-10 and MS rice wine were 98.90 ppm and 108.70 ppm,
respectively. These are also close to our results in TCS-10 and MS
vinegar. Thus, we could assume that there was no signicant effect
906 C.-W. Hsieh et al. / Industrial Crops and Products 50 (2013) 904908

Fig. 1. Typical 2 H NMR spectra of Taikeng 9 rice vinegar. Signals A, B, C, D and E refer to the working standard of CH2 DCH2 OCH2 CH3 , CH2 DCOOH, TMU, CH3 CH2 OCH2 CH2 D
and H2 O, respectively.

Table 1 135
(D/H)CH3 values of rice vinegar, molasses spirit vinegar and synthetic vinegar.
130
TK-9a TCS-10b TN-11c MSd Synthetic
vinegar 125
(D/H)CH3 (ppm) 98.62c 98.45c 98.62c 108.46b 131.58a
Standard deviation 0.15 0.15 0.22 0.25 0.34 120
(D/H)CH3 (ppm)

The online letters (ac) in the same row followed by different letters are signicantly 115
different (p < 0.05).
a
TK-9: Taikeng 9 vinegar. 110
b
TCS-10: Taichung sen 10 vinegar.
c
TN-11: Tainan 11 vinegar. 105
d
MS: Molasses spirit vinegar.
100

on the conversion process by acetic acid bacteria in our study. 95

According to the (D/H)CH3 values, we assumed it could be applied 0

to distinguish the source of vinegar. 0 20 40 60 80 100

Mix ratio (%)

3.2. Model test for rice vinegars mixed with acetic acids from Fig. 2. Change in (D/H)CH3 values of the three varieties of rice vinegar with different
other sources ratios of synthetic vinegar.

3.2.1. Model I: rice vinegar mixed with synthetic vinegar 3.2.3. Model III: vinegar obtained by fermenting rice wine mixed
Various ratios of synthetic vinegar were added to the 3 kinds of with molasses spirit
rice vinegar (TK-9, TCS-10 and TN-11), respectively. The (D/H)CH3 The 3 kinds of rice were fermented to rice wine (7% of alcohol),
values were measured by 2 H-NMR, and the results are shown mixed with various ratios of MS (7% of alcohol), and then fermented
in Fig. 2. In the TK-9 group, the (D/H)CH3 values ranged from
98.62 ppm to 131.54 ppm and revealed a linear correlation with 110
the volume of synthetic vinegar (TK-9: R2 > 0.9922). This situation
was observed in TCS-10 and TN-11 as well (TCS-10: R2 > 0.9885; 108
TN-11: R2 > 0.9954). According to these ndings, the SNIF-NMR
method revealed its potential to determine adulteration in rice 106
vinegar.
(D/H)CH3 (ppm)

104
3.2.2. Model II: rice vinegar mixed with MS-vinegar
TK-9
Various ratios of MS vinegar were added to the 3 kinds of rice 102
y = 0.0974x + 98.517
vinegar (TK-9, TCS-10 and TN-11), respectively. The (D/H)CH3 val- R2 = 0.9903
TSC-10
ues were measured by 2 H-NMR, and the results are shown in Fig. 3. 100
y = 0.0991x + 98.427
In the TCS-10 group, the (D/H)CH3 values ranged from 98.45 ppm R2 = 0.9980
to 108.46 ppm and revealed a linear correlation with the volume of TN-11
98
y = 0.0972x + 98.494
MS vinegar (TCS-10: R2 > 0.998). The linear correlation was similar R2 = 0.9882
to the study of Hsieh et al. (2005) that measured the adulteration in 0
0 20 40 60 80 100
rice wine by SNIF-NMR (R2 > 0.96). The correlation of determination
in the TK-9 and TN-11 groups was 0.9903 and 0.9882, respectively. Mix ratio (%)
The (D/H)CH3 values of the 3 kinds of rice vinegar showed no sig-
Fig. 3. Change in (D/H)CH3 values of the three varieties of rice vinegar with different
nicant difference in Model II. ratios of molasses spirit vinegar.
 C.-W. Hsieh et al. / Industrial Crops and Products 50 (2013) 904908 907

110 Table 3
Recovery calculations based on (D/H)CH3 values for the spiked sample (20%, v/v of
molasses spirit vinegar or synthetic vinegar in rice vinegar).
108
Mixture (D/H)CH3 value Calculated Actual
addition (%) addition (%)
106
TK-9a 20% SVd 105.35 20.19 20
(D/H)CH3 (ppm)

TK-9 20% MSe 100.62 21.59 20

104
TK-9
102 y = 0.099x + 98.183
R2 = 0.9881
TSC-10
y = 0.0981x + 98.191
100
R2 = 0.9792
TN-11
98 y = 0.0953x + 98.524
R2 = 0.9953
0
0 20 40 60 80 100
Mix ratio (%)
Fig. 4. The (D/H)CH3 values of rice vinegar fermented from 3 kinds of rice wine,
mixed with various ratios of MS.
into vinegar. The (D/H)CH3 values were measured by 2 H-NMR, and
the results are shown in Fig. 4. In the TN-11 group, the (D/H)CH3
values ranged from 98.62 ppm to 108.42 ppm and revealed a linear
correlation with the ratio of MS (TN-11: R2 > 0.9953). This situa-
tion was observed in TK-9 and TCS-10 as well (TK-9: R2 > 0.9881;
TN-11: R2 > 0.9792). The (D/H)CH3 values of the 3 kinds of rice vine-
gar showed no signicant difference in Model III. According to the 4. Conclusions
results in Model II and Model III, the SNIF-NMR method could dis-
tinguish the source of rice vinegar from MS.
3.3. LOD of adulteration test
In the experiment, the LOD (limit of detection) of the 2 H-NMR
instrument we used was about 0.5 ppm and the instrument noise
was 3-fold. The linear regression was calculated, and the results are
shown in Table 2. The limit of detection volume was 4.99% (TK-9),
3.68% (TCS-10) and 3.35% (TN-11), respectively.
The LODs of SNIF-NMR in the 3 kinds of rice vinegar mixed with
molasses spirit were 6.19%, 5.28% and 6.44%. The LODs of SNIF-
NMR in rice wine mixed with molasses spirit were 9.46%, 7.73% and
6.23%. The results were close to those of the study of Hsieh et al.
(2005) for rice wine mixed with molasses spirit, 6.12%. In general,
more than 50% of acetic acid or molasses spirit is added during the
vinegar production process to lower the cost. The LOD of the SNIF-
NMR method from our results implies that the method could be
applied to evaluate adulteration in commercial rice vinegar.
3.4. Judgment of adulteration test
An increase in the amount of adulteration led to an increase
of (D/H)CH3 , and this trend revealed a good linear correlation. The
results are shown in Table 3. In the experiment, we applied this
property to estimate the amount of adulteration. In the synthetic
vinegar group, the results of (D/H)CH3 in the 3 kinds of rice vinegar
were 105.35 ppm, 104.26 ppm and 104.82 ppm, respectively, with
the estimated amount of adulteration at 18.23%, 19.07% and 20.64%,
TCS-10b 20% SV 104.26 19.07 20
TCS-10 20% MS 100.85 21.40 20
TN-11c 20% SV 104.82 20.64 20
TN-11 20% MS 100.36 19.20 20
a
TK-9, Taikeng 9 vinegar.
b
TCS-10, Taichung sen 10 vinegar.
c
TN-11, Tainan 11 vinegar.
d
SV, synthetic vinegar.
e
MS, molasses spirit vinegar.
respectively. In the molasses spirit group, the results of (D/H)CH3
in the 3 kinds of rice vinegar were 100.63 ppm, 100.85 ppm and
100.36 ppm, respectively, with the estimated amount of adulter-
ation at 21.58%, 21.40% and 19.20%, respectively. According to these
ndings, the SNIF-NMR method proved to be a good way to evalu-
ate adulteration in rice vinegar. Freddy and Eric (2009) also applied
the SNIF-NMR method to evaluate the amount of beet vinegar in
red wine vinegar. They mixed 20% of beet vinegar in red wine vine-
gar; after it was analyzed using the SNIF-NMR method, the amount
of beet vinegar was 22%.
The results of (D/H)CH3 showed signicant differences (p < 0.05)
for the different adulterants; however, the results showed that
(D/H)CH3 had no effect when alcohol was converted to acetic acid by
acetic acid bacteria. When rice vinegar was mixed to have different
ratios of adulteration, the results showed an increase in (D/H)CH3
value with the amount of adulteration and revealed a good linear
correlation with the coefcient R2 > 0.97. According to these nd-
ings, we inferred that the SNIF-NMR method could be effectively
used to identify adulteration and determine the extent of adulter-
ation in rice vinegar. The SNIF-NMR method conrmed its potential
in detecting adulteration in rice vinegar and measuring the extent
of adulteration when a single adulterant had been added to the
rice vinegar. However, if both molasses spirit vinegar and synthetic
vinegar are added during adulteration, the SNIF-NMR method could
only judge whether or not there was any adulteration.
The method of SNIF-NMR analyses had been developed for lab-
oratories from sample preparation to the interpretation of results
in the wine elds for many years. These laboratory experiences
can be directly applied to the identication of vinegar adulteration.
True and false identication of vinegar is relevant to the condence
of livelihood and the fairness of price. In the future, an isotopic
database of rice vinegar with more species of rice and different
materials could be established. Furthermore, if we could combine
the SNIF-NMR method with IRMS and 1 H-NMR, the authentication
process would be more accurate and help stop the illegal adulter-
ation of rice vinegar.
References

Freddy, T., Eric, J., 2009. 2 H NMR and 13 C-IRMS analyses of acetic acid from vinegar,
Table 2 18
O-IRMS analysis of water in vinegar: international collaborative study report.
LOD of adulteration in vinegar. Analytica Chimica Acta 649, 98105.
Haines, E.B., Montague, C.L., 1979. Food sources of estuarine invertebrates analyzed
TK-9 TCS-10 TN-11
using 13 C/12 C ratios. Ecology 60, 4856.
Synthetic vinegar (%) 4.99 3.68 3.35 Hsieh, C.W., Wang, H.J., Chang, C.M., Ko, W.C., 2005. Detection of molasses-spirit in
MSa vinegar (%) 6.19 5.28 6.44 rice-spirits from TK-8 and TCS-10 by SNIF-NMR method. Journal of Food and
Fermentation with MS (%) 9.46 7.74 6.23 Drug Analysis 13, 251255.
Ko, W.C., Cheng, J.Y., Chen, P.Y., Hsieh, C.W., 2013. Optimized extraction
a
MS: molasses spirit vinegar. method of acetic acid in vinegar and its effect on SNIF-NMR analysis to
908 C.-W. Hsieh et al. / Industrial Crops and Products 50 (2013) 904908
control the authenticity of vinegar. Food and Bioprocess Technology 6,
22022206.
Martin, G.J., Martin, M.L., Mabon, F., Michon, M.J., 1983. A new method for the identi-
cation of the origin of ethanol in grain and fruit spirits: high-eld quantitative
deuterium nuclear magnetic resonance at the natural abundance level. Journal
of Agricultural and Food Chemistry 31, 311315.
Martin, G.J., Zhang, B.L., Naulet, N., Martin, M.L., 1986. Deuterium transfer in the
bioconversion of glucose to ethanol studied by specic isotope labeling at
the natural abundance level. Journal of the American Chemical Society 108,
51165122.
Martin, G.G., Wood, R., Martin, G.J., 1996. Detection of added beet sugar in concen-
trated and single strength fruit juices by deuterium nuclear magnetic resonance
(SNIF-NMR1 method): collaborative study. Journal of AOAC International 79,
917928.
Martin, M.L., Martin, G.J., 1990. Deuterium NMR in the Study of Site-Specic Natural
Isotope Fractionation (SNIF-NMR). NMR Basic Principles and Progress. Springer-
Verlag, Heidelberg, pp. 161.
Mazza, S., Murooka, Y., 2009. Vinegar through the age. In: Solieri, L., Giudici, P. (Eds.),
Vinegars of the World. Spinger-Verlag, Miln, pp. 1739.
Ogrinc, N., Kosir, I.J., Kocjancic, M., Kidric, J., 2001. Determination of authentic-
ity, regional origin, and vintage of Slovenian wines using a combination of
IRMS and SNIF-NMR analyses. Journal of Agricultural and Food Chemistry 49,
14321440.
Remaud, G., Guillou, C., Vallet, C., Martin, G.J., 1992. A coupled NMR and MS iso-
topic method for the authentication of natural vinegars. Fresenius Journal of
Analytical Chemistry 342, 457461.
Rossmann, A., 2001. Determination of stable isotope ratios in food analysis. Food
Reviews International 17, 347381.
Vallet, C., Arendt, M., Martin, G.J., 1998. Site specic isotope fractionation of
hydrogen in the oxidation of ethanol into acetic acid. Application to vinegars.
Biotechnology Techniques 2, 8388.
Vignali, C., Caligiani, A., Palla, G., 2007. Quantitative 2 H NMR spectroscopy with 1 H
lock extender. Journal of Magnetic Resonance 187, 120125.