Fil e ing he NMR Spec a o Mix u es by Coo dina ion o
Pa amagne ic Cu2+
Juan Co ea, Ana Ga cia-Ba andela, Llo encSocias-Pin o, and Edua do Fe nandez-Megia*
Ci e This: Anal. Chem. 2022, 94, 10907−10911
Read Online
ACCESS Me ics & Mo e A icle Recommenda ions *
sı Suppo ing In o ma ion
ABSTRACT: The pa amagne ic spin elaxa ion (PSR) il e
allows he selec i e NMR signal supp ession o componen s in
mix u es acco ding o hei complexa ion abili y o a pa amagne ic
ion. I elies on he as e elaxa ion o nuclei in pa amagne ic
en i onmen s and hus is complemen a y o classical di usion and
elaxa ion il e s. So a , he PSR il e has es ablished Gd3+ as he
sole PSR agen , es ic ing he pa amagne ic il e ing epe oi e.
He ein, we p esen Cu2+ as a obus PSR agen wi h cha ac e is ic
il e ing p ope ies. While Gd3+ depends on unspeci ic ion-pai
in e ac ions wi h anionic componen s, Cu2+ s ands ou o il e ing
species ia o de ed coo dina ion complexes. An e alua ion o he
pa amagne ic e ec o Cu2+ o e mo e han 50 small molecules and polyme s has un eiled di e en sensi i i ies o Cu2+ (especially
high o py idines, diamines, polyamines, and amino alcohols) and p ecise il e ing condi ions o mix u es (1H, COSY, and HMQC)
ha we e challenged wi h a es bed o comme cial d ugs. The ad an age o in eg a ing Cu2+ and Gd3+ o he s epwise PSR il e ing
o complex mix u es is also shown.
As na u e seldom p o ides pu e compounds, chemis s ha e
de o ed g ea e o s o he sepa a ion o complex
mix u es. Fo una ely, he NMR analysis o mix u es sides eps
he necessi y o physical sepa a ions unde ce ain condi ions.
NMR il e s ake ad an age o di e ences in he di usion
coe icien s and elaxa ion imes o he componen s o he
selec i e signal supp ession o small molecules and mac o-
molecules, espec i ely.
1,2
Wi h he aim o widening he NMR
il e ing po olio beyond molecula weigh limi s, ou g oup
has desc ibed he pa amagne ic spin elaxa ion (PSR) il e ,
3,4
which elies on he as e elaxa ion o nuclei in pa amagne ic
en i onmen s.
5,6
The addi ion o minu e concen a ions o
Gd3+ (pa amagne ic) o mix u es allows he selec i e
supp ession o pa icula componen s om he 1D and 2D
spec a, acco ding o hei Gd3+ complexa ion abili y (Figu e
1). Since Gd3+ has he la ges spin momen (S= 7/2) and a
high elec onic co ela ion ime (τsca. 10−8s), he Solomon−
Bloembe gen−Mo gan equa ions p edic a selec i e dec ease
o ans e se elaxa ion imes (T2) o species in as chemical
exchange wi h Gd3+.
7−9
The in e se p opo ionali y be ween
T2and he spec al line wid h
10
leads o hei selec i e signal
supp ession, wi hou a ec ing he esolu ion and chemical shi
o o he componen s in he mix u e. Since complexa ion o
Gd3+ is mainly elec os a ic (ion-pai ), he PSR il e a ec s
mo e anionic species han neu al and ca ionic species.
Success ul applica ions o he PSR il e include he sequen ial
NMR il e ing o mix u es o in e es in he pha maceu ical and
ood indus ies
4,11
and he as sc eening o DNA ligands.
12
In e es ingly, he PSR il e bene i s om a a he low
sensi i i y o size and molecula weigh ha makes i
compa ible wi h classical elaxa ion and di usion il e s. The
ad an age o his is aken o he il e ing o lowe PSR-
sensi i e species, which leads o line-b oadening a he han a
ull signal embedmen in he baseline, by implemen ing a
complemen a y sho T2- il e , such as he Ca −Pu cell−
Meiboom−Gill (CPMG).
13,14
Recei ed: May 6, 2022
Accep ed: July 25, 2022
Published: July 27, 2022
Figu e 1. PSR il e s based on Gd3+ (ion-pai ) and Cu2+
(coo dina ion) complexes.
Le e pubs.acs.o g/ac
© 2022 The Au ho s. Published by
Ame ican Chemical Socie y 10907
h ps://doi.o g/10.1021/acs.analchem.2c01983
Anal. Chem. 2022, 94, 10907−10911
Downloaded ia UNIV DE SANTIAGO DE COMPOSTELA on Sep embe 1, 2022 a 07:15:24 (UTC).
See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles.
While he PSR il e based on ion-pai Gd3+-complexes is
highly e icien in simpli ying he NMR analysis o complex
mix u es, he de elopmen o PSR il e s wi h o he pa a-
magne ic ions displaying al e na i e complexa ion modes
would g ea ly ex end he u ili y o his echnology. He ein,
we desc ibe ou e o s owa d a PSR il e based on he mo e
coo dina ing Cu2+ (S= 1/2, τsca. 10−9s), a pa amagne ic ion
aimed a selec i ely il e ing he NMR signals o species ia
coo dina ion complexes (Figu e 1).
The easibili y o a Cu2+ PSR il e was con i med by he
selec i e supp ession o glucosamine in he p esence o glucose
(Figu e 2C), a supp ession un easible o ep oduce in he
p esence o Gd3+ (Figu e S4) because o he simila sensi i i y
o bo h componen s o his ion.
4
Con e sely, he coo dina ing
1,2-amino alcohol moie y o glucosamine ensu es a selec i e
il e ing in he p esence o Cu2+.
The scope o Cu2+ as coo dina ing PSR agen was assessed
by analyzing he pa amagne ic e ec on he 1H NMR spec a
o a collec ion o mo e han 50 small molecules and polyme s
o in e es in he pha maceu ical and ood indus ies, which
display a la ge a ie y o unc ional g oups. Depending on he
ex en o signal b oadening ( om no e ec o comple e
supp ession), hese species we e assigned o se en g oups
(Table 1), wi h he mo e sensi i e ones comp ising highly
coo dina ing compounds. 1H NMR spec a o ep esen a i e
molecules in he uppe , medium, and bo om pa s o Table 1,
eco ded in he absence/p esence o Cu2+, a e shown in
Figu es S1−S3: (1S,2S)-2-amino-1-phenyl-1,3-p opanediol,
adenosine, suc ose. As a ule o humb, he b oadening e ec
o Cu2+ on he 1H NMR spec a o py idines, diamines,
polyamines, and amino alcohols was conside ably highe han
wi h Gd3+,
4
in consis ency wi h a highe coo dina ion abili y.
Nex , he possibili y o pe o ming selec i e supp essions by
Cu2+ among he se en g oups in Table 1 was assessed in wo-
componen mix u es (Figu e 2A). I was con i med ha he
mo e dis an he g oups, he easie he selec i e supp essions.
Also, he impossibili y o pe o ming supp essions wi hin a
single g oup and be ween some neighbo ing g oups. As a
esul , he ini ial g oups ( e lec ing he pa amagne ic b oad-
ening e ec ) we e educed o jus h ee ca ego ies (designa ed
as Blue, Yellow, and Red), acco ding o hei ease o
supp ession by Cu2+. In addi ion, om da a in Figu e 2A,
gene al condi ions o selec i e 1H, COSY, and HMQC
supp essions be ween ca ego ies we e de e mined (concen-
a ion o Cu2+/leng h o a complemen a y CMPG T2- il e ;
Figu e 2B):
Figu e 2. (A) Success ul and ailed PSR supp essions in wo-componen mix u es (D2O, 500 MHz). (B) PSR il e ing condi ions o selec i e 1H,
COSY, and HMQC supp essions. Rep esen a i e examples o selec i e supp essions be ween Blue-Yellow-Red ca ego ies. 1H NMR spec a (D2O,
500 MHz, 300 K) o a mix u e o he ollowing: (C) glucosamine (2 mg/mL) and glucose (2 mg/mL) be o e (a) and a e (b) he addi ion o Cu2+
(2 mM), (D) 2-amino-1-phenyl-1,3-p opanediol (1 mg/mL) and adenosine (3 mg/mL) be o e (c) and a e (d) he addi ion o Cu2+ (0.16 mM) +
T2- il e (CPMG, 30 ms), and (E) adenosine (1.2 mg/mL) and glucose (1.6 mg/mL) be o e (e) and a e ( ) he addi ion o Cu2+ (13 mM) + T2-
il e (CPMG, 100 ms).
Analy ical Chemis y pubs.acs.o g/ac Le e
h ps://doi.o g/10.1021/acs.analchem.2c01983
Anal. Chem. 2022, 94, 10907−10911
10908
•Blue-Red: > 2.0 mM Cu2+
•Blue-Yellow: < 2.0 mM Cu2+, < 50 ms CPMG
•Yellow-Red: > 2.0 mM Cu2+, > 50 ms CPMG
Figu e 2 depic s ep esen a i e examples o success ul
supp essions wi hin hese h ee ca ego ies. Fo ins ance, he
a o emen ioned glucosamine/glucose supp ession can be easily
a ionalized now, conside ing hei espec i e inclusion in o
he Blue and Red ca ego ies (Figu e 2C). Simila ly, Figu e
2D,E show selec i e supp essions ha exploi he use o
complemen a y T2- il e s be ween componen s o con iguous
ca ego ies. Rema kably, none o hese supp essions could be
ealized wi h Gd3+ as PSR ion (Figu es S4−S6), con i ming he
coo dina ion abili y o Cu2+ as esponsible o he selec i i y
achie ed. O he ep esen a i e spec a o success ul (di e en
ca ego ies) and un ui ul (same ca ego y) supp essions in he
wo-componen mix u es shown in Figu e 2A a e included in
he SI (Figu es S7−S12). Addi ional ad an ages ha eme ged
on he use o Cu2+ s Gd3+ as PSR agen include he possibili y
o wo king in a wide pH- ange (Gd3+ ends o p ecipi a e a
pH > 7.0) and a smalle b oadening e ec o e he esidual
HOD signal.
The eliabili y o Table 1 o p edic Cu2+ PSR il e s in
complex mix u es was challenged wi h a es bed o comme cial
d ugs, namely (i) an amoxicillin/cla ulanic acid an ibio ic
(amoxicillin, cla ulanic acid, PEG, PVP); (ii) Ca iban, a d ug
o ea nausea and omi ing in p egnancy (doxylamine,
py idoxine, suc ose); (iii) he an ibio ic P ode ma (doxycy-
cline, suc ose); and (i ) A epodin, a medicine o he
ea men and diagnosis o sup a en icula achyca dia
(adenosine iphospha e, glycine, benzyl alcohol).
We s a ed analyzing Amoxicillin/Cla ulanic acid Cin amed,
an an ibio ic ha con ains wo ac i e ing edien s, he
penicillin-like an ibio ic amoxicillin (Blue) and he be a-
lac amase inhibi o cla ulanic acid (no included in Table 1
bu expec ed o be Blue by simila i y). NMR- isible excipien s
comp ise poly(e hylene glycol) (PEG) and poly inylpy oli-
done (PVP), bo h Red species. A ending o he Blue and Red
ca ego ies o he cons i uen s, a selec i e PSR supp ession was
an icipa ed o amoxicillin and cla ulanic acid in he p esence
o Cu2+.Figu e 3 shows hei selec i e il e ing, con i ming he
p edic i e cha ac e o Table 1, and he h ee colo ca ego ies
p oposed. When lowe concen a ions o Cu2+ we e assessed, i
was e en possible o a ain a s epwise supp ession o he Blue
componen s, il e ing i s he mo e coo dina ing amoxicillin.
In e es ingly, he use o Gd3+ did no a o d a clean supp ession
o any componen , con i ming Cu2+ as a PSR agen wi h
cha ac e is ic il e ing p ope ies.
Then, we p oceeded o analyze h ee comme cial d ugs
(Ca iban, P ode ma, A epodin) con aining Yellow and Red
componen s, whe e a complemen a y CPMG il e was
expec ed o success ul supp essions. Ca iban is a medicine
used o ea nausea and omi ing in p egnan women ha
con ains doxylamine (an ihis amine) and py idoxine ( i amin
B6) as ac i e ing edien s, bo h subs i u ed py idines ha
belong o he Yellow ca ego y. The mix u e also includes
suc ose (Red) as NMR- isible excipien . As p edic ed, he only
addi ion o Cu2+ did no esul in a clean il e ing o
doxylamine and py idoxine. Howe e , concomi an applica ion
o a sho CPMG il e a o ded hei clean supp ession, lea ing
una ec ed he esolu ion and chemical shi o he suc ose
signals (Figu e 4). Rema kably, he ideli y o he PSR-CPMG
il e was also demons a ed in 2D COSY and HMQC
expe imen s, whe e he CPMG sequence was used as an
exci a ion block eplacing he i s exci a ion pulse.
15
No
unexpec edly, he use o Gd3+ as PSR agen was again
unsuccess ul, ei he in he absence o p esence o CPMG
il e s.
The an ibio ic P ode ma con ains wo NMR- isible
componen s, he ac i e ing edien doxycycline (Yellow) and
suc ose (Red) as excipien . He e again, he di ec il e ing o
he mos sensi i e Yellow componen was un easible by he
Table 1. Pa amagne ic B oadening E ec (G oups 1−7) and
Ease o Supp ession by Cu2+ (Blue, Yellow, Red Ca ego ies)
in 1H NMR (4 mg/mL in D2O, 500 MHz, 300 K)
a
a
G oup 7: supp ession o all signals a <1 mM Cu2+, G oup 6:
supp ession o all signals a >1 mM Cu2+, G oup 5: b oadening o all
signals a >1 mM Cu2+, G oup 4: supp ession o some signals a >1
mM Cu2+, G oup 3: b oadening o some signals a >1 mM Cu2+,
G oup 2: educed signal esolu ion a >1 mM Cu2+, G oup 1: no
e ec on signal esolu ion a >1 mM Cu2+.
Figu e 3. 1H NMR spec a (D2O, 500 MHz, 300 K) o Amoxicillin/
Cla ulanic acid Cin amed (10 mg/mL) be o e (a) and a e he
addi ion o 4 and 17 mM Cu2+ (b and c, espec i ely).
Analy ical Chemis y pubs.acs.o g/ac Le e
h ps://doi.o g/10.1021/acs.analchem.2c01983
Anal. Chem. 2022, 94, 10907−10911
10909
sole addi ion o Cu2+. Howe e , implemen a ion o a
simul aneous sho CPMG il e allowed he clean supp ession
o doxycycline (Figu e 5). Ve y simila il e ing condi ions also
allowed he e icien il e ing o he Yellow componen s
(adenosine iphospha e and glycine) o A epodin (Figu e
S13).
Ha ing es ablished he u ili y o Cu2+ as PSR agen wi h
il e ing p ope ies dependen on he coo dina ion abili y o
he componen s in a mix u e ( a he han ion-pai in e ac ions
o Gd3+), we decided o assess he in eg a ion o bo h
pa amagne ic ions in he il e ing o complex mix u es. To his
end, we selec ed Ace ilcis eina Mylan, a comme cial mucoly ic
d ug composed o ace ylcys eine (CuYellow/GdYellow) as ac i e
ing edien , ci ic acid (CuBlue/GdBlue) as excipien , and wo
swee ene s, D-manni ol (CuRed/GdYellow) and sodium sac-
cha in (CuRed/GdRed). A ending o he Blue-Yellow-Red
ca ego y o he componen s owa d Gd3+, we ha e p e iously
epo ed he sequen ial supp ession o ci ic acid in a i s s ep,
ollowed by he simul aneous supp ession o ace ylcys eine and
D-manni ol (bo h GdYellow componen s).
4
He ein, he
s onge Cu2+-coo dina ion o ace ylcys eine han D-manni ol
(Table 1) has been exploi ed o he s epwise supp ession o
he h ee componen s in a way una ainable wi h a single PSR
agen . Thus, as shown in Figu e 6, a e an ini ial supp ession
o ci ic acid wi h Gd3+, ace ylcys eine was il e ed wi h Cu2+,
ollowed by a inal supp ession o D-manni ol wi h Gd3+.
Applica ion o he PSR il e wi h Cu2+ s a s wi h he
assignmen o he indi idual componen s in a mix u e o he
Blue-Yellow-Red ca ego ies. As a i s app oach, use s a e
Figu e 4. 1H, 1H−1H COSY, and 1H−13C HMQC spec a (D2O, 500
MHz, 300 K) o Ca iban (72 mg/mL) be o e (a, c, e) and a e (b, d,
) he addi ion o Cu2+ (13 mM) + T2- il e (CPMG, 75 ms).
Figu e 5. 1H NMR spec a (D2O, 500 MHz, 300 K) o P ode ma (4
mg/mL) be o e (a) and a e (b) he addi ion o Cu2+ (4 mM) + T2-
il e (CPMG, 75 ms).
Figu e 6. NMR- isible componen s o Ace ilcis eina Mylan and hei
espec i e Blue-Yellow-Red ca ego ies owa d Cu2+ and Gd3+.1H
NMR spec a (D2O, 500 MHz, 300 K) o Ace ilcis eina Mylan (25
mg/mL) supplemen ed wi h saccha in (12.5 mg/mL) be o e (a) and
a e he addi ion o (b) Gd3+ (25 μM) + T2- il e (CPMG, 25 ms),
(c) Cu2+ (2.5 mM) + T2- il e (CPMG, 250 ms), and (d) Gd3+ (0.2
mM) + T2- il e (CPMG, 300 ms).
Analy ical Chemis y pubs.acs.o g/ac Le e
h ps://doi.o g/10.1021/acs.analchem.2c01983
Anal. Chem. 2022, 94, 10907−10911
10910
ad ised o ind s uc u al simila i ies be ween he species in a
mix u e o in e es and hose in Table 1. Ne e heless, o a
p ope inclusion o species in he Blue-Yellow-Red ca ego ies,
he pa amagne ic e ec o Cu2+ on hei 1H NMR spec a
should be de e mined as desc ibed in Table 1 (ex en o signal
b oadening as a unc ion o he concen a ion o Cu2+). Once
he species ha e been assigned o he h ee ca ego ies, selec i e
supp essions could be expec ed by applica ion o he PSR
condi ions shown in Figu e 2B. While clean supp essions
ope a e o Blue species in he p esence o Red ones by he
simple addi ion o mM concen a ions o Cu2+, he selec i e
il e ing o species om con iguous ca ego ies (Blue-Yellow
and Yellow-Red) a e un easible by he sole addi ion o Cu2+,
being necessa y he implemen a ion o simul aneous CPMG
il e s. Al hough he selec i e il e ing o species wi hin a
ca ego y migh wo k in speci ic examples using inc easing
concen a ions o Cu2+ o uning he leng h o CPMG il e s,
his will no be o gene al applica ion because he 1HT2 alues
o he species in a ca ego y will le el down in he p esence o a
PSR agen , making unlikely hei selec i e supp ession.
In conclusion, Cu2+ is p esen ed as a obus PSR agen wi h
cha ac e is ic NMR il e ing p ope ies di e en han Gd3+, he
a che ypal PSR agen so a . No only do he pa amagne ic
p ope ies change be ween nuclei, bu also hei complexa ion
modes di e , o e ing he oppo uni y o une he ou come o
he PSR il e . While Gd3+ elies on he ion-pai complexa ion
abili y o he componen s in a mix u e (mainly anionic
species), Cu2+ s ands ou because o a g ea e capaci y o
il e ing species ha pa icipa e in coo dina ion complexes,
such as py idines, diamines, polyamines, and amino alcohols.
An e alua ion o he pa amagne ic e ec o Cu2+ o e mo e
han 50 small molecules and polyme s has un eiled h ee
ca ego ies o compounds (Blue-Yellow-Red ca ego ies acco d-
ing o hei ease o supp ession by Cu2+) and p ecise il e ing
condi ions o 1H, COSY, and HMQC be ween hem. The
in eg a ion o he speci ic il e ing p ope ies o Cu2+ and Gd3+
as PSR agen s o he analysis o complex mix u es has been
also demons a ed, widening he ho izons o he PSR
echnology o quali y con ol, na u al p oduc ex ac s, o he
me abolic p o iling o biological samples. Finally, ha ing
demons a ed he u ili y o Cu2+ as PSR agen , a mo e p ecise
assignmen o species o he Blue-Yellow-Red ca ego ies is
en isaged using he ans e se elaxa ion enhancemen (R2p),
as p e iously done o Gd3+.
4
This app oach, which in ol es
he analysis o he1HT2 alues o he species o in e es in he
absence and p esence o Cu2+, will be he ocus o ou
in es iga ions in he u u e and epo ed in due ime.
■ASSOCIATED CONTENT
*
sı Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acs.analchem.2c01983.
Ma e ials, me hods, and NMR spec a (PDF)
■AUTHOR INFORMATION
Co esponding Au ho
Edua do Fe nandez-Megia −Cen o Singula de In es igación
en Química Biolóxica e Ma e iais Molecula es (CIQUS),
Depa amen o de Química O gánica, Uni e sidade de
San iago de Compos ela, 15782 San iago de Compos ela,
Spain; o cid.o g/0000-0002-0405-4933;
Email: [email p o ec ed]
Au ho s
Juan Co ea −Cen o Singula de In es igación en Química
Biolóxica e Ma e iais Molecula es (CIQUS), Depa amen o
de Química O gánica, Uni e sidade de San iago de
Compos ela, 15782 San iago de Compos ela, Spain
Ana Ga cia-Ba andela −Cen o Singula de In es igación en
Química Biolóxica e Ma e iais Molecula es (CIQUS),
Depa amen o de Química O gánica, Uni e sidade de
San iago de Compos ela, 15782 San iago de Compos ela,
Spain
Llo encSocias-Pin o −Cen o Singula de In es igación en
Química Biolóxica e Ma e iais Molecula es (CIQUS),
Depa amen o de Química O gánica, Uni e sidade de
San iago de Compos ela, 15782 San iago de Compos ela,
Spain
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acs.analchem.2c01983
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
This wo k was suppo ed by he Spanish Minis y o Science
and Inno a ion (RTI2018-102212-B-I00 and PID2021-
127684OB-I00), he Xun a de Galicia (ED431C 2018/30,
and Cen o singula de in es igación de Galicia acc edi a ion
2019−2022, ED431G2019/03), Axencia Galega de Inno ación
(IN845D 2020/09), and he Eu opean Union (Eu opean
Regional De elopmen Fund-ERDF).
■REFERENCES
(1) No oa-Ca ballal, R.; Fe nandez-Megia, E.; Jimenez, C.; Rigue a,
R. Na . P od. Rep. 2011,28, 78−98.
(2) Bingol, K.; B uschweile -Li, L.; Li, D.; Zhang, B.; Xie, M.;
B uschweile , R. Bioanalysis 2016,8, 557−573.
(3) Fe nandez-Megia, E.; Co ea, J.; No oa-Ca ballal, R.; Rigue a, R.
J. Am. Chem. Soc. 2007,129, 15164−15173.
(4) Co ea, J.; Pin o, L. F.; Rigue a, R.; Fe nandez-Megia, E. Anal.
Chem. 2015,87, 760−767.
(5) Be ini, I.; Luchina , C.; Aime, S. Coo d. Chem. Re . 1996,150,
77−110.
(6) Helm, L. P og. Nucl. Magn. Reson. Spec osc. 2006,49, 45−64.
(7) Kowalewski, J.; No denskiöld, L.; Bene is, N.; Wes lund, P.-O.
P og. Nucl. Magn. Reson. Spec osc. 1985,17, 141−185.
(8) Solomon, I. Phys. Re . 1955,99, 559−565.
(9) Bloembe gen, N. J. Chem. Phys. 1957,27, 572−573.
(10) La Ma , G. N.; de Ropp, J. S. In Biological Magne ic Resonance;
Be line , L. J., Reuben, J., Eds.; Plenum P ess: New Yo k, 1993; Vol.
12, pp 1−78.
(11) Co ea, J.; Pin o, L. F.; Zhao, L.; Rigue a, R.; Fe nandez-Megia,
E. Chem. Eu . J. 2018,24, 19236−19242.
(12) Pin o, L. F.; Co ea, J.; Zhao, L.; Rigue a, R.; Fe nandez-Megia,
E. ACS Omega 2018,3, 2974−2983.
(13) Meiboom, S.; Gill, D. Re . Sci. Ins um. 1958,29, 688−691.
(14) Ca , H. Y.; Pu cell, E. M. Phys. Re . 1954,94, 630−638.
(15) Williams, P. G.; Saunde s, J. K.; Dyne, M.; Moun o d, C. E.;
Holmes, K. T. Magn. Reson. Med. 1988,7, 463−471.
Analy ical Chemis y pubs.acs.o g/ac Le e
h ps://doi.o g/10.1021/acs.analchem.2c01983
Anal. Chem. 2022, 94, 10907−10911
10911