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Accurate quantification of DTX1 standard by quantitative Nuclear Magnetic Resonance
Tsuyoshi Kato, Mika Nagae, Tomoji Igarashi and Takeshi YasumotoJapan Food Research Laboratories 6-11-10 Nagayama, 206-0025, Tama, Japan
Table 3 A typical parameter of qNMR experments
Off
25 ℃ or 7 ℃
90°
5 ppm±20 ppm
1H
Value
Decoupling nuclei
Decoupling method
Data aquisition
Relaxation delay
Acquisition time
Parameter
4 sNuclei
60 sSpectral width
8 timesPluse angle
MPF8Temperature
13Cspinning
ValueParameter
SummarySummarySummarySummaryWe compared qNMR and weighing methods for accuracy to quantify DTX1 and OA.
The 1HNMR spectra of DTX1 and OA show signals of many protons. The signals of oxymethine, oxymetylene, and olefin protons were more or less separated from
each other and judged suitable for use in quantification.
The results of quantitation were nearly equivalent between Method B and Method C, and between Method A and Method B. Method A has an advantage over the
others in the simplicity of manipulation and in having low risks of contamination.
Uncertainty of the quantified results could be improved by choosing signals with higher signal to noise (S/N) ratio. Manually operated phase correction led to high
uncertainty, depending on the number and position of the signals. Signals F, H, and J produced good repeatability. They belong to oxymethine or oxymetylene and are
composed of 2 or 3 protons. About 1% uncertainty was achieved when 4 mg of DTX1 was used and the above-mentioned proton signals were employed for calculation.
The difference between the qNMR and weighing was relatively small (Table 6). The uncertainty of weighing depends on the sample size, larger the size smaller the
uncertainty. We prepared 25mg of DTX1 and 60mg of OA for this study. The qNMR suits for determining small or hygroscopic samples but need a good spectrometer
and careful hands of an analytical chemist.
Fig. 5 Calculation formula for purity
P : purity M : molecular weight W : mass of sampling weight
S : area of the signal H : number of 1H nuclei
Psample ====Ssample
Sstandard
Msample
Mstandard
Wstandard
Wsample
Hstandard
Hsample×××××××× Pstandard×××××××× ×××××××× ××××××××
Fig. 1 The MS spectra of DTX1 and OA by infusion analysisBoth toxin-standards produced, respectively, essentially single peaks, indicating
the insignificance of impurities.
DTX1DTX1
OAOA
Table 6 Comparison of the results between weighing and qNMR methods
Weighing and qNMRWeighing and qNMRWeighing and qNMRWeighing and qNMRqNMR method*qNMR method*qNMR method*qNMR method*Weighing methodWeighing methodWeighing methodWeighing method
8.208.208.208.20
4.474.474.474.47
0.640.640.640.64
ExpandedExpandedExpandedExpanded
UncertaintyUncertaintyUncertaintyUncertainty
(RSD%) k=2(RSD%) k=2(RSD%) k=2(RSD%) k=2
97.697.697.697.6
97.197.197.197.1
97.297.297.297.2
PurityPurityPurityPurity
1.181.181.181.18
0.330.330.330.33
0.640.640.640.64
ExpandedExpandedExpandedExpanded
UncertaintyUncertaintyUncertaintyUncertainty
(RSD%) k=2(RSD%) k=2(RSD%) k=2(RSD%) k=2
4.174.174.174.17
8.738.738.738.73
19.519.519.519.5
Measured valueMeasured valueMeasured valueMeasured value
(mg)(mg)(mg)(mg)
AAAA
AAAA
BBBB
methodmethodmethodmethod
0.020.020.020.02Ultra microUltra microUltra microUltra micro20.120.120.120.1OkadaicOkadaicOkadaicOkadaic acidacidacidacid
4.454.454.454.45Semi microSemi microSemi microSemi micro8.998.998.998.99
8.128.128.128.12Semi microSemi microSemi microSemi micro4.274.274.274.27DTX1DTX1DTX1DTX1
ExpandedExpandedExpandedExpanded
UncertaintyUncertaintyUncertaintyUncertainty
(RSD%) k=2**(RSD%) k=2**(RSD%) k=2**(RSD%) k=2**
Balance typeBalance typeBalance typeBalance typeWeighing sizeWeighing sizeWeighing sizeWeighing size
(mg)(mg)(mg)(mg)
AnalyteAnalyteAnalyteAnalyte
* The signals F, H, and J were used for quantification.
** k is a coverage factor. k = 2 defines an interval having a level of confidence of approximately 95 %.
The data produced by qNMR were essentially the same as those by weighing. The uncertainty of weighing method may increase depending on the sampling size
and the type of the balance used. On the other hand, the uncertainty in the qNMR was smaller than in weighing method when an ordinary semi-micro balance was
used.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
rep
eata
bili
ty R
SD
(%)
1transient1transient1transient1transient8transients8transients8transients8transients16384transients16384transients16384transients16384transients
A B C D E F G H I J K L M N O P Q R S
Fig. 4 Validation of the processing of the NMR spectra using DTX1Three spectra of varied transient numbers were processed 20 times for qNMR.
The repeatability was improved with the increase of S/N ratio but the extent of
improvement varied significantly, probably due to the variance originating from
phase correction. Nevertheless, the signals F, H, and J arising from multiple
protons residing on oxycarbons showed good repeatability, making them
suitable for use in quantitation.
processing of the NMR specra1, Phase correction2,Baseline correction3,Drift control4,Integration
C44H68O13 MW : 805.0 OA
JJ
CCHH AA
BB
EEJJ FF
GG
FF
KKH,IH,I
C,DC,DFF
Pyridine(IS)
A BC D
E F GHI
J
K
Fig. 3 The NMR spectrum of OA measured by method B
Method BMethod BMethod BMethod BMethodAMethodAMethodAMethodANumberNumberNumberNumber
SignalSignalSignalSignal N=9N=9N=9N=9N=9N=9N=9N=9ofofofof
S.D.S.D.S.D.S.D.PurityPurityPurityPurityS.D.S.D.S.D.S.D.PurityPurityPurityPurityprotonsprotonsprotonsprotons
0.230.230.230.23
0.970.970.970.97
0.210.210.210.21
0.140.140.140.14
0.230.230.230.23
0.150.150.150.15
0.250.250.250.25
0.130.130.130.13
0.200.200.200.20
0.120.120.120.12
0.190.190.190.19
0.190.190.190.19
0.120.120.120.12
1.221.221.221.22
0.410.410.410.41
0.100.100.100.10
0.160.160.160.16
0.100.100.100.10
0.280.280.280.28
0.070.070.070.07
0.400.400.400.40
0.170.170.170.17
0.250.250.250.25
0.0.0.0.28282828
97.597.597.597.597.197.197.197.1AllAllAllAllAverageAverageAverageAverage
96.896.896.896.896.996.996.996.91111KKKK
97.197.197.197.1
97.097.097.097.0
98.098.098.098.0
97.297.297.297.2
99.499.499.499.4
97.297.297.297.2
97.297.297.297.2
100.1100.1100.1100.1
99.899.899.899.8
97.397.397.397.3
97.297.297.297.2F,H,JF,H,JF,H,JF,H,J
97.597.597.597.5
97.997.997.997.9
97.197.197.197.1
99.0 99.0 99.0 99.0
97.097.097.097.0
97.397.397.397.3
98.698.698.698.6
98.998.998.998.9
99996.56.56.56.5
1111BBBB
2222CCCC
2222
1111
3333
1111
3333
1111
1111
JJJJ
IIII
HHHH
GGGG
FFFF
EEEE
AAAA
Table 4 The calculated purities from OA measured by method A and B
The spectral feature, the trend in purity of signals, and uncertainty of signals were similar between OA and DTX1. The signal purity of OA determined by the
Method A was nearly equivalent to that by Method B (Table 4).
Maleic acid
ca. 2 mg
Maleic acid
ca. 2 mg
CRM
Methanol-d6
Ca. 5 mL
Methanol-d6
700 mg
Solvents
5 mm
5 mm
NMR tube
Pyridine
some dropB
CHD2OD in
methanolA
ISMethod
Pyridine / Methanol
1 mL
Methanol-d6
ca. 700 mg
IS solution
DTX1 portionPyridineB
OA ca. 20 mg
OA ca. 9 mg
DTX1 ca. 4 mgCHD
2ODA
SampleISMethod
Table 1 The detail of accurate quantification of IS use of CRM
CRMIS
ISSample
Accurate samplingChange to
solutionqNMR
Measurements
Spectrumprocessing
andCalculation
Fig. 3 The process of method A and B
Table 2 The detail of accurate quantification of DSP-toxins use of IS
Fig. 2 The calculated purities of signals on qNMR spectrum of DTX1 measured by Method ASignals in the alkane region (δ0.7 – 2.4ppm) were congested and overlapped and thus were judged unsuitable for purity calculation. Signals of protons on oxygenated
carbons (δ3.2-4.7 ppm) or olefinic carbons (δ5.5-5.8 ppm) were well or moderately well separated. They were judged to be suitable for purity calculation. Signals
composed of two or three protons gave lower purity score than those of single proton signals. Uncertainty was smaller when several isolated signals were used.
Results and Discussion Results and Discussion Results and Discussion Results and Discussion
A1H
B1H
C1H××××2
D1H
E1H
F3H
G1H H
3H II1H1H
J2H
K1H LL
1H1HMM1H1H
NN18H18H OO
5H5H
PP9H9H
QQ1H1H
RR6H6H
SS7H7H
HDOHDO
99.199.199.199.1
±±±±0.40.40.40.4
98.998.998.998.9
±±±±0.50.50.50.5
100.5100.5100.5100.5
±±±±0.30.30.30.3
93.793.793.793.7
±±±±0.40.40.40.4
96.996.996.996.9
±±±±0.10.10.10.1
104.1104.1104.1104.1
±±±±0.50.50.50.598.298.298.298.2
±±±±0.10.10.10.1
103.7103.7103.7103.7
±±±±0.40.40.40.497.797.797.797.7
±±±±0.20.20.20.297.897.897.897.8
±±±±0.40.40.40.4
107.9107.9±±±±±±±±0.50.5
99.799.7±±±±±±±±0.50.5
98.198.1±±±±±±±±0.10.1
99.699.6±±±±±±±±0.10.1
103.1103.1±±±±±±±±0.10.1
104.5104.5±±±±±±±±0.40.4
96.396.3±±±±±±±±0.10.1
100.7100.7±±±±±±±±0.20.2C45H70O13
MW : 819.0
DTX1N,ON,O
N,PN,PN,ON,O
JJ
CC NN
HH
LL
AA
BB
EE
M,OM,O
NN
NNNN
HH
JJFF
GG
FF
P,SP,S
NN
KKNN
N,PN,PQ,NQ,N
PPPP
OO
H,IH,I
SS
RR
C,DC,D
RR
NN
PPFF
SS
CHD2OD(IS)
Average of purity at signal AAverage of purity at signal AAverage of purity at signal AAverage of purity at signal A----KKKK
all signals : 99.1 % all signals : 99.1 % all signals : 99.1 % all signals : 99.1 % ±±±±3.0 %3.0 %3.0 %3.0 %
two or three protons signals (F,H,J) : 97.6 %two or three protons signals (F,H,J) : 97.6 %two or three protons signals (F,H,J) : 97.6 %two or three protons signals (F,H,J) : 97.6 % ±±±±0.6 %0.6 %0.6 %0.6 %
oxymethine or oxymetylene E-K 12Holefin A-D 5H alkane L-S 48H
CRM sampleRemovablesubstances IS
Fig. 2 The indirect method using removable substance as IS
Method A and BMethod A and BMethod A and BMethod A and B
Reference Reference Reference Reference Saito T, Ihara T, Koike M, Kinugasa S, Fujimine Y, Nose K, Hirai T (2009) Accred Qual Assur 14 : 79 - 86
Method CMethod CMethod CMethod CMethod BMethod BMethod BMethod B
2.392.392.392.39N/AN/AN/AN/AKKKK
N/AN/AN/AN/AN/AN/AN/AN/AJJJJ
N/AN/AN/AN/A2.532.532.532.53IIII
2.492.492.492.492.552.552.552.55HHHH
2.542.542.542.542.542.542.542.54GGGG
2.362.362.362.36
N/AN/AN/AN/A
2.442.442.442.44
2.472.472.472.47
2.402.402.402.40
2.182.182.182.18
Result (mg)Result (mg)Result (mg)Result (mg)
2.412.412.412.41EEEE
2.442.442.442.44DDDD
2.462.462.462.46CCCC
2.422.422.422.42BBBB
2.392.392.392.39AAAA
2.492.492.492.49FFFF
SignalSignalSignalSignal
Table 5 Compare the result of DTX1 using Method B and C
The quantitative value of qNMR using method B was nearly
equivalent to the value of method C.
Certified Reference Material (CRM) is important to quantify target analytes in a
unit traceable to the SI units. However, we chose not to use CRM for Internal
Standards (IS) to avoid contamination of the precious toxins. Instead, we tested
an indirect method using two removable substances as IS. The ISs are quantified
by qNMR using CRM.
Method A : Use of the residual proton signal in deuterated methanol as Internal Standard (IS)Method B : Use of a volatile substance as an Internal Standard
Method C : Use of an external standard in a co-axial double NMR tube
Materials and methodsMaterials and methodsMaterials and methodsMaterials and methods
MaterialsMaterialsMaterialsMaterialsDTX1 (25mg) and OA (60mg) were purified in our laboratory. They were dried in
a vacuum desiccator before qNMR measurements. Maleic acid or 1,4-BTMSB
of a CRM grade was used as an internal standard for qNMR, depending on the
conditions.
qNMR Measurements
Spectrumprocessing
andCalculation
CRM solution1,4-BTMSB / CDCl3
1 mg / mL
DTX1 solutionPortion / CD3OD
Fig. 4 The process of method C
In addition, we tried another method which separate DSP-toxins sample from
IS by using an external standard in a co-axial double NMR tube.
Method CMethod CMethod CMethod C
IntroductionIntroductionIntroductionIntroductionQuantification of the Quantification of the diarrheticdiarrhetic shellfishshellfish poisoning toxins (DSP-toxins) is desired
to shift from the mouse bioassayto shift from the mouse bioassay (MBA) to LC-MS or other instrumental
analysis.analysis.
The reference toxins for use in instrumental analysis are to be of proven purity
and quantity traceable to International System of Units (SI units, e.g. kg). Though
quantitative 1HNMR (qNMR) is a powerful tool to quantify contaminants,
including other shellfish toxins, the protons to be used for quantification should
be selected carefully to minimize the uncertainty, taking into consideration the
structural features that affect the spectra, e.g. signal overlap and conformational
diversity. In the present study, we chose DTX1 and OA as target toxins and
compared three methods of measurements for the performance: easiness,
practicality, and accuracy.