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.