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Accurate quantification of DTX1 standard by quantitative Nuclear Magnetic Resonance Tsuyoshi Kato, Mika Nagae, Tomoji Igarashi and Takeshi Yasumoto Japan 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 1 H Value Decoupling nuclei Decoupling method Data aquisition Relaxation delay Acquisition time Parameter 4 s Nuclei 60 s Spectral width 8 times Pluse angle MPF8 Temperature 13C spinning Value Parameter Summary Summary Summary Summary We compared qNMR and weighing methods for accuracy to quantify DTX1 and OA. The 1 HNMR 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 1 H nuclei Psample ＝ Ssample Sstandard Msample Mstandard Wstandard Wsample Hstandard Hsample × Pstandard × × × Fig. 1 The MS spectra of DTX1 and OA by infusion analysis Both toxin-standards produced, respectively, essentially single peaks, indicating the insignificance of impurities. DTX1 DTX1 OA OA Table 6 Comparison of the results between weighing and qNMR methods Weighing and qNMR Weighing and qNMR Weighing and qNMR Weighing and qNMR qNMR method* qNMR method* qNMR method* qNMR method* Weighing method Weighing method Weighing method Weighing method 8.20 8.20 8.20 8.20 4.47 4.47 4.47 4.47 0.64 0.64 0.64 0.64 Expanded Expanded Expanded Expanded Uncertainty Uncertainty Uncertainty Uncertainty (RSD%) k=2 (RSD%) k=2 (RSD%) k=2 (RSD%) k=2 97.6 97.6 97.6 97.6 97.1 97.1 97.1 97.1 97.2 97.2 97.2 97.2 Purity Purity Purity Purity 1.18 1.18 1.18 1.18 0.33 0.33 0.33 0.33 0.64 0.64 0.64 0.64 Expanded Expanded Expanded Expanded Uncertainty Uncertainty Uncertainty Uncertainty (RSD%) k=2 (RSD%) k=2 (RSD%) k=2 (RSD%) k=2 4.17 4.17 4.17 4.17 8.73 8.73 8.73 8.73 19.5 19.5 19.5 19.5 Measured value Measured value Measured value Measured value (mg) (mg) (mg) (mg) A A B method method method method 0.02 0.02 0.02 0.02 Ultra micro Ultra micro Ultra micro Ultra micro 20.1 20.1 20.1 20.1 Okadaic Okadaic Okadaic Okadaic acid acid acid acid 4.45 4.45 4.45 4.45 Semi micro Semi micro Semi micro Semi micro 8.99 8.99 8.99 8.99 8.12 8.12 8.12 8.12 Semi micro Semi micro Semi micro Semi micro 4.27 4.27 4.27 4.27 DTX1 DTX1 DTX1 DTX1 Expanded Expanded Expanded Expanded Uncertainty Uncertainty Uncertainty Uncertainty (RSD%) k=2** (RSD%) k=2** (RSD%) k=2** (RSD%) k=2** Balance type Balance type Balance type Balance type Weighing size Weighing size Weighing size Weighing size (mg) (mg) (mg) (mg) Analyte Analyte Analyte Analyte * 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 repeatability RSD(%) 1transient 1transient 1transient 1transient 8transients 8transients 8transients 8transients 16384transients 16384transients 16384transients 16384transients 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 DTX1 Three 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 specra 1, Phase correction 2,Baseline correction 3,Drift control 4,Integration C 44 H 68 O 13 MW : 805.0 OA J C H A B E J F G F K H,I H,I C,D C,D F Pyridine(IS) A B CD E F G H I J K Fig. 3 The NMR spectrum of OA measured by method B Method B Method B Method B Method B MethodA MethodA MethodA MethodA Number Number Number Number Signal Signal Signal Signal N=9 N=9 N=9 N=9 N=9 N=9 N=9 N=9 of of of of S.D. S.D. S.D. S.D. Purity Purity Purity Purity S.D. S.D. S.D. S.D. Purity Purity Purity Purity protons protons protons protons 0.23 0.23 0.23 0.23 0.97 0.97 0.97 0.97 0.21 0.21 0.21 0.21 0.14 0.14 0.14 0.14 0.23 0.23 0.23 0.23 0.15 0.15 0.15 0.15 0.25 0.25 0.25 0.25 0.13 0.13 0.13 0.13 0.20 0.20 0.20 0.20 0.12 0.12 0.12 0.12 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.12 0.12 0.12 0.12 1.22 1.22 1.22 1.22 0.41 0.41 0.41 0.41 0.10 0.10 0.10 0.10 0.16 0.16 0.16 0.16 0.10 0.10 0.10 0.10 0.28 0.28 0.28 0.28 0.07 0.07 0.07 0.07 0.40 0.40 0.40 0.40 0.17 0.17 0.17 0.17 0.25 0.25 0.25 0.25 0. 0. 0. 0.28 28 28 28 97.5 97.5 97.5 97.5 97.1 97.1 97.1 97.1 All All All All Average Average Average Average 96.8 96.8 96.8 96.8 96.9 96.9 96.9 96.9 1 K 97.1 97.1 97.1 97.1 97.0 97.0 97.0 97.0 98.0 98.0 98.0 98.0 97.2 97.2 97.2 97.2 99.4 99.4 99.4 99.4 97.2 97.2 97.2 97.2 97.2 97.2 97.2 97.2 100.1 100.1 100.1 100.1 99.8 99.8 99.8 99.8 97.3 97.3 97.3 97.3 97.2 97.2 97.2 97.2 F,H,J F,H,J F,H,J F,H,J 97.5 97.5 97.5 97.5 97.9 97.9 97.9 97.9 97.1 97.1 97.1 97.1 99.0 99.0 99.0 99.0 97.0 97.0 97.0 97.0 97.3 97.3 97.3 97.3 98.6 98.6 98.6 98.6 98.9 98.9 98.9 98.9 96.5 6.5 6.5 6.5 1 B 2 C 2 1 3 1 3 1 1 J I H G F E A 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-d 6 Ca. 5 mL Methanol-d 6 700 mg Solvents 5 mm 5 mm NMR tube Pyridine some drop B CHD2OD in methanol A IS Method Pyridine / Methanol 1 mL Methanol-d 6 ca. 700 mg IS solution DTX1 portion Pyridine B OA ca. 20 mg OA ca. 9 mg DTX1 ca. 4 mg CHD 2 OD A Sample IS Method Table 1 The detail of accurate quantification of IS use of CRM CRM IS IS Sample Accurate sampling Change to solution qNMR Measurements Spectrum processing and Calculation 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 A Signals 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 A 1H B 1H C 1H×2 D 1H E 1H F 3H G 1H H 3H I I 1H 1H J 2H K 1H L L 1H 1H M M 1H 1H N N 18H 18H O O 5H 5H P P 9H 9H Q Q 1H 1H R R 6H 6H S S 7H 7H HDO HDO 99.1 99.1 99.1 99.1 ±0.4 0.4 0.4 0.4 98.9 98.9 98.9 98.9 ±0.5 0.5 0.5 0.5 100.5 100.5 100.5 100.5 ±0.3 0.3 0.3 0.3 93.7 93.7 93.7 93.7 ±0.4 0.4 0.4 0.4 96.9 96.9 96.9 96.9 ±0.1 0.1 0.1 0.1 104.1 104.1 104.1 104.1 ±0.5 0.5 0.5 0.5 98.2 98.2 98.2 98.2 ±0.1 0.1 0.1 0.1 103.7 103.7 103.7 103.7 ±0.4 0.4 0.4 0.4 97.7 97.7 97.7 97.7 ±0.2 0.2 0.2 0.2 97.8 97.8 97.8 97.8 ±0.4 0.4 0.4 0.4 107.9 107.9 ±0.5 0.5 99.7 99.7 ±0.5 0.5 98.1 98.1 ±0.1 0.1 99.6 99.6 ±0.1 0.1 103.1 103.1 ±0.1 0.1 104.5 104.5 ±0.4 0.4 96.3 96.3 ±0.1 0.1 100.7 100.7 ±0.2 0.2 C 45 H 70 O 13 MW : 819.0 DTX1 N,O N,O N,P N,P N,O N,O J C N H L A B E M,O M,O N N N H J F G F P,S P,S N K N N,P N,P Q,N Q,N P P O H,I H,I S R C,D C,D R N P F S CHD2OD (IS) Average of purity at signal A Average of purity at signal A Average of purity at signal A Average of purity at signal A-K 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 12H olefin A-D 5H alkane L-S 48H CRM sample Removable substances IS Fig. 2 The indirect method using removable substance as IS Method A and B Method A and B Method A and B Method 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 C Method C Method C Method C Method B Method B Method B Method B 2.39 2.39 2.39 2.39 N/A N/A N/A N/A K N/A N/A N/A N/A N/A N/A N/A N/A J N/A N/A N/A N/A 2.53 2.53 2.53 2.53 I 2.49 2.49 2.49 2.49 2.55 2.55 2.55 2.55 H 2.54 2.54 2.54 2.54 2.54 2.54 2.54 2.54 G 2.36 2.36 2.36 2.36 N/A N/A N/A N/A 2.44 2.44 2.44 2.44 2.47 2.47 2.47 2.47 2.40 2.40 2.40 2.40 2.18 2.18 2.18 2.18 Result (mg) Result (mg) Result (mg) Result (mg) 2.41 2.41 2.41 2.41 E 2.44 2.44 2.44 2.44 D 2.46 2.46 2.46 2.46 C 2.42 2.42 2.42 2.42 B 2.39 2.39 2.39 2.39 A 2.49 2.49 2.49 2.49 F Signal Signal Signal Signal 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 methods Materials and methods Materials and methods Materials and methods Materials Materials Materials Materials DTX1 (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 Spectrum processing and Calculation CRM solution 1,4-BTMSB / CDCl 3 1 mg / mL DTX1 solution Portion / CD 3 OD 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 C Method C Method C Method C Introduction Introduction Introduction Introduction Quantification of the Quantification of the diarrhetic diarrhetic shellfish shellfish poisoning toxins (DSP-toxins) is desired to shift from the mouse bioassay to 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 1 HNMR (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.
