Limited impact of presaturation time on the accuracy of 1H qNMR using a NOESY with high-power presaturation

Limited impact of presaturation time on the accuracy of 1H qNMR using a NOESY with high-power presaturation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Limited impact of presaturation time on the accuracy of 1 H qNMR using a NOESY with high-power presaturation Naoki Saito This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9252005/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 17 You are reading this latest preprint version Abstract A one-dimensional NOESY sequence with presaturation (NOESY-presat) is widely used for water suppression in 1 H NMR-based metabolomics. While presaturation strengths below approximately 100 Hz are generally recommended to maintain accuracy of quantitative 1 H NMR ( 1 H qNMR), higher presaturation strengths may be employed in practice to suppress large H 2 O signals and improve signal-to-noise ratios of 1 H NMR spectra. However, the accuracy of 1 H qNMR using NOESY-presat under such non-ideal conditions has not been systematically evaluated, particularly with respect to presaturation time. In this study, the effect of presaturation time on analyte concentration biases was examined under high presaturation strength of 356 to 370 Hz. Two H 2 O/D 2 O (90/10 vol %) solutions containing glycine (Gly), maleic acid (MA), and 3-(trimethylsilyl)-1-propanesulfonic acid- d 6 (DSS- d 6 ), or L-phenylalanine (Phe), L-valine (Val), MA, and DSS- d 6 were measured by 1 H qNMR using NOESY-presat. The results showed that large concentration biases of − 49% to − 8% for analyte signals located near the presaturation frequency corresponding to the bulk H 2 O signal were not reduced by altering the presaturation time over a wide range of 50 to 60,000 ms. These findings indicate that presaturation time is not an effective parameter for reducing concentration biases under high presaturation strengths. Therefore, presaturation strength is the dominant factor affecting accuracy of 1 H qNMR using NOESY-presat experiments. When high presaturation strengths are unavoidable, the resulting biases should be recognized as systematic and evaluated for each analyte signal, consistent with the established understanding for low presaturation strengths in 1 H qNMR. accuracy 1D NOESY presaturation time qNMR water suppression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Solution quantitative nuclear magnetic resonance (qNMR) spectroscopy has been advancing rapidly. In particular, high-sensitivity solution 1 H, 19 F, and 31 P qNMR are now versatile, broadly applicable techniques for determining concentrations or purities of organic compounds [ 1 – 9 ]. QNMR can theoretically quantify various analytes using reference materials, which are different from the analytes [ 10 – 12 ]; the integral areas of the qNMR spectra can accurately reflect the molar concentrations of the NMR nuclei, independently of the molecular structure. Comparing the integral area of an accurate quantity of reference material as internal standard with analyte integral areas from an unknown molar concentration of NMR nuclei allows the molar concentration of the analyte to be determined accurately. The concentration obtained can be converted mathematically to the purity of the analyte. The developments in solution qNMR have also advanced chemical analyses, such as metabolomic analyses. 1 H NMR human metabolomics is the systematic profiling of small-molecule metabolites in biofluids such as urine [ 13 , 14 ], serum [ 15 , 16 ], plasma [ 17 , 18 ], saliva [ 19 , 20 ], and semen [ 21 , 22 ]. In principle, 1 H qNMR can quantify metabolites of interest in a profile accurately without reference materials of the metabolites. Accurate concentrations of specific metabolites may be indicators of human diseases, including early stage cancer [ 23 , 24 ], the SARS-CoV-2 infection severity and phase [ 25 , 26 ]. 1 H NMR and 1 H qNMR approaches require minimal sample pretreatments, typically involving only buffer, internal standard, and deuterium oxide (D 2 O) addition to the supernatant obtained after centrifugation of a human sample [ 27 ]. These NMR approaches are easier than those using high-performance liquid chromatography-mass spectrometry [ 28 , 29 ] or gas chromatography-mass spectrometry [ 30 , 31 ]. However, one of the major challenges in 1 H qNMR for human samples is the presence of abundant light water (H 2 O). Effective water suppression is essential to avoid baseline distortion and dynamic range limitations, which can compromise the accuracy of solute signal areas on 1 H qNMR spectra. Inappropriate water suppression conditions introduce systematic bias in analyte concentrations. A one-dimensional NOESY sequence with presaturation (NOESY-presat) is widely used for water suppression in 1 H NMR and 1 H qNMR, particularly in human metabolomics applications [ 32 – 34 ]. For water suppression, suppression of the 1 H signal of faraway water, which is far from RF coil center, is crucial [ 35 ]. RF B 1 inhomogeneity means that presaturation alone cannot reduce the faraway water signal [ 36 ], whereas NOESY-presat can with a combination of presaturation and phase cycling. Quantitative accuracy can generally be preserved under recommended conditions, typically using presaturation strengths below approximately 100 Hz. However, higher presaturation strengths may be required in practice to sufficiently suppress large water signals, improve spectral quality, or accommodate limitations in receiver dynamic range, especially when using old probes and/or spectrometers. But the accuracy of 1 H qNMR using NOESY-presat under such non-ideal conditions has not been systematically evaluated [ 37 , 38 ]. In the author’s previous work, the effects of NOESY-presat parameters of evolution time, mixing time, and presaturation strength on quantitative accuracy were examined [ 39 ]. Analyte concentration biases arising from evolution and mixing times were attributed to off-resonance effects and longitudinal relaxation, respectively, and were minimized under appropriate conditions. Significant concentration bias was not observed for presaturation strengths below 100 Hz. In contrast, substantial negative biases in measured concentrations were observed at higher presaturation strengths (e.g., up to approximately 400 Hz), although such conditions improved baseline distortion and signal-to-noise ratios (SNRs). These results indicate a trade-off between spectral quality and quantitative accuracy under high-power presaturation. However, the presaturation time was fixed and its contribution to concentration bias remained unclear in the previous studies [ 39 , 40 ]. Systematic investigations of presaturation time effects are still limited [ 35 , 37 – 40 ]. The present work examined the effect of presaturation time on analyte concentration bias under high presaturation strengths (Fig. 1 ). The work focuses on practical cases in which higher presaturation strengths are employed rather than challenging the established recommendation of using low presaturation strengths for 1 H qNMR. The concentration biases were evaluated accurately by using certified reference materials (CRMs) with well-defined purities. The results provide practical insight into the limitations of 1 H qNMR using NOESY-presat under non-ideal presaturation conditions and clarify whether optimization of presaturation time can mitigate the bias induced by high presaturation strength. Materials and methods Materials and apparatus The samples were glycine (Gly; CRM number 6022-a, National Metrology Institute of Japan (NMIJ), Ibaraki, Japan; purity 0.999 ± 0.002 kg kg − 1 , where the value after “±” indicates expanded uncertainty of the coverage factor k = 2), maleic acid (MA; Trace Sure, FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan; purity 0.998 ± 0.005 kg kg − 1 , k = 2), L-phenylalanine (Phe; CRM number 6014-a, NMIJ; purity 0.999 ± 0.002 kg kg − 1 , k = 2), and L-valine (Val; CRM number 6015-a, NMIJ; purity 0.998 ± 0.002 kg kg − 1 , k = 2). The sample purities were verified using conventional 1 H qNMR in previous work [ 40 ]. The internal standard was 3-(trimethylsilyl)-1-propanesulfonic acid- d 6 (DSS- d 6 ; Trace Sure, FUJIFILM Wako Pure Chemical Co., Ltd.; purity 0.923 ± 0.008 kg kg − 1 , k = 2). Solvents were D 2 O (ISOTEC Inc., Miamisburg, OH, USA; 99.9 atom % D) and pure H 2 O. High-grade 7 in. NMR tubes (FUJIFILM Wako Pure Chemical Co., Ltd.), aluminum weighing dishes (FUJIFILM Wako Pure Chemical Co., Ltd.; φ8 mm, 0.05 mL), and clean screw vials (AS ONE Corp., Osaka, Japan; 20 mL) were used. Analytical balances were a microbalance with 0.001 mg resolution (MX5, Mettler-Toledo Inc., Greifensee, Switzerland) and a semi-microbalance with 0.1 mg resolution (ME204E, Mettler-Toledo Inc.). A 500 MHz 1 H NMR spectrometer was used (ECA 500, JEOL Ltd., Tokyo, Japan; 11.75 Tesla, Delta ver. 5.0.6 software) with a 5 mm auto-tunable broadband probe (TH5AT/FG, JEOL Ltd.). Spectral software was Mnova vers. 7.0.2 and 14.1.2 (Mestrelab Research, Santiago de Compostela, Spain). Sample preparations A sample solution prepared in the previous work was used [ 39 ]. The solution was a H 2 O/D 2 O (90/10 vol %) solution containing 275.4 mg kg − 1 Gly, 268.2 mg kg − 1 MA, and 892.9 mg kg − 1 DSS- d 6 . Expanded uncertainties for the Gly and MA concentrations were 0.6 and 1.4 mg kg − 1 , respectively. The expanded uncertainties were determined by multiplying combined standard uncertainties by a coverage factor k of 2, corresponding to a confidence level of approximately 95%. The combined standard uncertainties are shown in Fig. SI1. A sample solution was prepared, containing Phe (5.034 mg), Val (5.064 mg), MA (5.352 mg), and DSS- d 6 (5.420 mg), which were precisely weighed using the aluminum weighing dishes and the microbalance with 0.001 mg resolution. The samples with the dishes were put in a screw vial. H 2 O/D 2 O (90/10 vol %) solution (5 mL, 5159.6 mg) was added to the screw vial and weighed using the semi-microbalance with 0.1 mg resolution. Concentrations of the prepared solutions were 970.8 mg kg − 1 Phe, 975.6 mg kg − 1 Val, 1031.0 mg kg − 1 MA, and 965.7 mg kg − 1 DSS- d 6 . Expanded uncertainties ( k = 2) for the Phe, Val and MA concentrations were 2.1, 2.1 and 5.2 mg kg − 1 , respectively. The expanded uncertainties were determined using the combined standard uncertainties shown in Fig. SI1. NMR measurements As with the previous work [ 39 ], a previously installed NOESY sequence “single_pulse_noesy.jxp” was used but a scramble pulse (random phase pulse) to reduce remanent magnetization was removed to use the most popular NOESY sequence. All NMR measurements were performed using the following common parameters: spectral width of 500 ppm without digital filter; acquisition time of 4.2 s; probe temperature of 25°C; 32 scans; two dummy scans; relaxation delay time of 60 s; evolution time of 1 µs; mixing time of 0 s; DANTE presaturation interval of 0 s; DANTE presaturation offset the same as the frequency offset; auto receiver gain; 13 C decoupling and sample spin off. The spectral width of 500 ppm was used to avoid bias in signal area that may vary periodically due to the digital filter [ 41 ]. CW presaturation mode was conducted at the DANTE presaturation [ 42 ] interval of 0 s. For measurements of the H 2 O/D 2 O (90/10 vol %) solution containing Gly, MA, and DSS- d 6 , the frequency offset was 4.647 ppm of the bulk H 2 O signal. Presaturation time was arrayed over a maximum range from 1 to 60,000 ms. The presaturation attenuation was 40 dB, which was equivalent to an RF irradiation strength (γB 1 /2π) of 356.3 Hz. The RF irradiation strength was calculated by the reciprocal of the 360° pulse width converted using presaturation attenuation of 40 dB in addition to the 90° pulse width of 13.84 µs and 90° pulse attenuation of 5.9 dB (Table SI1). For measurements of the H 2 O/D 2 O (90/10 vol %) solution containing Phe, Val, MA and DSS- d 6 , the frequency offset was 4.666 ppm of the bulk H 2 O signal. The presaturation time was arrayed over a maximum range from 50 to 60,000 ms. The presaturation attenuation was 40 or 55 dB, which was equivalent to RF irradiation strengths of 370.2 or 65.8 Hz, respectively. The RF irradiation strengths were calculated by the reciprocals of 360° pulse widths converted using presaturation attenuation of 40 or 55 dB in addition to a 90° pulse width of 13.32 µs and 90° pulse attenuation of 5.9 dB (Table SI2). The H 2 O/D 2 O (90/10 vol %) solution containing Phe, Val, MA, and DSS- d 6 was also measured using a presaturation sequence. The frequency offset and presaturation time range were the same as for the NOESY-presat sequence. The presaturation attenuation was 40 dB. Spectral analyses The 1 H NMR spectra were referenced to the DSS- d 6 signal, which was set to 0 ppm. The spectral phase was manually corrected. Integral ranges for analyte signals and the internal standard were set manually so that their 13 C satellite signals were included. An integral range was set for each signal, although the integral range of the phenyl signals of Phe included all aromatic CH signals because they overlapped with each other. The spectral baseline was manually corrected using a linear segment or cubic spline algorithm. The measured concentrations were calculated for each analyte signal using Eq. 1. C a = ( S a / S IS )·( H IS / H a )·( W IS / W SS )·( M a / M IS )· P IS ·10 6 (1) Here, C is concentration (mg kg − 1 ), S is 1 H signal area, H is the number of 1 H nuclei on one molecule contributing to the 1 H signal area, W is the weight (mg), M is molar mass (g mol − 1 ), and P is purity (kg kg − 1 ). Subscripts a, IS, and SS refer to the analyte, internal standard, and sample solution, respectively. Results and discussion Receiver gain and SNR profiles Figure 2 shows a 1 H NMR spectrum of a H 2 O/D 2 O (90/10 vol %) solution containing Gly, MA, and DSS- d 6 measured using a NOESY-presat sequence. The presaturation attenuation was 40 dB, which was equivalent to the presaturation strength of 356.3 Hz to maximize the SNR under the experimental conditions. When the presaturation time increased from 1 to 1,000 ms, the receiver gain also increased from 8 to 38 dB because the SNR of the bulk H 2 O signal decreased dramatically (Figs. 3 a, 3 b). Consequently, the SNRs of the DSS- d 6 , Gly, and MA signals increased (Figs. 3 c, 3 d). For presaturation times from 1,000 to 60,000 ms, which was the same as the relaxation delay time, the receiver gain was the maximum constant value of 38 dB (Figs. 3 a, 3 b). The bulk H 2 O signal must have become saturated after the presaturation time of 1,000 ms because the SNR of the signal did not change significantly. Effect of presaturation time on measurement accuracy The following analysis focuses on high presaturation strength conditions that are not generally recommended for 1 H qNMR using NOESY-presat but may be encountered in practical measurements. The measured concentrations of Gly and MA did not respond significantly to the presaturation time from 1 to 60,000 ms (Figs. 3 e, 3 f). The concentrations are expressed as the deviation from the prepared concentration in the sample on the vertical axes (The unconverted concentration data are shown in Table SI3). The concentrations with presaturation times of less than 1,000 ms showed wider scattering than those with presaturation times of more than 1,000 ms, probably because of low measurement repeatability caused by insufficient receiver gains. Average biases of − 22% and − 8% were observed for the measured concentrations of Gly and MA, respectively. The biases were consistent with previous work [ 39 ], in which the presaturation attenuation of 40 dB was necessary to maximize the SNRs of Gly and MA. Decreasing the concentration bias caused by the presaturation attenuation of 40 dB by optimizing the presaturation time would increase the concentrations accuracy, and the NOESY-presat sequence would be more effective for 1 H qNMR human metabolomics. However, the presaturation time did not decrease the concentration biases of Gly and MA. To verify the effect of the presaturation time using other samples, a H 2 O/D 2 O (90/10 vol %) solution containing Phe, Val, MA, and internal standard DSS- d 6 was measured (Fig. 4 ). The prepared concentrations of the solution were higher than those of the H 2 O/D 2 O (90/10 vol %) solution containing Gly, MA, and DSS- d 6 to improve measurement repeatability. A common sample of MA was added to both solutions to compare the results. Figure 5 shows the responses of the measured concentrations of Phe, Val, and MA to the presaturation time from 50 to 60,000 ms. The concentrations are expressed as the deviation from the prepared concentration in the sample on the vertical axes (The unconverted concentration data are shown in Table SI4). The graphs in Figs. 5 a to 5 g are displayed in ascending order of absolute chemical shift difference between the analyte signal and presaturation offset. There was no change in MA concentrations (Fig. 5 d), and the average concentration bias was − 10%, which was consistent with the bias measured using the solution containing Gly, MA, and DSS- d 6 . Similarly, there was no change in Phe concentrations obtained using NCH and CH 2 signals (Figs. 5 a, 5 c), and the average concentration biases were − 49% and − 15%, respectively. The Val concentrations obtained using NCH signal did not change (Fig. 5 b), and the average concentration bias was − 24%. The differences in concentration bias size must have been caused by the absolute chemical shift difference between the analyte signal and presaturation offset (Fig. 6 ). The closer the chemical shift of the analyte was to the presaturation frequency corresponding to the bulk H 2 O signal, the higher the concentration bias. These results agreed with previous studies [ 39 , 40 ]. These results indicate that the high presaturation strength of 370.2 Hz, which exceeds commonly recommended conditions, is the dominant factor causing the concentration biases. Non-linear trends of measured concentrations There were non-linear trends in concentration values obtained using the aromatic CH signals of Phe and the CH and CH 3 signals of Val (Figs. 5 e to 5 g). These signals were far from the presaturation frequency corresponding to the bulk H 2 O signal. Reducing the effect of the high presaturation strength may have revealed potential effects of the presaturation time. However, the non-linear trends in the concentrations probably involved off-resonance effects caused by using the NOESY-presat sequence because the frequency offsets were the same as the presaturation frequency corresponding to the bulk H 2 O signal. To reduce the off-resonance effects sufficiently, the H 2 O/D 2 O (90/10 vol %) solution containing Phe, Val, MA, and DSS- d 6 was measured again using a presaturation sequence. Figure 7 shows measured concentration trends for the aromatic CH signals of Phe and the CH and CH 3 signals of Val using the presaturation sequence. The presaturation strength and time were same as for the NOESY-presat sequence. Subtracting the data in Fig. 7 from those in Fig. 5 obtained using the NOESY-presat sequence produced residues that were independent of the presaturation time (Fig. 8 ). The average concentration biases measured using the aromatic CH signals of Phe and the CH and CH 3 signals of Val were + 3%, + 2%, and + 1%, respectively. The biases arose from the off-resonance effect caused by a mixing time of 0 ms and evolution time of 1 µs in the NOESY-presat sequence, according to a previous study [ 39 ]. As shown in Fig. 7 without the off-resonance effect, the concentrations obtained using the aromatic CH signals of Phe and the CH signal of Val increased with presaturation time and then became steady. The increase in the presaturation time contributed to decreasing the concentration biases. The concentration values obtained using the CH 3 signal of Val were generally steady and then decreased as the presaturation time increased, and decreasing the presaturation time helped to reduce the concentration biases. However, the mechanisms for these trends remain uncertain and warrant further investigation. The concentration biases measured using the aromatic CH signals of Phe and the CH and CH 3 signals of Val were − 1%, − 1%, and − 4%, respectively, at a presaturation time of 60,000 ms. The absolute chemical shift difference from the integral range center of the aromatic CH signals of Phe to the presaturation frequency corresponding to the bulk H 2 O signal was similar to that from the integral range center of the CH signal of Val to the presaturation offset. In contrast, that from the integral range center of the CH 3 signal of Val to the presaturation offset was larger than those for the aromatic CH signals of Phe and the CH signal of Val. Increasing the absolute chemical shift difference from an analyte signal to the presaturation offset did not decrease the measured concentration bias. In the previous work, larger the absolute chemical shift difference from an analyte signal to the presaturation offset was, the lower the measured concentration bias [ 40 ]. However, D 2 O solutions containing Phe or Val were measured in the previous work. Because H 2 O/D 2 O (90/10 vol %) solution was used in the present work, the non-linear trends shown in Fig. 7 may have been caused by the presaturation affecting the abundant H 2 O signal, for example, via the intermolecular 1 H- 1 H nuclear Overhauser effect [ 43 ] accompanied by the three spin effect [ 44 ]. The findings of this work highlight that high presaturation strengths introduce significant and systematic concentration biases that cannot be mitigated by adjusting presaturation time alone within the examined range, although they can improve baseline distortion and SNR in practical cases. Therefore, such conditions should be regarded as inappropriate for quantitative analyses. When high presaturation strengths are unavoidable in practice, the magnitude and direction of the bias should be carefully evaluated for each analyte signal. These observations are consistent with the established understanding that low presaturation strengths are preferable for maintaining quantitative accuracy. Conclusions The effect of presaturation time in a NOESY-presat sequence was evaluated under high presaturation strengths (up to approximately 400 Hz), which may be used in practice despite not being generally recommended for 1 H qNMR. Large concentration biases of − 49% to − 8% observed for analyte signals located near the presaturation frequency corresponding to the bulk H 2 O signal were not mitigated by adjusting presaturation time, indicating that presaturation time is not an effective parameter for reducing such biases. Therefore, presaturation strength should be carefully controlled to ensure the accuracy of 1 H qNMR using NOESY-presat. When high presaturation strengths are unavoidable, the resulting biases should be recognized as systematic and evaluated for each analyte signal, consistent with the established understanding for low presaturation strengths. The NOESY-presat sequence consists of a presaturation interval, followed by a first 90° pulse. After a short switching period, referred to as the evolution time (t 1 ) for phase cycling, a second 90° pulse is applied. This is followed by a mixing time (t m ) during presaturation to enhance the nuclear Overhauser effect, and finally a third 90° pulse. The interval time of the presaturation was altered, whereas presaturation during the mixing time was not applied because t m was set to 0 s for improving quantitative accuracy. The prepared concentrations were 275.4 mg kg − 1 Gly, 268.2 mg kg − 1 MA, and 892.9 mg kg − 1 DSS- d 6 . Total uncertainty budgets of the Gly and MA concentrations are shown in Fig. SI1. The chemical shift reference was the DSS- d 6 signal at 0 ppm. The faraway H 2 O is the water component far from the RF coil center in the NMR probe [ 35 ]. Full responses of a) receiver gain (open diamonds) and SNRs of bulk H 2 O (blue diamonds), c) DSS- d 6 (open squares), Gly (red triangles), and MA (red circles), and e) concentrations of Gly (red triangles) and MA (red circles) to presaturation times. Expanded responses to presaturation times of 1,000 to 5,000 ms in figures a), c), and e) are shown in figures b), d), and f), respectively. The colors of the vertical axes correspond to those of the legend icons. The SNRs of bulk H 2 O are absolute values. The prepared concentrations were 970.8 mg kg − 1 Phe, 975.6 mg kg − 1 Val, 1031.0 mg kg − 1 MA, and 965.7 mg kg − 1 DSS- d 6 . Total uncertainty budgets of the Phe, Val and MA concentrations are shown in Fig. SI1. The chemical shift reference was the DSS- d 6 signal at 0 ppm. Responses to presaturation times of 50 to 60,000 ms of concentrations of a) Phe obtained using the NCH signal, b) Val obtained using the NCH signal, c) Phe obtained using the CH 2 signal, d) MA, e) Val obtained using the CH signal, f) Phe obtained using the CH signal, and g) Val obtained using the CH 3 signal. Bars are the standard deviation obtained by three repeated measurements. The data are same as those shown in Fig. 5 . The absolute chemical shift differences from the presaturation offset are 0.672 (Phe NCH), 1.047 (Val NCH), 1.547 (Phe CH 2 ), 1.576 (MA), 2.473 (Val CH), 2.606 (Phe CH), and 3.751 ppm (Val CH 3 ) in ascending order. The differences were obtained as the differences from the presaturation offset of 4.666 ppm to the integral range centers of the analyte signals on the 1 H NMR spectra before chemical shift corrections. The dotted curve line is a linear trendline converted from an exponential trendline. Responses of concentrations of a) Val obtained using the CH signal, b) Phe obtained using the CH signal, and c) Val obtained using the CH 3 signal to presaturation times of 50 to 60,000 ms. Bars are the standard deviation obtained by three repeated measurements. The full version including all analyte signal data is shown in Fig. SI2. The unconverted concentration data are shown in Table SI5. Responses to presaturation times of 50 to 60,000 ms of concentrations of a) Val obtained using the CH signal, b) Phe obtained using the CH signal, and c) Val obtained using the CH 3 signal. The full version including all analyte signal data is shown in Fig. SI3. The unconverted concentration data are shown in Table SI6. Declarations Conflict of interest The author declares no conflicts of interest. Author contributions Naoki Saito: writing – review and editing, writing – original draft, visualization, validation, methodology, investigation, formal analysis, data curation and conceptualization. Data availability Data can be made available upon request to the corresponding author. Acknowledgments The present work was supported by JSPS KAKENHI Grant Number JP24K17709. The used NMR spectrometer was one of the Fundamental Instruments for Measurement and Analysis (FIMA), National Institute for Environmental Studies. I thank Dr. Hiroaki Sasakawa in JEOL Ltd. for useful discussion. The work was presented in part at the 64 th Annual Meeting of the NMR society of Japan, Okinawa, Japan, in November 2025. References Nishizaki Y, Sugimoto N, Miura T, Asakura K, Suematsu T, Korhonen SP, Lehtivarjo J, Niemitz M, Pauli GF (2024) Quantum mechanical quantitative nuclear magnetic resonance enables digital reference standards at all magnetic fields and enhances qNMR sustainability. 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Sci Rep 9:4786. https://doi.org/10.1038/s41598-019-41216-y Tristán AI, Luna CJ, Abreu AC, Campos FMA, Salmerón ADM, Rodríguez FI, Maresca MAR, García AB, Melguizo C, Prados J, Fernández I (2024) Metabolomic profiling of COVID-19 using serum and urine samples in intensive care and medical ward cohorts. Sci Rep 14:23713. https://doi.org/10.1038/s41598-024-74641-9 Matpan E, Baykal AT, Telci L, Kundak T, Serteser M (2025) Time-series metabolomic profiling of SARS-CoV-2 infection: possible prognostic biomarkers in patients in the ICU by 1 H-NMR analysis. PLoS ONE 20:e0327244. https://doi.org/10.1371/journal.pone.0327244 Emwas AH, Luchinat C, Turano P, Tenori L, Roy R, Salek RM, Ryan D, Merzaban JS, Daouk RK, Zeri AC, Gowda GAN, Raftery D, Wang Y, Brennan L, Wishart DS (2015) Standardizing the experimental conditions for using urine in NMR-based metabolomic studies with a particular focus on diagnostic studies: a review. Metabolomics 11:872–894. https://doi.org/10.1007/s11306-014-0746-7 Ciborowski M, Lipska A, Godzien J, Ferrarini A, Korsak J, Radziwon P, Tomasiak M, Barbas C (2012) Combination of LC–MS- and GC–MS-based metabolomics to study the effect of ozonated autohemotherapy on human blood. J Proteome Res 11:6231–6241. https://doi.org/10.1021/pr3008946 Vokuev MF, Baygildiev TM, Plyushchenko IV, Ikhalaynen YA, Ogorodnikov RL, Solontsov IK, Braun AV, Savelieva EI, Rybalchenko IV, Rodin IA (2021) Untargeted and targeted analysis of sarin poisoning biomarkers in rat urine by liquid chromatography and tandem mass spectrometry. Anal Bioanal Chem 413:6973–6985. https://doi.org/10.1007/s00216-021-03655-3 Zlatkis A, Bertsch W, Lichtenstein HA, Tishbee A, Shunbo F, Liebich HM, Coscia AM, Fleischer N (1973) Profile of volatile metabolites in urine by gas chromatography-mass spectrometry. Anal Chem 45:763–767. https://doi.org/10.1021/ac60326a036 Duarte GHB, Fernandes AMAP, Silva AAR, Obando HRZ, Amaral AG, Mesquita AS, Filho JS, Lima VCC, Costa FD, Andrade VP, Porcari AM, Eberlin MN, Simionato AVC (2020) Gas chromatography-mass spectrometry untargeted profiling of non-Hodgkin’s lymphoma urinary metabolite markers. Anal Bioanal Chem 412:7469–7480. https://doi.org/10.1007/s00216-020-02881-5 McKay RT (2011) How the 1D-NOESY suppresses solvent signal in metabonomics NMR spectroscopy: an examination of the pulse sequence components and evolution. Concepts Magn Reson A 38A:197–220. https://doi.org/10.1002/cmr.a.20223 Emwas AH, Roy R, McKay RT, Ryan D, Brennan L, Tenori L, Luchinat C, Gao X, Zeri AC, Gowda GAN, Raftery D, Steinbeck C, Salek RM, Wishart DS (2016) Recommendations and standardization of biomarker quantification using NMR-based metabolomics with particular focus on urinary analysis. J Proteome Res 15:360–373. https://doi.org/10.1021/acs.jproteome.5b00885 Vignoli A, Ghini V, Meoni G, Licari C, Takis PG, Tenori L, Turano P, Luchinat C (2019) High-throughput metabolomics by 1D NMR. Angew Chem Int Ed 58:968–994. https://doi.org/10.1002/anie.201804736 Mo H, Raftery D (2008) Pre-sat180, a simple and effective method for residual water suppression. J Magn Reson 190:1–6. https://doi.org/10.1016/j.jmr.2007.09.016 Szántay C (1998) Analysis and implications of transition-band signals in high-resolution NMR. J Magn Reson 135:334–352. https://doi.org/10.1006/jmre.1998.1599 Akira K, Nohara A, Utsunomiya Y, Mitome H, Watanabe S, Tanaka M, Tanaka A (2021) Application of quantitative NMR spectroscopy to the quality evaluation of diclofenac gargles as hospital preparations. Chem Pharm Bull 69:721–726. https://doi.org/10.1248/cpb.c21-00079 Garrido BC, Carvalho LJ, Burton IW, McCarron P (2025) High-accuracy quantitative nuclear magnetic resonance using improved solvent suppression schemes. Anal Chem 97:21240–21248. https://doi.org/10.1021/acs.analchem.5c01139 Saito N (2024) Basic accuracy of a 1D NOESY with presaturation method using standard solutions of amino and maleic acids. Anal Bioanal Chem 416:5721–5731. https://doi.org/10.1007/s00216-024-05491-7 Saito N (2023) Fresh dual presaturation method for analyzing H 2 O-rich samples using quantitative 1 H NMR. Anal Chem 95:7855–7862. https://doi.org/10.1021/acs.analchem.2c05639 Saito T, Yamazaki T, Numata M (2019) Development of nuclear magnetic resonance as a tool of quantitative analysis for organic materials. Metrologia 56:054002. https://doi.org/10.1088/1681-7575/ab348d Morris GA, Freeman R (1978) Selective excitation in Fourier transform nuclear magnetic resonance. J Magn Reson 29:433–462. https://doi.org/10.1016/0022-2364(78)90003-3 Dalvit C, Fogliatto G, Stewart A, Veronesi M, Stockman B (2001) WaterLOGSY as a method for primary NMR screening: practical aspects and range of applicability. J Biomol NMR 21:349–359. https://doi.org/10.1023/A:1013302231549 Keeler J, Neuhaus D, Williamson MP (1987) The nuclear Overhauser effect in strongly coupled spin systems. J Magn Reson 73:45–68. https://doi.org/10.1016/0022-2364(87)90224-1 Additional Declarations No competing interests reported. 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Saito","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIie3Qv0vDQBTA8XcImZ7cesEh/8JzFLT5V3IU7FpwdLlH4LpUsiaz/0BHx5RbI66FOiQInSOuoqLGqXi1m5T7LgfH+3A/AEKhf5kw3ysKboGGTeUnbAaS0x/JzzEI0Y7BoWTmZm0Pj4lEZ6+n0/MU5G0NZ3e/E2o0cwmb0+qG7bqkS23UJoO48RDQnB+/ObF4EHaN5DJQDUFsPRcrOs4RXPpJrpDe050EVvqL6MU92yOkWhg59xNadVyV4MbVfJmfII21VVFW+96SFJO278FdFDjpXvB1lErplk+x58e2i1QGLjb7EJA1iOf9SCgUCh12H9zUUbXxo/ahAAAAAElFTkSuQmCC","orcid":"","institution":"National Institute for Environmental Studies","correspondingAuthor":true,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Saito","suffix":""}],"badges":[],"createdAt":"2026-03-28 10:40:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9252005/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9252005/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106244702,"identity":"4c8182f5-91c7-4bd5-a416-7fcb0d65529c","added_by":"auto","created_at":"2026-04-06 15:41:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119663,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental scheme to examine the effect of presaturation time under high presaturation strength.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/2754001bca99e5ca28051988.png"},{"id":106244573,"identity":"11626fda-400c-4912-96c7-9913ff386cae","added_by":"auto","created_at":"2026-04-06 15:41:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectrum of a H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Gly, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/8d319e56e4439cca6215b8be.png"},{"id":106244567,"identity":"5eee0b7d-1f07-4f99-9963-e325df0eb4ef","added_by":"auto","created_at":"2026-04-06 15:41:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":846157,"visible":true,"origin":"","legend":"\u003cp\u003eResponses of receiver gain, SNR, and analyte concentration to presaturation time in a NOESY-presat sequence.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/3030129c833a4a009c385949.png"},{"id":106244724,"identity":"2307965b-56e0-4f4e-969c-4aad509cdbb1","added_by":"auto","created_at":"2026-04-06 15:41:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":172329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectrum of a H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Phe, Val, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/cdd80e2e639cc54ce8f3617e.png"},{"id":106403344,"identity":"40be6169-cf1a-40a7-9e4d-807d4a5e59f7","added_by":"auto","created_at":"2026-04-08 09:14:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1154138,"visible":true,"origin":"","legend":"\u003cp\u003eResponses of analyte concentration to presaturation time in a NOESY-presat sequence.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/14408c40be049b6e7cb2111d.png"},{"id":106244698,"identity":"c2c88c80-09ad-4680-a889-ee79e18f6e5a","added_by":"auto","created_at":"2026-04-06 15:41:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":138008,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between chemical shift difference from presaturation offset and analyte concentration bias.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/84f5d80dfbffc38c76d3d9e7.png"},{"id":106244570,"identity":"dce39cb3-13b4-4bcd-aa2f-7ac6464d9abf","added_by":"auto","created_at":"2026-04-06 15:41:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":500500,"visible":true,"origin":"","legend":"\u003cp\u003eResponses of analyte concentration to presaturation time in a presaturation sequence.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/b694fba1fa622f892489bde0.png"},{"id":106244572,"identity":"36e32ede-b556-470d-81c8-13b0e33cc9e8","added_by":"auto","created_at":"2026-04-06 15:41:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":511096,"visible":true,"origin":"","legend":"\u003cp\u003eResidues obtained by subtracting presaturation sequence data from NOESY-presat sequence data.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/c6fef7e2c560b35c4a1f4fd5.png"},{"id":106414950,"identity":"e1a24c52-c12f-46f8-8289-4c757001538e","added_by":"auto","created_at":"2026-04-08 10:31:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4160815,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/c0f2b6d1-09d8-4dd7-8798-a100ac2b2284.pdf"},{"id":106244696,"identity":"f194e6b2-7395-46b0-9634-4a1f18c699dd","added_by":"auto","created_at":"2026-04-06 15:41:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14474,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/fd78943a0b41102b1ba827f2.docx"},{"id":106403658,"identity":"6b0a359d-5f60-4099-94b4-3bdb0f664bbb","added_by":"auto","created_at":"2026-04-08 09:14:42","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":842044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryInformationACQUALNaokiSaito.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9252005/v1/d3ea6405ac9f617a78d2a59d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eLimited impact of presaturation time on the accuracy of \u003csup\u003e1\u003c/sup\u003eH qNMR using a NOESY with high-power presaturation\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSolution quantitative nuclear magnetic resonance (qNMR) spectroscopy has been advancing rapidly. In particular, high-sensitivity solution \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e19\u003c/sup\u003eF, and \u003csup\u003e31\u003c/sup\u003eP qNMR are now versatile, broadly applicable techniques for determining concentrations or purities of organic compounds [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. QNMR can theoretically quantify various analytes using reference materials, which are different from the analytes [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]; the integral areas of the qNMR spectra can accurately reflect the molar concentrations of the NMR nuclei, independently of the molecular structure. Comparing the integral area of an accurate quantity of reference material as internal standard with analyte integral areas from an unknown molar concentration of NMR nuclei allows the molar concentration of the analyte to be determined accurately. The concentration obtained can be converted mathematically to the purity of the analyte.\u003c/p\u003e \u003cp\u003eThe developments in solution qNMR have also advanced chemical analyses, such as metabolomic analyses. \u003csup\u003e1\u003c/sup\u003eH NMR human metabolomics is the systematic profiling of small-molecule metabolites in biofluids such as urine [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], serum [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], plasma [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], saliva [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and semen [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In principle, \u003csup\u003e1\u003c/sup\u003eH qNMR can quantify metabolites of interest in a profile accurately without reference materials of the metabolites. Accurate concentrations of specific metabolites may be indicators of human diseases, including early stage cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the SARS-CoV-2 infection severity and phase [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e1\u003c/sup\u003eH qNMR approaches require minimal sample pretreatments, typically involving only buffer, internal standard, and deuterium oxide (D\u003csub\u003e2\u003c/sub\u003eO) addition to the supernatant obtained after centrifugation of a human sample [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These NMR approaches are easier than those using high-performance liquid chromatography-mass spectrometry [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] or gas chromatography-mass spectrometry [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, one of the major challenges in \u003csup\u003e1\u003c/sup\u003eH qNMR for human samples is the presence of abundant light water (H\u003csub\u003e2\u003c/sub\u003eO). Effective water suppression is essential to avoid baseline distortion and dynamic range limitations, which can compromise the accuracy of solute signal areas on \u003csup\u003e1\u003c/sup\u003eH qNMR spectra. Inappropriate water suppression conditions introduce systematic bias in analyte concentrations.\u003c/p\u003e \u003cp\u003eA one-dimensional NOESY sequence with presaturation (NOESY-presat) is widely used for water suppression in \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e1\u003c/sup\u003eH qNMR, particularly in human metabolomics applications [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. For water suppression, suppression of the \u003csup\u003e1\u003c/sup\u003eH signal of faraway water, which is far from RF coil center, is crucial [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. RF B\u003csub\u003e1\u003c/sub\u003e inhomogeneity means that presaturation alone cannot reduce the faraway water signal [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], whereas NOESY-presat can with a combination of presaturation and phase cycling. Quantitative accuracy can generally be preserved under recommended conditions, typically using presaturation strengths below approximately 100 Hz. However, higher presaturation strengths may be required in practice to sufficiently suppress large water signals, improve spectral quality, or accommodate limitations in receiver dynamic range, especially when using old probes and/or spectrometers. But the accuracy of \u003csup\u003e1\u003c/sup\u003eH qNMR using NOESY-presat under such non-ideal conditions has not been systematically evaluated [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the author\u0026rsquo;s previous work, the effects of NOESY-presat parameters of evolution time, mixing time, and presaturation strength on quantitative accuracy were examined [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Analyte concentration biases arising from evolution and mixing times were attributed to off-resonance effects and longitudinal relaxation, respectively, and were minimized under appropriate conditions. Significant concentration bias was not observed for presaturation strengths below 100 Hz. In contrast, substantial negative biases in measured concentrations were observed at higher presaturation strengths (e.g., up to approximately 400 Hz), although such conditions improved baseline distortion and signal-to-noise ratios (SNRs). These results indicate a trade-off between spectral quality and quantitative accuracy under high-power presaturation. However, the presaturation time was fixed and its contribution to concentration bias remained unclear in the previous studies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Systematic investigations of presaturation time effects are still limited [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present work examined the effect of presaturation time on analyte concentration bias under high presaturation strengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The work focuses on practical cases in which higher presaturation strengths are employed rather than challenging the established recommendation of using low presaturation strengths for \u003csup\u003e1\u003c/sup\u003eH qNMR. The concentration biases were evaluated accurately by using certified reference materials (CRMs) with well-defined purities. The results provide practical insight into the limitations of \u003csup\u003e1\u003c/sup\u003eH qNMR using NOESY-presat under non-ideal presaturation conditions and clarify whether optimization of presaturation time can mitigate the bias induced by high presaturation strength.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and apparatus\u003c/h2\u003e \u003cp\u003eThe samples were glycine (Gly; CRM number 6022-a, National Metrology Institute of Japan (NMIJ), Ibaraki, Japan; purity 0.999\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 kg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, where the value after \u0026ldquo;\u0026plusmn;\u0026rdquo; indicates expanded uncertainty of the coverage factor \u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2), maleic acid (MA; Trace Sure, FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan; purity 0.998\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 kg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2), L-phenylalanine (Phe; CRM number 6014-a, NMIJ; purity 0.999\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 kg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2), and L-valine (Val; CRM number 6015-a, NMIJ; purity 0.998\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 kg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2). The sample purities were verified using conventional \u003csup\u003e1\u003c/sup\u003eH qNMR in previous work [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The internal standard was 3-(trimethylsilyl)-1-propanesulfonic acid-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e (DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e; Trace Sure, FUJIFILM Wako Pure Chemical Co., Ltd.; purity 0.923\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 kg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2). Solvents were D\u003csub\u003e2\u003c/sub\u003eO (ISOTEC Inc., Miamisburg, OH, USA; 99.9 atom % D) and pure H\u003csub\u003e2\u003c/sub\u003eO. High-grade 7 in. NMR tubes (FUJIFILM Wako Pure Chemical Co., Ltd.), aluminum weighing dishes (FUJIFILM Wako Pure Chemical Co., Ltd.; φ8 mm, 0.05 mL), and clean screw vials (AS ONE Corp., Osaka, Japan; 20 mL) were used. Analytical balances were a microbalance with 0.001 mg resolution (MX5, Mettler-Toledo Inc., Greifensee, Switzerland) and a semi-microbalance with 0.1 mg resolution (ME204E, Mettler-Toledo Inc.). A 500 MHz \u003csup\u003e1\u003c/sup\u003eH NMR spectrometer was used (ECA 500, JEOL Ltd., Tokyo, Japan; 11.75 Tesla, Delta ver. 5.0.6 software) with a 5 mm auto-tunable broadband probe (TH5AT/FG, JEOL Ltd.). Spectral software was Mnova vers. 7.0.2 and 14.1.2 (Mestrelab Research, Santiago de Compostela, Spain).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample preparations\u003c/h3\u003e\n\u003cp\u003eA sample solution prepared in the previous work was used [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The solution was a H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing 275.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Gly, 268.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MA, and 892.9 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e. Expanded uncertainties for the Gly and MA concentrations were 0.6 and 1.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The expanded uncertainties were determined by multiplying combined standard uncertainties by a coverage factor \u003cem\u003ek\u003c/em\u003e of 2, corresponding to a confidence level of approximately 95%. The combined standard uncertainties are shown in Fig. SI1. A sample solution was prepared, containing Phe (5.034 mg), Val (5.064 mg), MA (5.352 mg), and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e (5.420 mg), which were precisely weighed using the aluminum weighing dishes and the microbalance with 0.001 mg resolution. The samples with the dishes were put in a screw vial. H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution (5 mL, 5159.6 mg) was added to the screw vial and weighed using the semi-microbalance with 0.1 mg resolution. Concentrations of the prepared solutions were 970.8 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Phe, 975.6 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Val, 1031.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MA, and 965.7 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e. Expanded uncertainties (\u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2) for the Phe, Val and MA concentrations were 2.1, 2.1 and 5.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The expanded uncertainties were determined using the combined standard uncertainties shown in Fig. SI1.\u003c/p\u003e\n\u003ch3\u003eNMR measurements\u003c/h3\u003e\n\u003cp\u003eAs with the previous work [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], a previously installed NOESY sequence \u0026ldquo;single_pulse_noesy.jxp\u0026rdquo; was used but a scramble pulse (random phase pulse) to reduce remanent magnetization was removed to use the most popular NOESY sequence. All NMR measurements were performed using the following common parameters: spectral width of 500 ppm without digital filter; acquisition time of 4.2 s; probe temperature of 25\u0026deg;C; 32 scans; two dummy scans; relaxation delay time of 60 s; evolution time of 1 \u0026micro;s; mixing time of 0 s; DANTE presaturation interval of 0 s; DANTE presaturation offset the same as the frequency offset; auto receiver gain; \u003csup\u003e13\u003c/sup\u003eC decoupling and sample spin off. The spectral width of 500 ppm was used to avoid bias in signal area that may vary periodically due to the digital filter [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. CW presaturation mode was conducted at the DANTE presaturation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] interval of 0 s.\u003c/p\u003e \u003cp\u003eFor measurements of the H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Gly, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, the frequency offset was 4.647 ppm of the bulk H\u003csub\u003e2\u003c/sub\u003eO signal. Presaturation time was arrayed over a maximum range from 1 to 60,000 ms. The presaturation attenuation was 40 dB, which was equivalent to an RF irradiation strength (γB\u003csub\u003e1\u003c/sub\u003e/2π) of 356.3 Hz. The RF irradiation strength was calculated by the reciprocal of the 360\u0026deg; pulse width converted using presaturation attenuation of 40 dB in addition to the 90\u0026deg; pulse width of 13.84 \u0026micro;s and 90\u0026deg; pulse attenuation of 5.9 dB (Table SI1). For measurements of the H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Phe, Val, MA and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, the frequency offset was 4.666 ppm of the bulk H\u003csub\u003e2\u003c/sub\u003eO signal. The presaturation time was arrayed over a maximum range from 50 to 60,000 ms. The presaturation attenuation was 40 or 55 dB, which was equivalent to RF irradiation strengths of 370.2 or 65.8 Hz, respectively. The RF irradiation strengths were calculated by the reciprocals of 360\u0026deg; pulse widths converted using presaturation attenuation of 40 or 55 dB in addition to a 90\u0026deg; pulse width of 13.32 \u0026micro;s and 90\u0026deg; pulse attenuation of 5.9 dB (Table SI2).\u003c/p\u003e \u003cp\u003eThe H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Phe, Val, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e was also measured using a presaturation sequence. The frequency offset and presaturation time range were the same as for the NOESY-presat sequence. The presaturation attenuation was 40 dB.\u003c/p\u003e\n\u003ch3\u003eSpectral analyses\u003c/h3\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectra were referenced to the DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e signal, which was set to 0 ppm. The spectral phase was manually corrected. Integral ranges for analyte signals and the internal standard were set manually so that their \u003csup\u003e13\u003c/sup\u003eC satellite signals were included. An integral range was set for each signal, although the integral range of the phenyl signals of Phe included all aromatic CH signals because they overlapped with each other. The spectral baseline was manually corrected using a linear segment or cubic spline algorithm. The measured concentrations were calculated for each analyte signal using Eq.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC\u003c/em\u003e \u003csub\u003ea\u003c/sub\u003e = (\u003cem\u003eS\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e/\u003cem\u003eS\u003c/em\u003e\u003csub\u003eIS\u003c/sub\u003e)\u0026middot;(\u003cem\u003eH\u003c/em\u003e\u003csub\u003eIS\u003c/sub\u003e/\u003cem\u003eH\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e)\u0026middot;(\u003cem\u003eW\u003c/em\u003e\u003csub\u003eIS\u003c/sub\u003e/\u003cem\u003eW\u003c/em\u003e\u003csub\u003eSS\u003c/sub\u003e)\u0026middot;(\u003cem\u003eM\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e/\u003cem\u003eM\u003c/em\u003e\u003csub\u003eIS\u003c/sub\u003e)\u0026middot;\u003cem\u003eP\u003c/em\u003e\u003csub\u003eIS\u003c/sub\u003e\u0026middot;10\u003csup\u003e6\u003c/sup\u003e (1)\u003c/p\u003e \u003cp\u003eHere, \u003cem\u003eC\u003c/em\u003e is concentration (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cem\u003eS\u003c/em\u003e is \u003csup\u003e1\u003c/sup\u003eH signal area, \u003cem\u003eH\u003c/em\u003e is the number of \u003csup\u003e1\u003c/sup\u003eH nuclei on one molecule contributing to the \u003csup\u003e1\u003c/sup\u003eH signal area, \u003cem\u003eW\u003c/em\u003e is the weight (mg), \u003cem\u003eM\u003c/em\u003e is molar mass (g mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and \u003cem\u003eP\u003c/em\u003e is purity (kg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Subscripts a, IS, and SS refer to the analyte, internal standard, and sample solution, respectively.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReceiver gain and SNR profiles\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows a \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of a H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Gly, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e measured using a NOESY-presat sequence. The presaturation attenuation was 40 dB, which was equivalent to the presaturation strength of 356.3 Hz to maximize the SNR under the experimental conditions. When the presaturation time increased from 1 to 1,000 ms, the receiver gain also increased from 8 to 38 dB because the SNR of the bulk H\u003csub\u003e2\u003c/sub\u003eO signal decreased dramatically (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Consequently, the SNRs of the DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, Gly, and MA signals increased (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). For presaturation times from 1,000 to 60,000 ms, which was the same as the relaxation delay time, the receiver gain was the maximum constant value of 38 dB (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The bulk H\u003csub\u003e2\u003c/sub\u003eO signal must have become saturated after the presaturation time of 1,000 ms because the SNR of the signal did not change significantly.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of presaturation time on measurement accuracy\u003c/h3\u003e\n\u003cp\u003eThe following analysis focuses on high presaturation strength conditions that are not generally recommended for \u003csup\u003e1\u003c/sup\u003eH qNMR using NOESY-presat but may be encountered in practical measurements. The measured concentrations of Gly and MA did not respond significantly to the presaturation time from 1 to 60,000 ms (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The concentrations are expressed as the deviation from the prepared concentration in the sample on the vertical axes (The unconverted concentration data are shown in Table SI3). The concentrations with presaturation times of less than 1,000 ms showed wider scattering than those with presaturation times of more than 1,000 ms, probably because of low measurement repeatability caused by insufficient receiver gains. Average biases of \u0026minus;\u0026thinsp;22% and \u0026minus;\u0026thinsp;8% were observed for the measured concentrations of Gly and MA, respectively. The biases were consistent with previous work [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], in which the presaturation attenuation of 40 dB was necessary to maximize the SNRs of Gly and MA. Decreasing the concentration bias caused by the presaturation attenuation of 40 dB by optimizing the presaturation time would increase the concentrations accuracy, and the NOESY-presat sequence would be more effective for \u003csup\u003e1\u003c/sup\u003eH qNMR human metabolomics. However, the presaturation time did not decrease the concentration biases of Gly and MA.\u003c/p\u003e \u003cp\u003eTo verify the effect of the presaturation time using other samples, a H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Phe, Val, MA, and internal standard DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e was measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The prepared concentrations of the solution were higher than those of the H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Gly, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e to improve measurement repeatability. A common sample of MA was added to both solutions to compare the results. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the responses of the measured concentrations of Phe, Val, and MA to the presaturation time from 50 to 60,000 ms. The concentrations are expressed as the deviation from the prepared concentration in the sample on the vertical axes (The unconverted concentration data are shown in Table SI4). The graphs in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg are displayed in ascending order of absolute chemical shift difference between the analyte signal and presaturation offset. There was no change in MA concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), and the average concentration bias was \u0026minus;\u0026thinsp;10%, which was consistent with the bias measured using the solution containing Gly, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e. Similarly, there was no change in Phe concentrations obtained using NCH and CH\u003csub\u003e2\u003c/sub\u003e signals (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), and the average concentration biases were \u0026minus;\u0026thinsp;49% and \u0026minus;\u0026thinsp;15%, respectively. The Val concentrations obtained using NCH signal did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), and the average concentration bias was \u0026minus;\u0026thinsp;24%. The differences in concentration bias size must have been caused by the absolute chemical shift difference between the analyte signal and presaturation offset (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The closer the chemical shift of the analyte was to the presaturation frequency corresponding to the bulk H\u003csub\u003e2\u003c/sub\u003eO signal, the higher the concentration bias. These results agreed with previous studies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These results indicate that the high presaturation strength of 370.2 Hz, which exceeds commonly recommended conditions, is the dominant factor causing the concentration biases.\u003c/p\u003e\n\u003ch3\u003eNon-linear trends of measured concentrations\u003c/h3\u003e\n\u003cp\u003eThere were non-linear trends in concentration values obtained using the aromatic CH signals of Phe and the CH and CH\u003csub\u003e3\u003c/sub\u003e signals of Val (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). These signals were far from the presaturation frequency corresponding to the bulk H\u003csub\u003e2\u003c/sub\u003eO signal. Reducing the effect of the high presaturation strength may have revealed potential effects of the presaturation time. However, the non-linear trends in the concentrations probably involved off-resonance effects caused by using the NOESY-presat sequence because the frequency offsets were the same as the presaturation frequency corresponding to the bulk H\u003csub\u003e2\u003c/sub\u003eO signal.\u003c/p\u003e \u003cp\u003eTo reduce the off-resonance effects sufficiently, the H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution containing Phe, Val, MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e was measured again using a presaturation sequence. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows measured concentration trends for the aromatic CH signals of Phe and the CH and CH\u003csub\u003e3\u003c/sub\u003e signals of Val using the presaturation sequence. The presaturation strength and time were same as for the NOESY-presat sequence. Subtracting the data in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e from those in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e obtained using the NOESY-presat sequence produced residues that were independent of the presaturation time (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The average concentration biases measured using the aromatic CH signals of Phe and the CH and CH\u003csub\u003e3\u003c/sub\u003e signals of Val were +\u0026thinsp;3%, +\u0026thinsp;2%, and +\u0026thinsp;1%, respectively. The biases arose from the off-resonance effect caused by a mixing time of 0 ms and evolution time of 1 \u0026micro;s in the NOESY-presat sequence, according to a previous study [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e without the off-resonance effect, the concentrations obtained using the aromatic CH signals of Phe and the CH signal of Val increased with presaturation time and then became steady. The increase in the presaturation time contributed to decreasing the concentration biases. The concentration values obtained using the CH\u003csub\u003e3\u003c/sub\u003e signal of Val were generally steady and then decreased as the presaturation time increased, and decreasing the presaturation time helped to reduce the concentration biases. However, the mechanisms for these trends remain uncertain and warrant further investigation. The concentration biases measured using the aromatic CH signals of Phe and the CH and CH\u003csub\u003e3\u003c/sub\u003e signals of Val were \u0026minus;\u0026thinsp;1%, \u0026minus;\u0026thinsp;1%, and \u0026minus;\u0026thinsp;4%, respectively, at a presaturation time of 60,000 ms. The absolute chemical shift difference from the integral range center of the aromatic CH signals of Phe to the presaturation frequency corresponding to the bulk H\u003csub\u003e2\u003c/sub\u003eO signal was similar to that from the integral range center of the CH signal of Val to the presaturation offset. In contrast, that from the integral range center of the CH\u003csub\u003e3\u003c/sub\u003e signal of Val to the presaturation offset was larger than those for the aromatic CH signals of Phe and the CH signal of Val. Increasing the absolute chemical shift difference from an analyte signal to the presaturation offset did not decrease the measured concentration bias. In the previous work, larger the absolute chemical shift difference from an analyte signal to the presaturation offset was, the lower the measured concentration bias [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, D\u003csub\u003e2\u003c/sub\u003eO solutions containing Phe or Val were measured in the previous work. Because H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solution was used in the present work, the non-linear trends shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e may have been caused by the presaturation affecting the abundant H\u003csub\u003e2\u003c/sub\u003eO signal, for example, via the intermolecular \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH nuclear Overhauser effect [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] accompanied by the three spin effect [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings of this work highlight that high presaturation strengths introduce significant and systematic concentration biases that cannot be mitigated by adjusting presaturation time alone within the examined range, although they can improve baseline distortion and SNR in practical cases. Therefore, such conditions should be regarded as inappropriate for quantitative analyses. When high presaturation strengths are unavoidable in practice, the magnitude and direction of the bias should be carefully evaluated for each analyte signal. These observations are consistent with the established understanding that low presaturation strengths are preferable for maintaining quantitative accuracy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe effect of presaturation time in a NOESY-presat sequence was evaluated under high presaturation strengths (up to approximately 400 Hz), which may be used in practice despite not being generally recommended for \u003csup\u003e1\u003c/sup\u003eH qNMR. Large concentration biases of \u0026minus;\u0026thinsp;49% to \u0026minus;\u0026thinsp;8% observed for analyte signals located near the presaturation frequency corresponding to the bulk H\u003csub\u003e2\u003c/sub\u003eO signal were not mitigated by adjusting presaturation time, indicating that presaturation time is not an effective parameter for reducing such biases. Therefore, presaturation strength should be carefully controlled to ensure the accuracy of \u003csup\u003e1\u003c/sup\u003eH qNMR using NOESY-presat. When high presaturation strengths are unavoidable, the resulting biases should be recognized as systematic and evaluated for each analyte signal, consistent with the established understanding for low presaturation strengths.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe NOESY-presat sequence consists of a presaturation interval, followed by a first 90\u0026deg; pulse. After a short switching period, referred to as the evolution time (t\u003csub\u003e1\u003c/sub\u003e) for phase cycling, a second 90\u0026deg; pulse is applied. This is followed by a mixing time (t\u003csub\u003em\u003c/sub\u003e) during presaturation to enhance the nuclear Overhauser effect, and finally a third 90\u0026deg; pulse. The interval time of the presaturation was altered, whereas presaturation during the mixing time was not applied because t\u003csub\u003em\u003c/sub\u003e was set to 0 s for improving quantitative accuracy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe prepared concentrations were 275.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Gly, 268.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MA, and 892.9 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e. Total uncertainty budgets of the Gly and MA concentrations are shown in Fig. SI1. The chemical shift reference was the DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e signal at 0 ppm. The faraway H\u003csub\u003e2\u003c/sub\u003eO is the water component far from the RF coil center in the NMR probe [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFull responses of a) receiver gain (open diamonds) and SNRs of bulk H\u003csub\u003e2\u003c/sub\u003eO (blue diamonds), c) DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e (open squares), Gly (red triangles), and MA (red circles), and e) concentrations of Gly (red triangles) and MA (red circles) to presaturation times. Expanded responses to presaturation times of 1,000 to 5,000 ms in figures a), c), and e) are shown in figures b), d), and f), respectively. The colors of the vertical axes correspond to those of the legend icons. The SNRs of bulk H\u003csub\u003e2\u003c/sub\u003eO are absolute values.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe prepared concentrations were 970.8 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Phe, 975.6 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Val, 1031.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MA, and 965.7 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e. Total uncertainty budgets of the Phe, Val and MA concentrations are shown in Fig. SI1. The chemical shift reference was the DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e signal at 0 ppm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResponses to presaturation times of 50 to 60,000 ms of concentrations of a) Phe obtained using the NCH signal, b) Val obtained using the NCH signal, c) Phe obtained using the CH\u003csub\u003e2\u003c/sub\u003e signal, d) MA, e) Val obtained using the CH signal, f) Phe obtained using the CH signal, and g) Val obtained using the CH\u003csub\u003e3\u003c/sub\u003e signal. Bars are the standard deviation obtained by three repeated measurements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe data are same as those shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The absolute chemical shift differences from the presaturation offset are 0.672 (Phe NCH), 1.047 (Val NCH), 1.547 (Phe CH\u003csub\u003e2\u003c/sub\u003e), 1.576 (MA), 2.473 (Val CH), 2.606 (Phe CH), and 3.751 ppm (Val CH\u003csub\u003e3\u003c/sub\u003e) in ascending order. The differences were obtained as the differences from the presaturation offset of 4.666 ppm to the integral range centers of the analyte signals on the \u003csup\u003e1\u003c/sup\u003eH NMR spectra before chemical shift corrections. The dotted curve line is a linear trendline converted from an exponential trendline.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResponses of concentrations of a) Val obtained using the CH signal, b) Phe obtained using the CH signal, and c) Val obtained using the CH\u003csub\u003e3\u003c/sub\u003e signal to presaturation times of 50 to 60,000 ms. Bars are the standard deviation obtained by three repeated measurements. The full version including all analyte signal data is shown in Fig. SI2. The unconverted concentration data are shown in Table SI5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResponses to presaturation times of 50 to 60,000 ms of concentrations of a) Val obtained using the CH signal, b) Phe obtained using the CH signal, and c) Val obtained using the CH\u003csub\u003e3\u003c/sub\u003e signal. The full version including all analyte signal data is shown in Fig. SI3. The unconverted concentration data are shown in Table SI6.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no conflicts of interest.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNaoki Saito: writing \u0026ndash; review and editing, writing \u0026ndash; original draft, visualization, validation, methodology, investigation, formal analysis, data curation and conceptualization.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData can be made available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present work was supported by JSPS KAKENHI Grant Number JP24K17709. The used NMR spectrometer was one of the Fundamental Instruments for Measurement and Analysis (FIMA), National Institute for Environmental Studies. I thank Dr. Hiroaki Sasakawa in JEOL Ltd. for useful discussion. The work was presented in part at the 64\u003csup\u003eth\u003c/sup\u003e Annual Meeting of the NMR society of Japan, Okinawa, Japan, in November 2025.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNishizaki Y, Sugimoto N, Miura T, Asakura K, Suematsu T, Korhonen SP, Lehtivarjo J, Niemitz M, Pauli GF (2024) Quantum mechanical quantitative nuclear magnetic resonance enables digital reference standards at all magnetic fields and enhances qNMR sustainability. 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J Magn Reson 73:45\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0022-2364(87)90224-1\u003c/span\u003e\u003cspan address=\"10.1016/0022-2364(87)90224-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"accreditation-and-quality-assurance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acqa","sideBox":"Learn more about [Accreditation and Quality Assurance](http://link.springer.com/journal/769)","snPcode":"769","submissionUrl":"https://submission.nature.com/new-submission/769/3","title":"Accreditation and Quality Assurance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"accuracy, 1D NOESY, presaturation time, qNMR, water suppression","lastPublishedDoi":"10.21203/rs.3.rs-9252005/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9252005/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA one-dimensional NOESY sequence with presaturation (NOESY-presat) is widely used for water suppression in \u003csup\u003e1\u003c/sup\u003eH NMR-based metabolomics. While presaturation strengths below approximately 100 Hz are generally recommended to maintain accuracy of quantitative \u003csup\u003e1\u003c/sup\u003eH NMR (\u003csup\u003e1\u003c/sup\u003eH qNMR), higher presaturation strengths may be employed in practice to suppress large H\u003csub\u003e2\u003c/sub\u003eO signals and improve signal-to-noise ratios of \u003csup\u003e1\u003c/sup\u003eH NMR spectra. However, the accuracy of \u003csup\u003e1\u003c/sup\u003eH qNMR using NOESY-presat under such non-ideal conditions has not been systematically evaluated, particularly with respect to presaturation time. In this study, the effect of presaturation time on analyte concentration biases was examined under high presaturation strength of 356 to 370 Hz. Two H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (90/10 vol %) solutions containing glycine (Gly), maleic acid (MA), and 3-(trimethylsilyl)-1-propanesulfonic acid-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e (DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e), or L-phenylalanine (Phe), L-valine (Val), MA, and DSS-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e were measured by \u003csup\u003e1\u003c/sup\u003eH qNMR using NOESY-presat. The results showed that large concentration biases of \u0026minus;\u0026thinsp;49% to \u0026minus;\u0026thinsp;8% for analyte signals located near the presaturation frequency corresponding to the bulk H\u003csub\u003e2\u003c/sub\u003eO signal were not reduced by altering the presaturation time over a wide range of 50 to 60,000 ms. These findings indicate that presaturation time is not an effective parameter for reducing concentration biases under high presaturation strengths. Therefore, presaturation strength is the dominant factor affecting accuracy of \u003csup\u003e1\u003c/sup\u003eH qNMR using NOESY-presat experiments. When high presaturation strengths are unavoidable, the resulting biases should be recognized as systematic and evaluated for each analyte signal, consistent with the established understanding for low presaturation strengths in \u003csup\u003e1\u003c/sup\u003eH qNMR.\u003c/p\u003e","manuscriptTitle":"Limited impact of presaturation time on the accuracy of 1H qNMR using a NOESY with high-power presaturation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-06 15:40:52","doi":"10.21203/rs.3.rs-9252005/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-15T07:21:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T03:48:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T08:39:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T22:29:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62339948091433830139936717047719276409","date":"2026-04-03T15:14:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T14:01:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165988723671975290738146620508052367634","date":"2026-04-03T10:15:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85591672175474143190501117700838978110","date":"2026-04-02T12:45:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15188558669417672716551232176206214334","date":"2026-04-02T12:23:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176605605620345953212351673364668479724","date":"2026-04-02T11:18:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137190582523811558665512946985172924283","date":"2026-04-02T10:54:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228739503181934518546204258314134447639","date":"2026-04-02T01:18:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263335681245899317593143553919490511930","date":"2026-04-01T19:13:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-01T15:09:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T14:04:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T11:59:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Accreditation and Quality Assurance","date":"2026-03-28T10:31:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"accreditation-and-quality-assurance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acqa","sideBox":"Learn more about [Accreditation and Quality Assurance](http://link.springer.com/journal/769)","snPcode":"769","submissionUrl":"https://submission.nature.com/new-submission/769/3","title":"Accreditation and Quality Assurance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"de6b6980-68e0-487a-8d75-f575749ff38e","owner":[],"postedDate":"April 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-15T07:26:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-06 15:40:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9252005","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9252005","identity":"rs-9252005","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}