Prostate diffusion‐weighted MRI scans can suffer from geometric distortions, signal pileup, and signal dropout attributed to differences in tissue susceptibility values at the interface between the prostate and rectal air. The aim of this work is to present and validate a novel model based reconstruction method that can correct for these distortions.0 field map and a set of EPI data acquired with blip‐up and blip‐down phase encoding gradients, we model the distortion correction problem linking the distortion‐free image to the acquired raw corrupted k‐space data and solve it in a manner analogous to the sensitivity encoding method. Both a quantitative and qualitative assessment of the proposed method is performed in vivo in 10 patients.In regions of severe signal pileup, standard techniques for distortion correction have difficulty recovering the underlying true signal. Furthermore, because of drifts and inaccuracies in the determination of center frequency, echo planar imaging (EPI) scans can be shifted in the phase‐encoding direction. In this work, using a B2, respectively. Compared to the Topup (distortion correction method commonly used for neuro imaging), the proposed method achieved Dice scores (0.87 and 0.85 versus 0.82 and 0.80) and better qualitative results in patients where signal pileup was present because of high rectal gas residue.Without distortion correction, mean Dice similarity scores between a reference T2W and the uncorrected EPI images were 0.64 and 0.60 for b‐values of 0 and 500 s/mmModel‐based reconstruction can be used for distortion correction in prostate diffusion MRI. This results in signal stretching in areas within the image where the gradient of B0 field has the same polarity as the phase‐encoding gradient. Conversely, signal compression or pileup occurs in regions where the B0 field gradient direction is opposite to that of the phase‐encoding gradient. This results in multiple pixels along the phase‐encoding direction being merged into a single pixel. The main source of B0 field inhomogeneities is the susceptibility differences that arise at the interface between tissues of different susceptibilities. Larger local susceptibility differences will create a stronger off‐resonance field that will result in more‐severe signal pileup or stretching within the image. For DWI prostate images, susceptibility differences result in severe distortions and poor depiction of zonal anatomy.In DWI, single‐shot echo planar imaging (SS‐EPI) has been the preferred k‐space read out technique because of its fast speed and robustness against motion artefacts. However, SS‐EPI has a disadvantage of low bandwidth along the phase‐encoding direction, making it sensitive to inhomogeneities in the magnetic field. In MRI, spatial encoding is achieved by using magnetic field gradients, and the measured signal represents the Fourier transform of the object. In the presence of magnetic field inhomogeneities, an additional off‐resonance field ,0 field inhomogeneities may be corrected directly during the reconstruction process using this full inverse problem solution, although a previous estimation of the B0 field is needed. The complex averaging used in the reconstruction avoids the bias from noise that can occur when magnitude images are combined.Several methods have been proposed to address the BIn this work, we propose a model‐based reconstruction framework to solve the full inverse problem of prostate DWI distortion correction. By using the EPI raw k‐space data from acquisitions with blip‐up and blip‐down phase‐encoding gradients, we model the distortion correction problem linking the corrupted k‐space data to the corrected image and solve it in a manner analogous to sensitivity encoding (SENSE),22.10 scan is acquired as a separate dual‐echo gradient echo scan for estimation of the B0 field.The proposed framework acquires 2 EPI data sets (blip‐up and blip‐down) with opposite phase‐encoding gradient directions. A B2.20 field inhomogeneities, the corrupted k‐space Yj corresponding to jth coil  can be related to the undistorted image x by the following model (Equation With BEquation :(1)Yjk,lm, n are image coordinate indices, M, N are image dimensions, k, l are k‐space coordinate indices, t is the sample time for location  in k‐space, ΔB0 is the B0 field in Hz that is estimated from a separate dual echo gradient echo scan, Cj is the jth coil sensitivity, and j = 1, 2,..,Jwhere f0), the model in Equation 1 is modified as Equation In the presence of a center frequency offset .In the above expression, the center frequency offset ∆2.3The proposed reconstruction framework has the following 3 steps.2.3.1Y1j and Y2j be the k‐space for blip‐up and blip‐down EPI scans for jth coil, respectively. Mathematically, we can write  from multicoil data are calculated as a multicoil combination, where the jth coil contribution is obtained by performing adjoint of encoding operators E1j and E2j onto the corresponding k‐space Y1j and Y2j, respectively.where xcp1 and xcp2 are expressed as Equation Mathematically, (·)H is the Hermitian operator.wherefopt0, is estimated as a parameter that maximizes the mutual information (MI) similarity measurexcp1 and xcp2) acquired with a b‐value of 0 s/mm2 by solving the following unconstrained problem Δf0,o0 field is corrected by updating it with estimated optimal center frequency offset ∆f0,opt. The definition of Mutual Information function used in our method is described in Appendix The B2.3.22), the phase of the blip‐up and blip‐down data in the image space might be different because of small physiological motion that may occur during the diffusion sensitization gradients. If data are combined from the 2 phase‐encoding directions without phase correction, this may result in phase cancellation leading to signal dropout in the final reconstructed image. To cater for this issue, we propose to calculate a phase correction ∆Ф obtained by taking the Hermitian inner product between the preliminary model‐based conjugate phase reconstructions (xcp1 and xcp2) of blip‐up and blip‐down EPI data using corrected B0 field Δϕ* denotes the complex conjugate, andY2j selected as a reference, phase correction was applied to Y1j by (1) first applying the inverse Fourier transform (IFT) to it, (2) multiplying by an exponential term containing the phase correction ΔФ to get the corrected image y~1j, and (3) transforming back to k‐space by the Fourier transform (FT) to get a final phase‐corrected k‐space Y~1j. Mathematically  in the current iteration becomes smaller than ϵ  giving the final distortion corrected output image x˙.The above minimization problem is strictly convex because the encoding operator Ei and EiH, respectively; i = 1 and i = 2 refer to blip‐up and blip‐down scans, respectively. The forward encoding operator Ei involves multiplication of input image x by individual coil sensitivities Cj that is followed by a modified Fourier transform operator G that maps the product of image and coil sensitivity from image to Fourier space, taking into account the susceptibility effects determined by B0 field and phase‐encoding direction dependent k‐space sampling times ti. The adjoint encoding operator EiH involves transforming each individual coil k‐space data to image space by adjoint of modified Fourier transform operator (HG) followed by multiplication by the corresponding complex conjugated coil sensitivity (Cj)* and the final summation over all the coils.Figure 2.4Ten male patients  were recruited from the clinical prostate imaging pathway and were consented for additional image acquisitions. No antispasmodic agent was administered. Patients were placed feet first into the scanner and imaging was carried out during free breathing for all patients. The study was approved by the ethics committee, and written signed consent was obtained from all patients for the research scans.3, field of view (FOV) = 180 to 220 × 180 to 220 × 55 to 90 mm3, partial Fourier acquisition with half scan factor of 0.75, TE/TR = 80 msec/2500 msec, phase‐encoding direction = anterior‐posterior (AP) axis with fat shift in the direction “P” for blip‐up and direction “A” for blip‐down scans, b‐values = 0 and 500 s/mm2, number of isotropic diffusion directions = 3, number of averages = 3, phase‐encode bandwidth per pixel = 10.4 to 11.2 Hz/pixel, and scan time = 30 sec. For calculation of the B0 field, a separate 3D dual‐echo gradient echo scan was acquired with the following parameters: resolution = 2 × 2 × 2 mm3, FOV = 200 to 250 × 200 to 250 × 70 to 120 mm3, flip angle = 6°, right to left phase‐encoding direction, SENSE acceleration factor = 1, TE difference (ΔTE) = 2.3 msec, TE1/TE2/TR = 4.6/6.9/8.7 msec, and scan time = 1 minute. For reference, axial T2W images were acquired using a turbo spin echo scan with the following parameters: resolution = 2 × 2 × 4 mm3, FOV = 180 to 220 × 180 to 220 × 55 to 90 mm3, SENSE acceleration factor = 2, TE/TR = 100/4700 msec, and scan time = 40 sec. Volume shimming was performed to cover the whole prostate and surrounding areas.Scanning was performed on a 3T scanner  equipped with 16 anterior + 16 posterior channel receive coil array. Single‐shot EPI data in both blip‐up and blip‐down phase‐encoding directions were acquired. The EPI scans had the following parameters: resolution = 2 × 2 × 4 mm2.5To save the raw data together with the relevant information needed for the reconstruction framework, a software patch was implemented using ReconFrame software . EPI phase correction was performed using the ReconFrame tool to correct for ghosts originating from alternating offsets of phase encode lines in k‐space. Subsequent postprocessing was implemented in MATLAB .0 field was calculated using the quantitative susceptibility mapping toolbox0 field in image domain to smooth out noisy components. The 3D B0 field and T2W images were resampled to match the EPI scan resolution and the associated FOV. The iterative optimization in Equation 6 to estimate the optimal center frequency offset Δf0,opt was carried out using the fminsearch function in MATLAB that uses the Nelder‐Mead simplex algorithm.0,opt was set to the value of Δf0 found in the last iteration.The B∊ for convergence of CG iterations in Equation 10 was set to 0.0025 in all our experiments. The model‐based corrected reconstruction was compared against uncorrected blip‐up and blip‐down reconstructions and the neuroimaging distortion correction Topup method.This was followed by phase correction (Equations 7 and 8) and model‐based reconstruction in Equation 10. The threshold 2.6For quantitative evaluation, reconstructed images were converted to DICOM format and exported to Horos software,2.72) can change dynamically because of small physiological motion occurring during the diffusion sensitization gradients. To evaluate the performance of the phase correction procedure used in our proposed framework, a mid‐prostate single slice was acquired for b‐value of 500 s/mm2 on 2 patients for 40 dynamics with blip‐up phase‐encoding gradient. Model‐based reconstruction was done with and without phase correction for each pair of dynamics  in each diffusion direction with dynamic 1 selected as a reference. The goal is to check whether the proposed phase correction can work in cases where the dynamics have significantly different phases from one another in the conjugate phase reconstructions.The phase of DWI data  for a mid‐prostate slice are shown in Figure 3.20 field, uncorrected blip‐up and blip‐down reconstructions, correction using blip‐up data only and blip‐down data only, and proposed model‐based reconstructions are shown for b‐values of 0 and 500 s/mm2, respectively. Because of susceptibility differences at the interface between different tissues in the prostate region, the variation in B0 was around 120 Hz that corresponds to a shift of 7 to 8 pixels at a bandwidth/pixel of ~15.80 Hz in phase‐encoding direction. This resulted in pileup in regions where the B0 field gradient magnitude was high and its direction was opposite to that of phase‐encoding gradient  for uncorrected blip‐up reconstructions and 0.68  and 0.62  for uncorrected blip‐down reconstructions. The proposed method achieved mean Dice similarity scores of 0.87 and 0.85 for b‐value of 0 and 500 s/mm2, respectively, and scored higher than the corresponding Dice similarity scores of the Topup method . Qualitative scores  for all 4 methods corresponding to b‐value of 0 s/mm2 and b‐value of 500 s/mm2 are given in Tables Box plots showing the Dice scores for different reconstruction methods are shown in Figure 3.32). Here, results are shown for a pair of dynamics  that had significant different phases in conjugate phase reconstructions. Without phase correction, signal cancellation occurred in the reconstructed image in each diffusion direction. By using the proposed phase correction (Equation 8), the signal was preserved in each diffusion direction. The magnitude average of the reconstructions in three diffusion directions (Mag Avg) is shown in the right column that had close correspondence to the reference T2W image.Figure 40 field may also be used as a method for overcoming uncertainties in coordinates that may happen in the process of reconstruction from raw data.A novel model‐based reconstruction framework is proposed that can correct for geometric distortions, signal pileup, and signal dropout in diffusion‐weighted prostate images. By using the power of complimentary encoding information within data from both blip‐up and blip‐down directions, the model‐based framework was able to correct most of the pileup in regions of severe distortions and performed better than the Topup method (commonly used for neuroimaging) or model‐based reconstructions using data from 1 phase‐encoding gradient direction only. The proposed mutual information maximization used for correction of center frequency offset in the B0 field to be static except for center frequency offset attributed to the frequency drift between B0 and EPI scans. In case of motion or changes in the rectal area adjacent to the prostate region between the EPI and B0 scans, the B0 field estimated from B0 scan may be mismatched, resulting in inaccurate distortion correction. Some of the remaining distortions in the proposed method reconstructions can be attributed to dynamic changes in B0 field because no antispasmodic agent drug was administered in our scans that would suppress bowel movements and/or rectal gas. Changes in B0 might be addressed by a joint estimation of both B0 and the corrected EPI images, starting with the initial B0 fields estimated from B0 scan.The proposed method assumes the B2), the low SNR in the reconstructed images might affect the inverse problem and the technique may benefit from preconditioning or additional regularization.For higher b‐values , the measurement of B0 field in that area might not be always possible, leading to noisy and unwrapped phases in the B0 field. To address this issue, B0 field might be modeled in this region using a susceptibility map distribution0 field inside the rectal‐air region. The PDF method calculates the B0 field inside an ROI by projection of known dipole fields from outside the ROI.Our proposed method uses a B0 distortion and motion corruption effects.Last, the proposed method does not include any physiological motion effects that may occur between the EPI blip‐up and blip‐down scans. In case of motion between blip‐up and blip‐down scans, both motion parameters and the image would need to be estimated simultaneously and the optimization in Equation 10 becomes nonconvex. The optimization may be simplified by estimating motion in a preceding step similar to the framework in a previous workProstate diffusion MRI is recognized as a potential biomarker for tumor detection, but, currently, it is unusable in some patients because of significant distortions. We proposed a novel model‐based reconstruction framework that can correct these distortions by using data from opposite phase‐encoding gradient directions. The proposed method was applied successfully in 10 clinical patients, despite no antispasmodic drug being administered. The proposed technique may offer potential to radiologists and clinicians by increasing the diagnostic value of prostate images for tumor detection, thus making prostate MRI a more reliable and reproducible biomarker in future.FIGURE S1 In vivo reconstruction results for patients 3 (P3) to patient 10 (P10) for data acquired at b‐value of 0. For EPI scans, the AP axis was selected as the phase‐encoding direction with fat shift in the direction “P” for blip‐up and “A” for blip‐down scans, respectively. Whole prostate (red) was delineated on reference T2W image (left column) and overlaid on uncorrected blip‐up (UC P), uncorrected blip‐down (UC A), Topup, and proposed distortion‐corrected (C AP) reconstructionsFIGURE S2 In vivo reconstruction results for patients 3 (P3) to patient 10 (P10) for data acquired at b‐value of 500 s/mm2. For EPI scans, the AP axis was selected as the phase‐encoding direction with fat shift in the direction “P” for blip‐up and “A” for blip‐down scans, respectively. Whole prostate (red) was delineated on reference T2W image (left column) and overlaid on uncorrected blip‐up (UC P), uncorrected blip‐down (UC A), Topup, and proposed distortion‐corrected (C AP) reconstructionsFIGURE S3 Plot of normalized residual error r=EHEx-EHY~2EHY~2 as a function of the CG iteration numberFIGURE S4 Qualitative assessment: mean percentage of improvement in qualitative scores  using model‐based reconstruction compared to uncorrected blip‐up (UC P), uncorrected blip‐down (UC‐A), and Topup methods. The results are shown for b‐values of 0 and 500 s/mm2. The improvements in all the qualitative scores for the proposed method were positive compared to the other reconstructionsClick here for additional data file.


This work showcases the 1,3‐haloboration reaction of alkynes in which boron and chlorine add to propargyl systems in a proposed sequential oxazoliumborate formation with subsequent ring‐opening and chloride migration. In addition, the functionalization of these propargyl esters with dimethyl groups in the propargylic position leads to stark differences in reactivity whereby a formal 1,1‐carboboration prevails to give the 2,2‐dichloro‐3,4‐dihydrodioxaborinine products as an intramolecular chelate. Density functional theory calculations are used to rationalize the distinct carboboration and haloboration pathways. Significantly, this method represents a metal‐free route to highly functionalized compounds in a single step to give structurally complex products. The activation of carbon–carbon double and triple bonds by main group compounds has been a staple motif in the synthesis of a plethora of new element–carbon bonds such as C−C,3 , or borocations developed by Ingleson et al. In the case of borocations, stereoselective control is observed to give predominantly the syn‐addition product.2][AlCl4] which, upon addition of various 1‐trimethylsilylalkynes, undergoes a subsequent 1,2‐carboboration to yield the diene product.More recent work in such elementoboration reactions are seen through the synthetically useful haloboration reaction whereby a halogen, predominantly chlorine, is installed typically through a 1,1‐ or 1,2‐haloboration to yield the corresponding halovinylboronic ester (Scheme 6F5)3, as well as others of a similar nature,Although a significant amount of research has focused on the sterically encumbered, strong Lewis acid, B revealed the proposed structure of 3 a, which interestingly is the product of a formal 1,3‐haloboration reaction. These encouraging initial results then led us to expand the substrate scope to a series of phenyl substituted propargyl esters , 18 h , and 48 h (3 d).Initial investigations of the commercially available PhBCl2Cl2/hexane solution of 3 c at −40 °C produced a crop of crystals suitable for X‐ray diffraction. The structure was unambiguously determined to be indeed the product of a formal 1,3‐haloboration agreeing with spectroscopic analyses  appeared to make no difference in reactivity with all showing almost quantitative conversion within 9 hours. Additionally, trialling other boron reagents such as BCl3 as well as the borocation [PhClB(2‐DMAP)][AlCl4]Conducting the reaction between PhBCl1H and 2H NMR spectroscopy whilst comparing the in situ data of the protic versus deuterated compounds shed light on the fate of the terminal alkynyl hydrogen atom and hence the reaction mechanism  in accordance with the literature.2 a–2 c . Additionally, when using compound 5, which features a combination of H and Me in the propargyl position, a more complex transformation is observed when monitoring the reaction coordinate over time. Analyzing the in situ 1H and 11B NMR spectra suggests that, after initial combination of PhBCl2 with 5 in a 1:1.2 ratio, the haloboration product 6 prevails, as observed by the characteristic broad singlet at δ=6.35 ppm alongside the formation of a resonance at ca. δ=5.2 ppm for the proposed vinyl and methylene protons, respectively (see the Supporting Information). Over time, these resonances reduce in intensity giving way to a new broad singlet at δ=3.36 ppm, consistent with the generation of the proton in the adjacent to the borane in the chelating structure 7. In addition, new resonances appear for the newly formed vinyl proton quartet at δ=5.45 ppm, and the methyl doublet at δ=1.90 ppm. This assertion is bolstered when observing the in situ 11B NMR spectra over time whereby the expected singlet at about δ=55.1 ppm forms after 1 h at ambient temperature, which reduces in intensity over time yielding another singlet resonance at ca. δ=9.1 ppm, again indicating the reversible formation of 6 en route to 7  is strongly thermodynamically preferred over haloboration (R2=H). Intriguingly, however, we found that the hypothetical carboboration product  for R2=H is >30 kcal mol−1 more stable than the R2=H haloboration product obtained experimentally, raising the question: why does the carboboration reaction not occur for R2=H?To shed light on the divergent reactivity realized in this work, DFT calculations were performed. The reaction pathways for haloboration (upper) and carboboration (lower) are displayed in Figure trans‐oxyboration and formation of the intermediate dioxolium intermediate I, two pathways are available: 1) chloride migration that results in a metastable haloboration product or 2) phenyl migration that yields the thermodynamically favored carboboration product. The carboboration reaction of 1 a is disfavored for two reasons: first, following the kinetically favored pathway, chloride migration occurs yielding the haloboration product and reversion back to the dioxolium I is strongly hindered by a high reverse barrier  from the product. Second, in order to yield the carboboration product from I, a prohibitively large barrier for phenyl migration  must be overcome . For comparison, the phenyl migration barrier for compounds 2 a (R2=Me) is 9 kcal mol−1 lower than for 1 a (R2=H). Hence, for 1 a (R2=H), the kinetically favored haloboration product is formed. Conversely, the haloboration product of 2 a (R2=Me) has a comparatively low reverse barrier from the product  and also a low barrier for phenyl migration  in comparison to that for 1 a (R2=H). Hence, any haloboration product formed from starting materials 2 a may revert to I fleetingly and phenyl migration will occur. Once phenyl migration occurs, there is a strong free energy incentive to form the thermodynamically preferred chelate 4, which has a very prohibitive >+70 kcal mol−1 barrier (ΔGsol) to revert to I.After 1,2‐5 to form haloboration product 6 and then the carboboration product 7 , explaining why both 3 and 6 are observed. The relatively low barriers for phenyl migration to form 4 and 7 with the strong thermodynamic driving force explain why the carboboration products occur for R2=Me and R2=Me, H. Overall, these calculations reveal a remarkably subtle interplay of kinetic and thermodynamic factors that are acutely sensitive to the R2 groups and which cause profoundly different reaction products.Intriguingly, in line with the observed reversible behavior of the mono‐methylate In summary, this work has shown both the formal 1,1‐carboboration as well as formal 1,3‐haloboration of alkynes can occur through simple tuning of the alkyne starting material being used. All of these multi‐step reactions proceed cleanly with high conversions and yields being noted in a one‐pot, atom efficient manner, garnering synthetically useful and functionally diverse compounds for further reactivity. In depth computational studies have helped elucidate the proposed mechanism that differentiates this divergent elementoboration.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The method includes the chemical separation of Zr (isobaric interference of 90Zr) from the samples followed by determination of 90Sr/88Sr abundance  sensitivity (2.1 × 10−10). The analytical performance of this method was assessed in the IAEA-TEL 2017-3 worldwide open proficiency test. For 90Sr determination, tap water and milk powder samples were distributed amongst the participant laboratories with reference values of 11.2 ± 0.3 Bq kg−1 (2.2 ± 0.1 fg g−1) and 99.9 ± 5.0 Bq kg−1 (19.5 ± 1.0 fg g−1), respectively. The stable Sr concentrations were 39.4 ± 0.9 ng g−1 and 2.5 ± 0.1 µg g−1 while the 90Sr/88Sr isotope ratios were 6.47 ± 0.17 × 10−8 and 9.04 ± 0.45 × 10−9 in the tap water and milk powder samples, respectively. For TIMS measurement, 50 mL water and 1 g milk powder samples were taken for analysis. This TIMS method demonstrated an impressive accuracy  and precision  when compared with radiometric techniques. For the first time in the history of inorganic mass-spectrometry, 90Sr analysis using a TIMS instrument is confirmed by an independent proficiency test.A novel method for the determination of ultra-trace level  The 90Sr (T1/2 = 28.8 y) is a pure beta emitter, produced by the fission of U and Pu isotopes. Low level (some Bq kg−1 or mBq kg−1) 90Sr contamination exists in different environmental matrices as a result of nuclear weapon tests and nuclear accidents. Basically, Sr element has no biological role in the human body, however it has the potential to incorporate into the bone structure because it is bio-chemically similar to calcium, thus the 90Sr isotope can cause long-term radiation dose. From the view point of public health and natural radiation protection, precise and accurate determination of 90Sr in various environmental, biological and radioactive waste samples is essential1.The 90Sr analysis, where beta particles are emitted by the radioactive decay of 90Sr which are detected using different types of detectors, such as, gas ionization detector, solid or liquid scintillators, etc. In recent years, owing to significant developments on sample introduction, detector and interference removal techniques, many successful 90Sr determinations have been reported using mass spectrometry instruments. For this purpose, inductively coupled plasma mass spectrometry (ICP-MS) instruments with collision/reaction cells or triple quadrupole system have been primarily applied5. Alternatively, thermal ionisation mass spectrometry (TIMS) is a robust technique for 90Sr analysis6. The main advantages of the mass spectrometry method over the radiometric are the shorter analysis time, higher sample throughput and smaller sample intake.In general, radiometric methods have been mainly used for 90Sr detection in environmental samples with mass spectrometry instrument is a challenging task. The first critical point is the isobaric interference of 90Zr as a consequence of significant amounts of Zr element in environmental samples. For example, in case of Japanese soil samples, the mean concentration of Zr element is around 90 µg g−1 (varying in the range from 0.6 to 430 µg g−1)7, which corresponds to ~46 µg g−1 of 90Zr since its isotopic abundance is 51.5%8. The typical 90Sr level in environmental samples is quite low, in the range of fg g−1, for contaminated Fukushima soil samples it is around 4 fg g−1 (20 Bq kg−1) or lower9. In order to successfully measure 90Sr using inorganic mass spectrometry either a high mass resolution  which is not yet available to resolve the isobaric 90Zr peak or an efficient chemical purification method for removal of Zr from Sr at ultra-trace levels is required11. For practical purposes, quite a high decontamination factor (in the range of 108 to 1011) is required for 90Zr isobaric interference removal  in case of soil samples12.The low level 90Sr determination is the peak tailing on the higher mass side from 88Sr. Although there exists reliable ultra-high vacuum condition (<10−8 Pa) in the source and analyser zone of the mass spectrometry instruments, scattering of ions occurs during their acceleration. This scattering process causes the energy spread of the ion beam and it is recognized as a peak tail in the mass spectra. In the case of 90Sr ion beam detection, undesired interference exists because of the peak tailing of the major abundant 88Sr isotope. To depict this interference the abundance sensitivity term is used and defined as:The second critical point for the application of mass spectrometry instruments for 90Sr analysis in environmental samples, abundance sensitivity in the range of 10−7 to 10−11 is required.For reliable 90Sr analysis, those methods are neither well established nor routinely used. Accreditation to an internal standard applying mass spectrometry method for 90Sr determination has not been well documented. The main reason for the less development is due to interdisciplinary challenges of this method. Traditionally, the determination of 90Sr is carried out by radio-chemists with a strong chemistry and nuclear measurement background while the application of mass spectrometry instrument is the domain of mass spectrometry experts with strong physics and/or engineering background.Although, there is a new trend to utilize mass spectrometry instruments for 90Sr determination after the Fukushima nuclear accident, a 90Sr analysis method was developed using the Phoenix X62 TIMS in the laboratory of the National Institutes for Quantum and Radiological Science and Technology (QST), Japan. During the validation process of this new method, detection limit, abundance sensitivity (90Sr/88Sr), accuracy and precision were determined with reference materials e.g. IRMM-426 (wild berry) and NIST-4354 (freshwater lake sediment) assay proving that the method is suitable for environmental level 90Sr analysis6.Considering the need of rapid and precise 90Sr analysis, which is the exclusive territory of radiometric methods.To establish a higher level of quality assurance, it is essential to participate in a proficiency test controlled by an internationally recognised reference laboratory. To fulfil this standard, our laboratory participated in the IAEA-TEL-2017 world-wide open proficiency test organized by the International Atomic Energy Agency. For the first time, an inorganic mass spectrometry method was employed in a proficiency test for 13. For this purpose, five different samples spiked with known amounts of target radionuclides were distributed. Three tap water samples  each around 500 g, one milk powder (Sample 4) around 180 g and one Ca-carbonate from spring water (Sample 5) around 40 g were allocated. The samples were prepared with various alpha-, beta- and gamma-emitter natural (radionuclides of 238U and 232Th decay series) and anthropogenic  radionuclides but our laboratory has interest only in the 90Sr determination.In 2017, the IAEA-TEL-2017 World-Wide Open Proficiency Test was organized by the Reference Materials Group of the IAEA Terrestrial Environment Laboratory, Seibersdorf, Austria, in order to monitor the analytical capabilities and reveal gaps in analytical methodologies of different radioanalytical laboratories90Sr determination, Sample 1, 3 and 4 were spiked with 90Sr. Sample 3 was a quality control sample with described radionuclide content. The details of the samples analysed are summarised in the Table For 90Sr analysis using mass spectrometry instruments. Naturally, the water contains less amounts of trace elements compared to any kind of mammal milk. Therefore, the water sample contains significantly lower Sr concentration (39.4 ng g−1) than the concentrated milk powder (2.5 µg g−1). In the water samples, the 90Sr concentrations were in the same range, 23.7 Bq kg−1 (4.6 fg g−1) and 11.2 Bq kg−1 (2.2 fg g−1) respectively. In the milk powder it was higher, 99.9 Bq kg−1 (19.5 fg g−1), although it corresponds to 13 Bq kg−1 (2.5 fg g−1) in the original milk sample since the average dry matter content of a raw cow milk is around 13%14. Thus, stable Sr and 90Sr concentrations were different in samples, 90Sr/88Sr isotope ratio also differed and varied in the range from 1.37 × 10−7 to 9.04 × 10−9. The measured 90Sr/88Sr isotope ratios and the calculated 90Sr concentrations along with the detection limits are shown in Tables As discussed in the introduction, Sr and Zr concentration can affect the reliability of k powder .5 µg g−1ation 39. ng g−1 t−1 for the tap water samples and 97.9 ± 5.5 Bq kg−1 for the milk powder (k = 1). The range of TIMS measurement results were very narrow, varied in the range of 11.6–11.8 Bq kg−1 for tap water samples and 96.6–99.9 Bq kg−1 for milk powder samples. Each measurement produced results with excellent accuracy and precision compared to the reference values of 11.2 ± 0.3 (2.2 ± 0.1 fg g−1) and 99.9 ± 5 Bq kg−1 (19.5 ± 1.0 fg g−1), respectively. These results demonstrated the robust analytical capacity of TIMS for 90Sr analysis.The quality control tap water samples were measured four times while the test samples five times. In the report submitted to IAEA, the mean decay corrected results were reported, 11.7 ± 0.4 Bq kg13. The results of tap water sample (Sample 1) are shown in Fig. −1. The TIMS result (marked with black quadrat) proved that the new TIMS method is competitive with radiometric methods for the measurement of 90Sr in water samples.The reported results of the proficiency test were published in the evaluation report−1) was slightly higher than the reference value (11.2 Bq kg−1) thus the relative bias (RB) statistical parameter, calculated by Eq. TIMS result  must be below  the MARB and over  the relative bias k = 2.58) as interpreted in Eq. 4.58 as inz-score statistics has been used to evaluate the performances of the participant laboratories and are shown in Fig. z-score calculation was carried out using the robust standard deviation described in Eqs z-score is a relative parameter, as robust standard deviation originates from the reported results and is affected by the performance of the participant laboratories. The reported results with a higher z-score of 2 not acceptable. The range of z-scores was from −7.7 to 10.3. Four laboratories reported exactly the same value as the reference one (z-score = 0) and seven more laboratories presented values with almost the same z-score as in the range from −0.01 to 0.01. In our case, the z-score of 0.44 was in the range from −0.5 to 0.5 which is quite impressive since only 37 laboratories of the 94 were able to accomplish this level of accuracy.The −1. Compared to tap water results, reliable 90Sr analysis in milk powder seems a more challenging task.The results of the milk powder sample (Sample 4) are shown in Fig. 90Sr in milk powder samples. In this case, TIMS result (97.9 Bq kg−1) was slightly lower than the reference value (99.9 Bq kg−1) thus the relative bias was also very good, −2.5% as presented in Fig. The TIMS result is marked here with a black quadrat and confirmed that this novel TIMS method is competitive with radiometric methods to determine The uncertainty of this TIMS measurement was 5.7% and the calculated P value was 7.6% therefore the precision of the TIMS method complied with the standards assigned by the IAEA proficiency test. The precision of the milk powder measurement is given in Fig. z-scores of the milk powder measurements are presented in the Fig. z-score acceptance limit (1.1) was much lower than in case of the water sample (2) because the MARB level for milk powder (15%) was lower, than for tap water (20%). In this case, the range of z-scores was quite wide, from −7.7 to 46.7. However, the z-score values of 31out of 80 laboratories reported were in the range from −0.5 to 0.5 as well as our laboratory with the excellent result of −0.17.The calculated 17. This precision performance is not possible for 90Sr analysis because the sample amount of 90Sr loaded onto a single degassed zone refined Re filament is very low, only a few fg6. Therefore, the generated 90Sr ion beam is quite weak and only few counts are detectable with a relatively high measurement error. In case of tap water sample (Sample 1), around 400 ng stable strontium was loaded along with 20 fg 90Sr, while for milk powder samples (Sample 4) around 1000 ng stable strontium was loaded along with 8 fg 90Sr. Consequently, the 90Sr beam intensity was higher for tap water samples, resulting in better relative standard deviation of 90Sr/88Sr isotope ratio (~2%) than for the milk powder (~5%) as can be seen in Tables TIMS is a highly precise instrument to measure accurately stable isotope ratios. For instance, stable Sr isotope ratios can be determined with a relative standard deviation of around 0.003%88Sr or 86Sr peaks play a significant role that needs special attention. At the beginning, a direct measurement of the 88Sr peak on a Faraday cup and simultaneously 90Sr on a Daly ion-counter was tested. 88Sr ion beam detection was not possible over 10 V using Faraday cup with resistor of 1011 Ω and the 90Sr ion beam remained undetectable on the Daly ion-counter if the 90Sr/88Sr isotope ratio was lower than 10−8. Since there was no way to expand the detection range of the Faraday cups, a less abundant Sr isotope, 86Sr was used to detect the reference ion beam instead of 88Sr. In Sr element the abundance of 88Sr is 82.58%, whereas the abundance of 86Sr is 9.86%. Moreover, the 86Sr/88Sr isotope ratio (0.1194) is a naturally stable ratio with fluctuation less than 0.1%18. Considering that the uncertainty of the 90Sr determination is more than 1% for the TIMS method, the uncertainty of 86Sr/88Sr isotope ratio has to be compared always with the overall measurement uncertainty for all kinds of environmental samples. The 87Sr isotope or 87Sr/88Sr isotope ratio cannot be utilized due to the radiogenic origin of the 87Sr (decay product of the 87Rb).The detection capacity limit of the Faraday cups during TIMS measurement for abundant 90Sr analysis for radiometric methods is affected by two major parameters. One is the background count rate and second the sample amount. Basically, the background count rate is a constant, caused by background radiation derived from 40K, U- and Th-decay series  and electric noise of the measurement system19. With increments of the sample intake, 90Sr concentration is increased in the analyte and the DL can be improved. However, the limitation lies in expenditure  and increase in preparation time along with the sample amount.The detection limit (DL) of 90Sr/88Sr abundance sensitivity, corresponding to a background effect of the peak tail of 88Sr and electric noise of the instrument. The abundance sensitivity is a kind of quality of a given mass spectrometry instrument which may decline with time as the pressure degrades mainly in the analyser house. However, the abundance sensitivity must be considered as a constant and needs to be checked regularly as a protocol of quality control.There are two affecting parameters also for the mass spectrometry method. One is the 90Sr contamination, the 90Sr/88Sr isotope ratio will be higher in the water sample than in the milk sample because water has a significantly lower stable Sr concentration than the milk. Therefore, if 90Sr/88Sr abundance sensitivity is a constant, the detection limit will be lower for water than milk samples. In this proficiency test, as it can be observed in Table −1) had two magnitudes lower stable Sr concentration than milk powder (2.5 ± 0.1 µg g−1). This explains the detection limit data in Tables −1) than for milk powder sample (2.3 Bq kg−1). Consequently, mass spectrometry is competitive with radiometric method if the stable Sr concentration is in the range of ng g−1 in environmental samples, such as in fresh water samples.The other parameter is not the sample intake itself but the stable Sr concentration of sample. For example, if one litre water and milk have the same level of 90Sr analysis with TIMS. Typically, 400 ng of stable Sr loaded on the filament is adequate for reliable analysis but larger sample amounts result in a more controllable beam and more measurement cycles. Obviously, to get the required amount of stable Sr, more water sample was used than milk powder for 90Sr analysis. Counting with some strontium loss during the sample preparation and separation, the sample intake was calculated with 2000 ng stable strontium. This stable strontium amount corresponded to 50 mL water sample and 1 g milk powder. The homogenous distribution of the added 90Sr in water sample is not an issue but in case of the milk powder non-homogeneity may occurs. To decrease the uncertainties derived from non-homogeneity, 50 g milk powder sample was recommended by the organizers of the proficiency test as a minimum sample intake. Since the sample preparation and strontium separation procedures of the TIMS method is optimized for 2000–4000 ng stable strontium, the risk was taken and only 1 g milk powder sample was digested to analyze 90Sr. As our measurement results demonstrated, the homogeneity of 90Sr in the milk powder sample was excellent and 1 g sample represented 90Sr distribution perfectly.The stable Sr concentration also influences the sample size that is necessary for 90Sr in soil matrix, 100 mg or less sample amount is sufficient for reliable 90Sr analysis using the TIMS instrument, however the detection limit must be considered.The sample homogeneity and stability are fundamental requirements for reference materials and proficiency test materials. In case of homogeneity test, the advantage of the mass spectrometry method is to be recognized. For example, to test the homogeneity of 90Sr measurement capacity but the limitations of the proficiency test must be considered. In case of the mass spectrometry methods, both the 90Sr activity concentration and the 90Sr/88Sr isotope ratios are important. As it can be observed in Fig. 90Sr/88Sr isotope ratio of the water and milk powder samples distributed for the IAEA proficiency test are relatively high, varies between 1.4 × 10−7 and 9.0 × 10−9 therefore 90Sr detection was rendered possible using TIMS. Moreover, the Zr interference was not a real challenge since Zr concentrations in the samples were quite low (pg g−1 range). As Fig. −1 range) and Zr (µg g−1 range) concentration in soil samples. In case of Fukushima soil samples wherein stable Sr concentration is over 100 µg g−1, 90Sr is not detectable around 100 Bq kg−1 but could be confidently measured at around 1000 Bq kg−1. Consequently, TIMS 90Sr measurement capacity is out of range to use the most preferred and frequently used IAEA-375 reference material (Chernobyl affected soil sample) for internal quality control.The TIMS method demonstrated a very precise and accurate 90Sr for mass spectrometry analysis (wide range of 90Sr/88Sr isotope ratios and Zr concentration) but interestingly for radiometric analysis it is lacking, considering the characteristics of the Fukushima accident. Fukushima accident affected environmental samples are mainly contaminated with volatile beta emitter fission products such as, I, Te and Cs isotopes. The contamination of non-volatile  and refractory elements  are very low in comparison because the primary concrete containment system mounted at the Fukushima Daichi reactors and the reactor pressure vessels were not damaged significantly by hydrogen explosions22. The 90Sr (and 89Sr) is a pure beta emitter radionuclide and beta particles have continuous spectrum unlike gamma rays therefore efficient separation of interfering beta emitter radionuclides is crucial before 90Sr measurement with radiometric instruments (every fission product is a beta emitter as well as gamma emitter except 90Sr and 89Sr). In Fukushima environmental samples the 137Cs (T1/2 = 30.2 y) has the highest significance for long-term radioactive contamination23. The 90Sr/137Cs activity ratio in Fukushima contaminated samples is around 1 × 10−4 which corresponds to a Cs decontamination factor of 105 24. As can be seen in Fig. 90Sr/137Cs activity ratio are not available, wherein presence of 137Cs significantly dominates 90Sr. The 90Sr/137Cs activity ratio in certified reference materials varies between 3.6 × 10−3 and 1.8 × 101 (only the IAEA-152 milk sample was in the range of 10−3 but acquisition of this sample is not possible as it is out of stock). Consequently, applicable internal quality control is not possible and valid proficiency tests are not available for laboratories where 90Sr determination is carried out in Fukushima samples using radiometric methods. One possible way to solve this problem, until the production of proper reference materials, is the cooperation of laboratories where mass spectrometry methods are applied for 90Sr analysis and selected Fukushima samples could be determined in both laboratories for the performance estimation (inter-laboratory comparison).As Fig. 3. 1 g milk powder was ashed at 600 °C and dissolved with 5 mL 8 M HNO3.50 mL water sample was evaporated and dissolved with 5 mL 8 M HNO3 solution preparation, analytical grade Tamapure AA-100 reagents (68% w/w) and Milli-Q2 purified water (>18 MΩ cm at 25 °C) were used.For HNO2O and preconditioned with 5 mL 8 M HNO3 with a flow rate of ~0.2 mL min−1. The samples dissolved in 5 mL 8 M HNO3 were loaded into the prepared columns and rinsed with 3 mL 8 M HNO3 and 3 mL 3 M HNO3. Finally, the Sr was stripped from Sr resin with 3 mL 0.05 M HNO3.For strontium separation 0.5 mL DGA and Sr extraction chromatography resin of 100–150 μm particle size  packed into polypropylene gravity columns  and washed with 10 mL H3 solution was evaporated to about 0.1 mL and mixed with 0.5 ml HNO3 and 0.5 mL H2O2. This solution was boiled for 10 minutes and evaporated to dryness. Thereafter, the residue containing Sr was dissolved in 0.25 ml 1 M HNO3 and evaporated to a tiny drop. The Sr sample was loaded onto a degassed single rhenium filament (99.999%) along with one microliter TaF5 activator.The 0.05 M HNO90Sr analysis using TIMS are given elsewhere12.The detailed information of reagents and materials, sample preparation, strontium separation and 6 but schematic diagram of the 90Sr measurement method  was used. The detailed strontium analysis method is described elsewhere8 to 1011), additional chemical separation of Zr is necessary. Applying two consecutive DGA and Sr resin extraction chromatography separations on environmental samples prior to the TIMS measurement, Zr decontamination factor of 1011 can be accomplished12.Owing to the evaporation temperature and ionization potential difference between Sr (~1380 °C and 5.7 eV) and Zr (~1900 °C and 6.6 eV), Zr separation can be accomplished during the thermal ionization process, however to reach higher decontamination level  and the analyzer (10−9 Pa or lower). The abundance sensitivity of the TIMS has been regularly checked and determined measuring NIST-987 standard reference material without any traces of 90Sr6. The NIST-987 has been purified by extraction chromatography to avoid 90Zr interference.The abundance sensitivity of the Phoenix X62 TIMS is 2.1 × 1090Sr activity concentration was calculated as the follows:A90 is the 90Sr activity concentration (Bq·kg−1), Rm is the measured 90Sr/88Sr isotope ratio, CSr is the stable Sr concentration of the sample (mg·kg−1), NA is the Avogadro constant, NA88 is the natural abundance of 88Sr (0.8258), MSr is the atomic mass of the Sr element (87.62 amu), T1/2 is the half-life of the 90Sr (9.1 × 108 s).The 26 but some important calculations are given here:RB is the relative bias, Am is the reported 90Sr activity concentration (Bq·kg−1) and Aref is the reference 90Sr activity concentration (Bq·kg−1), MARB is the maximum acceptable relative bias.P is the precision of the measurement, um is the combined uncertainty (k = 1) of the reported 90Sr activity concentration (Bq·kg−1) and uref is the combined uncertainty (k = 1) of the reference 90Sr activity concentration (Bq·kg−1).z is the z-score value, Am is the reported 90Sr activity concentration (Bq·kg−1) and Aref is the reference 90Sr activity concentration (Bq·kg−1), Robust sd is the robust standard deviation (Bq·kg−1).The statistical calculations discussed are presented elsewhereThe robust standard deviation was calculated as follows:90Sr determination. Considering the accuracy, precision and detection limit, the new mass spectrometry method is very competitive with the traditional radiometric methods and ready for accreditation. The outstanding sensitivity of Daly ion-counting detector and improved abundance sensitivity of the thermal ionization mass spectrometry widen the application not only stable isotope ratio but also radioactive isotope ratio analysis of radionuclides with a half-life less than 100 years. In case of a nuclear accident when the rapid analysis of 90Sr is critical, the usage of this new method is recommended.The results of this work demonstrate that our scientific efforts, to push the boundaries of inorganic mass spectrometry into a higher level, were successful. This is the first time that an inorganic mass spectrometry method fulfilled the requirements of a proficiency test in environmental level 90Sr analysis method in the future. It can also be a useful tool for reference material characterization, where employment of different methods is important for reliable results. Additionally, the low sample intake makes the new method capable to test radionuclide homogeneity in reference material.The new method needs further improvement to decrease the strontium amount necessary for analysis. This step can significantly reduce the sample intake along with the corrosive acid usage. Furthermore, the separation time can be also shortened promising the most rapid 90Sr/88Sr isotope ratio, the availability of reference materials is adequate to support the internal quality control process of laboratories where mass spectrometry method used for 90Sr analysis. However, from the viewpoint of 90Sr/137Cs activity ratio, reference material related to the Fukushima accident is not accessible.Considering the


Following collisional activation via electron capture from nitrogen gas, complexes for n in the range 2–6 exhibit a switch in reaction product as a function of n. For low values, solvated CaOH+ is the dominant fragment, but as n increases beyond 4, this is displaced by the appearance of solvated CaOR+. A separate study of unimolecular metastable decay by [Ca(ROH)n]2+ complexes found evidence of charge separation to form CaOH+(ROH)n−1 + R+. For two isomers of butanol, the n = 3 complexes were found to follow parallel, but different metastable pathways: one leading to the appearance of CaOH+ and another that resulted in proton abstraction to form ROH2+. These differences have been attributed to the precursor complexes adopting geometries where one ROH molecule occupies a secondary solvation shell. Comparisons were made with a previous study of magnesium complexes; [Mg(ROH)n]2+ show that the difference in second ionization energy Mg+ (15.09 eV) as opposed to Ca+ (11.88 eV) influences behavior. A complex between Ca2+ and 1-chloroethanol is shown to favor the formation of CaCl+ as opposed to CaOH+ as a unimolecular charge separation product, which is attributed to differences in bond energy in the precursor molecule.A comprehensive examination of how the identity of an alcohol molecule can change the behavior of a solvated, alkaline earth dication has been undertaken. The metal dication of CaThe online version of this article (10.1007/s13361-019-02263-x) contains supplementary material, which is available to authorized users. Observed chemical processes include unimolecular charge separation and the formation of CaOH+ and R+ and loss of the radicals H⋅ and R⋅, promoted by electron capture. Although there are common patterns of behavior within the range of molecules studied, there are also notable variations that appear to arise from differences in geometry.Advances in the techniques available for generating metal dication complexes in the gas phase have made it possible to study a wide range of their chemical and spectroscopic properties –3. Toget2+ complexes have been in association with water molecules and the determination of hydration numbers z+ were detected. Instead, the recorded mass spectra contained a wide range of both singly and doubly charged complexes formed from different combinations of calcium atoms with each of the alcohol molecules. Also present were very intense signals arising from (ROH)nH+ ions and their fragmentation products. Fortunately, the resolution of the mass spectrometer was sufficient to allow a clear separation of the signals of interest.Neutral clusters, some of which contained a single metal atom, then entered the ion source of a high-resolution reverse geometry double focusing mass spectrometer (VG-ZAB-E), where they were subjected to electron ionization at energies of between 70 and 100 eV. Since only ions rather than neutral complexes were detected, it is very likely that extensive ligand evaporation took place following ionization, which is believed to help reduce the internal energy content of complexes. Accordingly, under most experimental conditions, no ion complexes of the form [Ca(Ar)n]2+ complexes, extensive use has been made of the MIKES  technique 2+ complexes 2+ and [Mg(NH3)n]2+ complexes [3)n]2+ complexes 2+ system. Very rarely was n = 1 observed, which is attributed to instability due to Coulomb fission.Although the technique used to form complexes made it possible to generate dications with values of 3OH)n]2+ complexes as a function of size n is shown in Online Resource n = 4 does not necessarily imply any favored coordination number, but is more likely to arise as a consequence of the fragmentation patterns of larger complexes, combined with the observation that, in comparison to complexes where n > 4, [Ca(CH3OH)4]2+ has a higher binding energy 2+ showed evidence for the loss of CH3OH2+, but that no similar process could be identified for [Ca(CH3OH)2]2+, which instead loses CH3+. Very similar observations were made for Mg2+ [3OH)3]2+. The presence can be seen of both reactions (3OH)6]2+, where reaction   3OH)n]2+eactions  being in+(H2O)n and MOH+(H2O)n (M+ = Mg or Ca) with the latter becoming the sole cation beyond n = 5 [+ being more stable in the presence of large numbers of water molecules [3OH)4]2+ 2+ and [Mg(CH3OH)n]2+ complexes concluded that switching arises from a more rapid decline in the reaction barrier to the elimination of H from the OH group than for the elimination of CH3. As n increases, solvation helps to stabilize the more polarizable CaOCH3+ ion core [Similar switching reactions involving singly charged alkaline earth metal ions in association with both water and methanol molecules have been the subject of experimental and theoretical studies –39. Sincnd n = 5 . This trolecules . For theolecules –39. As p3OH)4]2+ , 19, whend Leary , 19, is nd Leary  of the iion core .2 and [Ca(C2H5OH)2]2+, where two separate series of fragment ions is observed and centered on the formation of either CaOC2H5+ or CaOH+. The strong peak at ~ 7800 eV includes several processes contributing to the formation of CaOH+(C2H5OH), and removal of the collision gas reveals the metastable peak shown in Figure 2H5+ as the complementary fragment ion. Earlier analysis of similar charge transfer processes made use of simple one-dimensional potential energy curves to account for the charged fragments 2+ complex. They concluded that the lowest-energy pathway was that leading to the formation of MgOH+ and CH3+ as charge transfer fragments 2+ shows very similar reaction products to those seen for [Ca(C2H5OH)2]2+ and includes a strong signal arising from the metastable loss of C2H5+ from the precursor ion. Figure 2H5OH)6]2+ following collisional activation, where it can be seen that, similar to the equivalent methanol complex, the reaction products are now entirely due to the formation of CaOC2H5+(C2H5OH)m, with no evidence of fragments containing CaOH+. Figure By applying a voltage to the collision cell , it is possible to separate reaction steps due to metastable decay from those initiated by collisional activation. Online Resource r nature , and the3H7OH)3]2+ ions with nitrogen. Once again, there is a mix of fragment ions corresponding to the formation of both CaOH+ and CaOC3H7+, with the former product ion no longer appearing once the precursor contains 6 1-propanol molecules. Both the n = 2 and n = 3 complexes exhibit strong metastable signals corresponding to the loss of C3H7+, and similar to that shown in Online Resource 3H7OH)n]2+, also noted the presence of a strong metastable signal for the appearance of SrOH+ when n = 2, but failed to appreciate the role played by electron transfer in processes induced by collisional activation in complexes where n ≥ 3 2+ complexes for small values of n resulted in sequences of product ions similar to those seen in previous examples, i.e., based on Ca+OC4H9 and CaOH+. Two of the complexes, n = 2 and 3, exhibited strong metastable signals, and an example recorded for [Ca(C4H9OH)3]2+ is shown in Figure The collisional activation of individual [Can]2+ complexes for n ≥ 4 resulted in product ions centered on the formation of CaOC4H9+, and no further metastable processes were observed.A similar observation was made on the corresponding Mg2+ 2+. The results provide evidence of two types of switching reactions, each being associated with separate fragmentation pathways and each having a clearly recognizable kinetic energy release profile. The first switching reaction is induced by electron capture from a collision gas (nitrogen) and proceeds via the generation of a singly charged Ca+(ROH)n species. This ion subsequently decays to give a charged core which takes the form of either CaOH+ or CaOR+, with the latter gaining in intensity as n increases. This pattern of behavior matches that seen for a wide range of singly charged alkaline earth/ROH complexes, including those where R=H [n]2+ ions that undergo unimolecular charge separation within the limited values of n as shown. These reactions give in each case two singly charged fragments either of the form CaOH+ as a core together with R+ or CaOR+ as a core together with ROH2+. This latter behavior could be attributed to complexes being formed where at least one molecule occupies a hydrogen-bonded site in the second solvation shell. The geometry of this arrangement would then facilitate the removal of a proton from a first-shell molecule to leave CaOR+ as a core. Examples of where a second solvation shell begins to develop before completion of the first shell have been identified previously in complexes with Mg2+ but not in any involving Ca2+ [2+ complexes. A qualitative trend observed for the latter was that for the generic metastable step:An extensive series of experiments have been undertaken to examine the influence different aliphatic alcohols have on the behavior of the dication complexes [Ca(ROH)here R=H –39. A seing Ca2+ , 14, 45.n required to stabilize each complex against unimolecular charge separation increased as the ionization energy of R declined; the relevant data are given in Table 2+, in that all of the complexes require four alcohol molecules to stabilize against charge separation. However, there are differences between Mg2+ and Ca2+, in that the higher ionization energy of the former requires five rather than four molecules of both 1-propanol and 1-butanol to stabilize against UCS.The value of ESM 1(PDF 212 kb)


The subsequent treatment with 1‐adamantanamine disassembles the cucurbit[7]uril/spermine complex and readily induces reconversion of B‐ into Z‐DNA. The DNA conformational change is unequivocally demonstrated using different independent methods. Direct evidence for supramolecular interactions involved in DNA conformational changes is further provided. These findings can therefore open a new route to control DNA helical structure in a reversible way.As a left‐handed helical structure, Z‐DNA is biologically active and it may be correlated with transcription and genome stability. Until recently, it remained a significant challenge to control the B/Z‐DNA transition under physiological conditions. The current study represents the first to reversibly control B/Z‐DNA transition using cucurbit[7]uril‐based supramolecular approach. It is demonstrated that cucurbit[7]uril can encapsulate the central butanediamine moiety [HN(CH Importantly, CB7 can encapsulate the central butanediamine moiety [HN(CH2)4NH] to form an inclusion complex (CB7/spm) and reverse Z‐DNA caused by spm back to B‐DNA , as reflected by very small changes in the shape and intensity of the CD bands . Moreover, the 6mer‐3G takes the B‐form at spm concentrations up to 2.0 × 10−3m.We next investigated the effects of spm on conformation of oligonucleotides containing three contiguous CpG dinucleotides. In these short stretches of DNA, the first or second dG is replaced by 8‐oxodG (6mer‐oxoG1 and 6mer‐oxoG2 in Table S1 in the Supporting Information). The CD spectra of 6mer‐oxoG1 indicated classical B‐DNA conformation. Upon addition of increasing amounts of spm, there was a gradual appearance of a negative peak at ≈290 nm and a positive peak at about 260 nm. From the observations, spm can efficiently induce conformational transition of 6mer‐oxoG1 from B‐ into Z‐DNA Figure3A. The−6m CB7 resulted in a significant decrease of Z‐DNA formation, and this effect was more evident with the 160 × 10−6 and 240 × 10−6m treatments. Moreover, the treatment with 300 × 10−6m CB7 almost entirely diminished the spm effects on Z‐DNA formation.Next, we examined whether CB7 can reverse the effects of spm in Z‐DNA induction for 6mer‐oxoG1. Importantly, the sequential addition of CB7 produced evident changes in the shape and intensity of the CD bands Figure B. There −6m AM treatment, and higher levels of enhancement were observed with the 200 × 10−6 and 250 × 10−6m treatments. Moreover, the 300 × 10−6m AM treatment largely diminished the CB7 effects and the CD spectrum was very close to that observed for the sample with only spm. From these observations, AM is able to counteract the effects of CB7 on spm and reinduce Z‐DNA in a dose‐dependent manner.We further studied the effects of AM on the double‐helical structures of 6mer‐oxoG1 Figure C. SpecifFigureWe next tested whether B/Z‐DNA transition can be controlled for multiple cycles. Hence, 6mer‐oxoG1 was subjected to the spm treatment, followed by sequential addition of CB7 and AM. After each addition, the sample was incubated further before the CD spectrum was recorded. From these observations, the presence of excess CB7 induced broad positive absorbance at 290 nm and strong negative bands at 253 nm, indicating the formation of right‐handed B‐DNA. Strikingly, continual addition of AM produced an inverted CD spectrum with a characteristic negative band at about 290 nm  into Z‐DNA in the absence of spm, as reflected by very small variations of the CD spectra . Moreover, high concentrations of AM did not markedly affect Z‐DNA structures induced by spm . Therefore, these results clearly indicated that the above B/Z‐DNA transition could not be attributed to the direct interaction between the DNA and regulator (CB7 or AM).2.41H‐NMR refinement have been performed. In B‐DNA conformation, the nucleobases are usually in anti orientation. However, in left‐handed Z‐DNA, purine bases generally adopt a syn orientation.1H‐NMR spectral assignment of DNA is usually complicated, especially during conformational transition. However, nonexchangeable proton 2D nuclear Overhauser effect spectroscopy (NOESY) provides a unique method for DNA conformational analysis in solution without prior knowledge of the spectral assignment.To unequivocally verify the helix transitions of 6mer‐oxoG1 between different conformations, 1D and 2D 1H‐NMR spectra of 6mer‐oxoG1 were recorded in the absence of spm. Since a full analysis of 2D results is beyond the scope of this study, we have focused on cross sections illustrating correlations between the base aromatic protons (7.95–7.15 ppm) and the deoxyribose H1′ fingerprint protons (6.45–4.90 ppm). Previous research has shown that there is a direct response to the B–Z transition in this region.] Figure S9A (Supporting Information) shows expanded plot of the aromatic proton region, in which five major proton resonances are observed. This is consistent with the existence of five bases with nonexchangeable aromatic protons in 6mer‐oxoG1. On the basis of the homonuclear correlation spectroscopy (COSY) spectrum , those signals between 7.7 and 7.2 ppm can be readily identified as deoxycytosine  H6/H5 protons since they represent the only aromatic scalar‐coupled protons with 3 bonds apart.[[qv: 29a]] From these observations, the dG H8 proton resonances were assigned vertically between 7.90 and 7.85 ppm.As the starting point, 1D and 2D anti or 2.5 Å if it is syn, and the distance between cytosine H6 and H5 is 2.5 Å.[[qv: 29a]] Figure S9C (Supporting Information) shows an expansion of NOESY spectrum of 6mer‐oxoG1 demonstrating no cross‐peak to the dG base protons between 7.9 and 7.8 ppm. Figure anti orientation of the dG base in 6mer‐oxoG1. The helical structure of 6mer‐oxoG1 can therefore be characterized as B‐DNA.According to previous studies, the distance between H8 and H1′ in the same dG is 3.7 Å if the glycosidic angle is 1H‐NMR results , the chemical shift of dG H8 was observed to gradually shift upfield with increasing amounts of spm. Another interesting finding was that evident shifts were observed for dC aromatic proton resonances. In the presence of 50 × 10−6m spm, dG H8 proton resonances were shifted at a considerable level (≈50%). spm at a higher dose (200 × 10−6m) shifts dG H8 proton resonance to a predominantly new position . Next, the COSY spectrum of 6mer‐oxoG1 was recorded in the presence of 200 × 10−6m spm . The three major proton resonances between 7.5 and 7.3 ppm were assigned to dC H6/H5 protons due to strong scalar couplings. The NOESY spectrum shows four cross‐peaks in the selected window . The stacked plot of the NOESY and COSY spectra displays that only dG H8 (7.75 ppm) and deoxyribose H1′ (6.2 ppm) were connected through space correlations . Such observed variations show almost completely opposite pattern to that with spm treatment, possibly due to the inclusion of spm into the CB7 cavity. In the presence of 800 × 10−6m CB7, dG H8 proton resonances were almost completely restored to original levels (≈7.9 ppm). We further recorded the 2D spectra of the same sample. The COSY spectrum shows six cross‐peaks, which are correspond to dC H6/H5 peaks over the range 7.7–7.2 ppm in 1D 1H‐NMR experiment . Figure S13C (Supporting Information) shows NOESY spectrum of the same sample. Based on the stacked plot of the NOESY and COSY spectra . This trend is in accordance with our expectations. In the presence of 800 × 10−6m AM, dG H8 proton resonance was almost completely shifted and very similar to that with only spm treatment . The COSY spectrum of this sample shows three well‐resolved cross‐peaks from dC H6/H5 scalar couplings . Importantly, a high‐intensity intraresidual NOE interaction was observed between the more downfield dG H8 signal (≈7.75 ppm) and deoxyribose H1′ resonance  as a function of temperature.3 nm as a2.71H‐NMR study was next performed to gain more insights into the host–guest chemistry behind reversible B/Z‐DNA transition.] Figure S21 (Supporting Information) shows all proton resonances of spm in different chemical environments. There are five chemically inequivalent methylenes, which are labeled with the a, b, c etc. . From our observations, the binding of CB7 to spm induced substantial shifts of all proton resonances. Importantly, the proton resonances of methylenes  belonging to H2N—(CH2)3—NH— moiety move downfield, indicative of their positioning outside of the CB7 cavity near the portals . In particular, the largest downfield shift was observed for the proton resonances of “methylene c” attached to secondary amine group and considerably smaller changes in the shifts were observed for the protons on neighboring groups (a and b). However, the proton resonances of methylenes (d and e) belonging to the —HN(CH2)4NH— moiety moved upfield, indicative of their positioning inside the shielding cavity of CB7. Therefore, it is concluded that CB7 encapsulates the central butanediamine moiety [HN(CH2)4NH] to form an inclusion complex (CB7/spm).The 1H‐NMR results and therefore the host–guest interactions are probably responsible for regulating B/Z‐DNA transition.In a subsequent experiment, evident shifts were observed for the proton resonances of spm methylenes upon addition of AM to the above preparation (CB7–spm) , indicating occurrence of specific decomplexation. This behavior suggests that the binding affinity of CB7 with AM is higher than that with spm. These observations are consistent with those expected from the CD and −6m) showed a weak fluorescence band at 493 nm, whereas fluorescence at this wavelength increased with increasing amount of CB7. These observations have established a 2:1 stoichiometry for the (CB7)2·ThT complex. Importantly, the emission intensity displayed a gradual decrease by adding spm into the above solution .3The precise control of DNA structures can help researchers investigate some important biological processes.6m−1).1H‐NMR study demonstrated that, in the absence of DNA, CB7 can bind spm at the central butanediamine moiety in a 1:1 stoichiometry. However, in the presence of DNA, a several‐fold molar excess of CB7 over spm was required to reverse the Z‐DNA back to B‐DNA.The current study represents the first to reversibly control B/Z‐DNA transition using CB7‐based supramolecular approach. Given previous findings that 8‐oxodG, an important oxidative DNA damage, is potentially epigenetic,1H‐NMR determination elegantly complemented CD measurements and further evidenced the existence of the well‐defined B/Z‐DNA transition. Importantly, COSY experiment was used to group and identify aromatic scalar‐coupled protons on a single base. Moreover, the glycosidic conformation was identified by NOESY experiment on the basis of spatial connectivities between protons coupled by dipolar interaction.] By use of these methods, structural information of residues was obtained without giving any information about their position in the sequence or protons on a one sugar. The 1H‐NMR experiments reveal that structural transition behaviors are quite reversible. In this context also, it can serve as an interesting starting point for future researchers looking to finish full NMR structural study. Since polyamines are widely dispersed in the cell, the current study further provides new and valuable insight into reversible and accurate control of DNA helical structure in vivo.In the current study, the CD was used as a reference technique to evidence DNA conformational changes.] Our results demonstrate that the structural transition behaviors of DNA with different handedness are quite reversible. Moreover, the 4In summary, the present study represents the first to reversibly control B/Z‐DNA transition using CB7‐based supramolecular approach. Using various methods, we provide direct evidence that the CB7‐driven supramolecular competition enables reversible on/off switching of B/Z‐DNA transition. Whether such a strategy is operative in vivo is yet to be understood. We believe that this approach offers a general and facile way toward reversible and accurate control of DNA helical structure.5Chemical Synthesis: The compound CB7 was synthesized according to the previous literatures.Circular Dichroism Study: The CD experiments were performed at room temperature using a Jasco‐810 spectropolarimeter  equipped with a Peltier temperature controller. All samples were measured in a quartz cell of 1 cm optical path length. The spectra were collected from 340 to 200 nm at a scanning speed of 200 nm min−1. The bandwidth was 5 nm and the response time was 2 s. All spectra were baseline‐corrected for signal contributions from the buffer and were the average of three runs.Supramolecular Control of B/Z‐DNA Transition: The desired buffer  was used throughout the investigation.The spm‐Induced Z‐DNA Formation: The DNA was dissolved to the desired concentration in the desired buffer, heated to 90 °C for 5.0 min, and then cooled slowly to room temperature. The desired spm concentration was obtained by adding appropriate amounts of stock solution, and the mixture was incubated for 15 min at room temperature. Subsequently, the CD spectra were determined and compared to a blank solution.The CB7‐Induced Z‐DNA Deformation: The starting sample was prepared in the desired buffer containing DNA and spm at desired concentrations. After a 15 min incubation at room temperature, CB7 was added sequentially to the starting preparation prior to measurement of CD spectra.The AM‐Induced Z‐DNA Reformation: The starting system was prepared in the desired buffer containing DNA, spm, and CB7 at desired concentrations. The following procedure was similar to the above one wherein AM was added sequentially to the starting preparation prior to measurement of CD spectra.1H‐NMR Study of DNA Conformation Transition: The 1D 1H‐NMR experiment was routinely performed at 298 K using a Bruker Avance III HD Ascend 850 MHz spectrometer equipped with a 5 mm triple‐resonance (HCN) cryoprobe. The spectra were acquired with a 2 s relaxation delay and 32 transients using watergate pulse sequence with gradients (W5) for water suppression. The MestReNova program was used to process 1D spectra obtained from the original data.1H‐NMR experiments were performed at 298 K using a Bruker Avance III HD Ascend 700 MHz spectrometer equipped with a 5 mm triple‐resonance (HCN) cryoprobe. The 2D 1H–1H COSY and NOESY were collected with spectral widths of 10 ppm and 2048 (40–160 transients) and 120 complex points in the direct and indirect dimensions, respectively. The mixing time for NOESY spectra was 150 ms. The signal assignments were based on the chemical shifts and intensity patterns.The 2D Thermal Stability and Reversibility Study: This assay was performed in a Jasco‐810 spectropolarimeter using a water‐jacketed quartz cell. The DNA samples were prepared under the conditions of the experiment shown. The CD spectra were recorded as a function of temperature from 5 to 85 °C.−1 and the absorbance values were collected every 1 °C. The temperature‐dependent CD change was recorded at 253 nm. The melting point corresponded to the mid‐transition temperature.The CD melting profiles were recorded using a heating rate of 0.2 °C minCompetitive Dye‐Binding Titrations: The titrations were performed at room temperature in the desired buffer .The CB7‐Induced Fluorescence Enhancement: The ThT solution was prepared at a final concentration of 80 × 10−6m. Subsequently, increasing amounts of CB7 (from 0 to 160 × 10−6m) were added sequentially with fast mixing. The fluorescent emission spectra (area: 450–800 nm) were then determined at room temperature using a LS55 fluorescence spectrometer . The solution was excited at 421 nm, and a 1 cm path‐length quartz cell was used. Slit width: excitation = 10 nm; emission = 20 nm. All spectra were baseline‐corrected for the signal contributions from the buffer. The plot of emission@493 nm was recorded as a function of the concentration of CB7 added.The spm‐Induced Fluorescence Change: The CB7/ThT complex was prepared in the desired buffer at desired concentrations. The following procedure was similar to the above one wherein spm was added sequentially to the starting preparation prior to fluorescence measurement. The plot of emission@493 nm was recorded as a function of the concentration of spm added.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


This paper presents an NMR crystallography study of three polymorphs of furosemide. Experimental magic‐angle spinning (MAS) solid‐state NMR spectra are reported for form I of furosemide, and these are assigned using density‐functional theory (DFT)‐based gauge‐including projector augmented wave (GIPAW) calculations. Focusing on the three known polymorphs, we examine the changes to the NMR parameters due to crystal packing effects. We use a recently developed formalism to visualise which regions are responsible for the chemical shielding of particular sites and hence understand the variation in NMR parameters between the three polymorphs. Although composed of the same molecular units, the molecules can interact with each other differently resulting in different properties. The polymorphs can vary in unit‐cell dimensions, symmetries, intermolecular bonds, stacking arrangements, and conformational changes in the molecular unit itself.Solid‐state nuclear magnetic resonance (NMR) is a highly sensitive probe of the local atomic environment, making it an effective tool for distinguishing between similar polymorphs.1H solid‐state NMR chemical shift.The influence of intermolecular effects on NMR chemical shifts can be studied by comparing the calculated chemical shifts from an isolated molecule extracted from the crystal structure to the calculated chemical shifts for the full crystal structure.Z′  =  2 and Z  =  4, and a recent study using GIPAW calculations that considered FURSEM01, FURSEM17, and a new form and solid‐state NMR experiments has determined that FURSEM17 is likely an inaccurate solution of form I.Furosemide Scheme  is an ac13C CP magic‐angle spinning (MAS), 1H–13C refocused INEPT, and 1H double quantum (DQ) MAS experiments. The GIPAW‐calculated NMR chemical shifts are analysed focusing on intermolecular hydrogen bonding and aromatic ring current effects. The analysis notes subtleties between solved structures of the same form, and different geometry optimization approaches.In this study, we analyse three solutions for form I: FURSEM01 (F01),22.11H Larmor frequency of 500.1 MHz (13C Larmor frequency of 125.8 MHz). A Bruker 4‐mm triple resonance MAS probe (in double‐resonance mode) was used in 1H–13C CP MAS and refocused INEPT experiments, and a Bruker 2.5‐mm double resonance MAS probe was used for the 1H DQ experiment. In 1H–13C CP MAS and refocused INEPT experiments, SPINAL64 1H heteronuclear decoupling1H nutation frequency for pulses and decoupling was 100 kHz.Furosemide was purchased from Sigma‐Aldrich . Solid‐state NMR experiments were performed using Bruker Avance III NMR spectrometers operating at a 1H (SQ‐DUMBO)–13C SQ refocused INEPT experiment can be found in Fig. 5 of Elena et al.,1H DQ MAS,1H DQ MAS experiment, a 16‐step phase cycle was used to select Δp  =  ±2 on the DQ excitation pulses (four steps) and Δp  =  ±1 (four steps) on the z‐filter 90° pulse, where p is the coherence order. For the 2D 1H–13C refocused INEPT experiment, eDUMBO‐122 homonuclear decoupling,1H evolution period and the spin‐echo durations. The 32‐μs eDUMBO‐122 cycle was divided into 320 steps of 100 ns. The STATES‐TPPI method was used to achieve sign determination in F1 in the refocused INEPT and DQ MAS experiments.A pulse sequence and coherence transfer pathway diagram for the 2.2π Å−1. All crystal structures were optimised using the Perdew‐Burke‐Ernzerhof (PBE)Calculations were performed using a developer version of the CASTEP code2.3δiso, are related to the calculated isotropic magnetic shieldings, σiso, by a reference value σref : Isotropic chemical shifts, Ifullcell) is of a single crystallographic unit cell. The second calculation (denoted Inomol) is performed on a unit cell of the crystal with the molecule that contains the atom of interest removed. Depending on the crystal structure, it may be necessary to simulate a supercell of the unit cell, such that the missing molecule is surrounded by all of its nearest neighbours. In this work, a 2 × 1 × 1 supercell was used for the NICS calculations of all three polymorphs.*α  <  β  <  γ, and α and γ are swapped. To account for this, a 1 × 1 × 2 supercell was used.FURSEM01 does not employ the convention The NICS, σNICS, is obtained from calculating the value of the magnetic shielding at the site of the atom in the missing molecule. The final calculation (denoted IIInemolo) is a vacuum supercell with only one molecule containing the atom of interest. The molecule to crystal change in chemical shifts Δδ is the change in shifts between calculations IIIonemol and Ifullcell. Within this setup, we can analyse all intermolecular effects. The values denoted as “H bond strength” correspond to the sum of the molecule to crystal change in chemical shift and the NICS; as such, this accounts for changes in the NMR chemical shift due to interactions between a specific molecule and its neighbours.The necessary magnetic shielding calculations to compute the molecule to crystal change in chemical shift and the NICS are illustrated in Fig. 1 of Zilka et al.33.113C CP MAS spectrum of furosemide form I, recorded at room temperature, is presented in Figure Z′  =  2), each with 12 carbon atoms, corresponding to, at most, 24 peaks in the 13C spectrum. The 13C CP MAS spectrum is consistent with previously reported data.V′). Moreover, the splitting in the values of the chemical shift between the two inequivalent molecules is also well reproduced. In Figure 13C chemical shifts are plotted against the GIPAW‐calculated absolute isotropic 13C chemical shielding of furosemide form I. The (negative) gradient A 2D 1H DQ MAS spectrum of furosemide form I recorded using fast MAS (30 kHz). The peaks correspond to pairs of protons with a significant Figure 3.2−1; however, after a full relaxation, this is reduced to 0.1 kJ mol−1. Form I is known to be the experimentally most stable polymorph, and indeed, it has the lowest total energy, with forms II and III, 1.3 and 2.6 kJ mol−1 higher, respectively. Note that Nyman and Day showed that, for an analysis of over 500 organic molecular polymorphs, 95% of polymorphs are within 7.2 kJ mol−1.Having considered a comparison of experimental NMR spectra and GIPAW calculations of form I of furosemide, we now use first‐principles calculations to assess the accuracy of the alternative XRD structures deposited for form I. We also examine the structures of the other two known forms of furosemide. Table NMR chemical shieldings have been calculated using the GIPAW method for all the geometry optimised furosemide structures. The full GIPAW calculated absolute chemical shielding for each distinct site in the asymmetric unit cells is presented in the 1H magnetic shielding between the form I structures. The full GIPAW calculated magnetic shieldings for each distinct site in the asymmetric unit cells are presented in the The energetic similarity of the three form I structures is reflected in the root‐mean‐square deviation of atomic position. This is shown in Table 1H chemical shift appears to be the angle between the N–C bond and the furan ring. This is 93° in form III as compared with 111° and 114° for the other cases. The molecule to crystal changes in chemical shift encompass the effect due to intermolecular interactions and ring currents combined. This can be further decomposed into differences due to hydrogen bonds and to long‐range packing interactions. All forms have an OH...O hydrogen bond as the strongest intermolecular bond. In addition, at least one NH...X (where X is N or O) hydrogen bond exists in all forms, and forms I and II have two NH...X hydrogen bonds. The differences in longer range packing interaction are apparent in the calculated NICS values. In each polymorph, a different hydrogen interacts with an aromatic ring. The effect of the ring currents is visualised through Magnetic Shielding Contribution Field mapsWe now examine the changes in chemical shifts between the three polymorphs. A full listing of the GIPAW calculated chemical shifts values of the furosemide polymorphs, form I, II, and III, can be found in Table 413C CP MAS, 1H–13C refocused INEPT and 1H DQ MAS NMR spectra for form I of furosemide. The spectra have been assigned by comparison with chemical shifts computed using DFT and the GIPAW approach. We have used DFT calculations to assess the quality of three of the deposited structures for form I of furosemide. After a full relaxation of the atomic positions and unit cell parameters, all three structures are essentially indistinguishable, giving differences in GIPAW calculated 1H chemical shifts that are well below the precision obtained in our experiments. Further calculations on forms II and III of furosemide have employed a recently developed approachWe have presented Table S1: Shielding values (in ppm) of three geometry optimised form I structures ‐ relaxed structures, molecule 1Table S2: Shielding values (in ppm) of three geometry optimised form I structures ‐ fixed structures, molecule 1Table S3: Shielding values of three geometry optimised form I structures ‐ relaxed structures, molecule 2Table S4: Shielding values of three geometry optimised form I structures ‐ fixed structures, molecule 2Table S5: Difference in shielding values between relaxed and fixed geometry optimised structures of form II and IClick here for additional data file.