Quantitative 1H NMR Spectroscopy Research Articles

Anthocyanins and the Antioxidant Capacities of Wild Berries that Grow in Shizuoka, Japan

ABSTRACT Fruits from five wild edible berries that grow in the eastern region of Shizuoka (Japan) were collected, and their antioxidant activities were evaluated using the ORAC, DPPH, FRAP, and ABTS assays to obtain basic scientific knowledge of these species. Among the Shizuoka wild berries tested, Rubus microphyllus L. fil. exhibited the highest antioxidant activity, which is equivalent to that of commercial raspberry (Rubus idaeus L.). Furthermore, anthocyanins from these wild berries were analyzed using quantitative 1H NMR spectroscopy. Cyanidin-3-glucoside and pelargonidin-3-glucoside were detected in Shizuoka wild berries, with the latter identified as the characteristic anthocyanin. The present study revealed that Shizuoka wild berries are rich in natural antioxidants and pigments and are therefore potentially beneficial for reducing the risks of associated with lifestyle-related diseases in consumers.

Quantitative 1H NMR Spectroscopy Method for Determination of Anthraquinone Derivatives in Extracts from Rubia tinctorum L. Roots and Rhizomes

Quantitative 1H NMR Spectroscopy Method for Determination of Anthraquinone Derivatives in Extracts from Rubia tinctorum L. Roots and Rhizomes

The Molecular Footprint of Peptides on the Surface of Ultrasmall Gold Nanoparticles (2 nm) Is Governed by Steric Demand.

Ultrasmall gold nanoparticles were functionalized with peptides of two to seven amino acids that contained one cysteine molecule as anchor via a thiol-gold bond and a number of alanine residues as nonbinding amino acid. The cysteine was located either in the center of the molecule or at the end (C-terminus). For comparison, gold nanoparticles were also functionalized with cysteine alone. The particles were characterized by UV spectroscopy, differential centrifugal sedimentation (DCS), high-resolution transmission electron microscopy (HRTEM), and small-angle X-ray scattering (SAXS). This confirmed the uniform metal core (2 nm diameter). The hydrodynamic diameter was probed by 1H-DOSY NMR spectroscopy and showed an increase in thickness of the hydrated peptide layer with increasing peptide size (up to 1.4 nm for heptapeptides; 0.20 nm per amino acid in the peptide). 1H NMR spectroscopy of water-dispersed nanoparticles showed the integrity of the peptides and the effect of the metal core on the peptide. Notably, the NMR signals were very broad near the metal surface and became increasingly narrow in a distance. In particular, the methyl groups of alanine can be used as probe for the resolution of the NMR spectra. The number of peptide ligands on each nanoparticle was determined using quantitative 1H NMR spectroscopy. It decreased with increasing peptide length from about 100 for a dipeptide to about 12 for a heptapeptide, resulting in an increase of the molecular footprint from about 0.1 to 1.1 nm2.

Characterization and antifungal properties against wood decaying fungi of hydrothermal liquefaction liquids from spent mushroom substrate and tomato residues

This study aimed to investigate the potential of converting bio-based residues from industrial production of mushrooms and tomatoes into more valuable chemicals with antifungal properties using hydrothermal liquefaction (HTL). Liquid fractions were obtained from HTL of spent substrate of Agaricus bisporus (Lange) Imbach and Pleurotus ostreatus (jacq.) P. kumm., recomposted Agaricus bisporus spent substrate, and tomato residues. The quantitative 1H NMR spectroscopy analysis revealed that the HTL liquids of all residues contained antifungal constituents like phenols and organic acids. The HTL liquids at dilutions of 10 % were able to inhibit the fungi by over 80 %. Interestingly, the fungus P. ostreatus showed tolerance to these constituents as its growth was promoted at the lowest concentration of all the HTL liquids. The HTL liquids had lower ecotoxicity than the commercial wood preservative. These results suggest that the tested residues could be a promising source of preservative chemical constituents for the wood industry.

Sequence-Encoded Differences in Phase Separation Enable Formation of Resilin-like Polypeptide-Based Microstructured Hydrogels.

Microstructured hydrogels are promising platforms to mimic structural and compositional heterogeneities of the native extracellular matrix (ECM). The current state-of-the-art soft matter patterning techniques for generating ECM mimics can be limited owing to their reliance on specialized equipment and multiple time- and energy-intensive steps. Here, a photocross-linking methodology that traps various morphologies of phase-separated multicomponent formulations of compositionally distinct resilin-like polypeptides (RLPs) is reported. Turbidimetry and quantitative 1H NMR spectroscopy were utilized to investigate the sequence-dependent liquid-liquid phase separation of multicomponent solutions of RLPs. Differences between the intermolecular interactions of two different photocross-linkable RLPs and a phase-separating templating RLP were exploited for producing microstructured hydrogels with tunable control over pore diameters (ranging from 1.5 to 150 μm) and shear storage moduli (ranging from 0.2 to 5 kPa). The culture of human mesenchymal stem cells demonstrated high viability and attachment on microstructured hydrogels, suggesting their potential for developing customizable platforms for regenerative medicine applications.

Utilization of a kinetic isotope effect to decrease decomposition of ceftriaxone in a mixture of D2O/H2O

The discovery of cephalosporin and demonstration of its improved stability in aqueous solution, as well as enhanced in vitro activity against penicillin-resistant organisms, were major breakthroughs in the development of β-lactam antibiotics. Although cephalosporins are more stable with respect to hydrolytic degradation than penicillins, they still experience a variety of chemical transformations. The present study offers an insight into the rates and mechanisms of ceftriaxone degradation at the therapeutic concentration in water, a mixture of water and deuterium oxide, and deuterium oxide itself at the neutral pH.Specific ceftriaxone degradation products were observed in aged samples (including a previously unreported dimer-type species), and by comparing the degradation rates in H2O and D2O, the observation of a kinetic isotope effect provided some valuable insight as to the nature of the initial ceftriaxone degradation. The effect of protium to deuterium isotope change on the degradation kinetics of ceftriaxone was evaluated using the method of initial rates based on HPLC analysis as well as by quantitative 1H NMR spectroscopy. Moreover, computational analysis was utilized to get a molecular insight into chemical processes governing the ceftriaxone degradation and to rationalize the stabilizing effect of replacing H2O with D2O.

Crystallization kinetics and equilibrium melting temperature of poly(ether ketone ketone) with high terephthalate content utilizing fast scanning calorimetry

Fast scanning calorimetry (FSC) was used to analyze the crystallization kinetics and determine the equilibrium melting temperature (Tmo) of KEPSTAN PEKK 8001 (PEKK 80/20) with a terephthalate to isophthalate (T/I) ratio of 80/20. Quantitative 1H, qualitative 13C and 2D HSQC NMR spectroscopy for KEPSTAN 8001 and KEPSTAN 7002 were collected to confirm linear topology and comonomer composition. Utilizing FSC, the isothermal crystallization kinetics for PEKK 80/20 are reported for the first time over a large temperature range from 200 °C to 310 °C utilizing an interrupted isothermal crystallization method. The crystallization kinetics of PEKK 80/20 exhibits parabolic behavior with the most rapid crystallization occurring at 260 °C. Compared to earlier studies of PEKK copolymers with lower T/I ratios (i.e., PEKK 70/30 and 60/40) using conventional DSC, PEKK 80/20 is observed to crystallize up to 1 to 2 orders of magnitude faster (i.e., at rates inaccessible with conventional DSC). The Tmo of PEKK 80/20 was determined utilizing the zero-entropy-production temperature (TZEP) allowing for the first time an accurate determination of the equilibrium melting temperature of 382 °C. This value was compared to a traditional Hoffman-Weeks linear extrapolation over a range of heating rates (500 K/s – 20,000 K/s) to arrest melt-reorganization upon heating. Due to unavoidable crystalline reorganization during slow heating scans with conventional DSC, this new value for Tmo using FSC is significantly higher than the commonly referenced literature value of 368 °C. The double melting peak behavior observed after isothermal crystallization of PEKK 80/20 is discussed and understood to be due to reorganization during heating at relatively slow heating rates, as commonly observed with conventional DSC.

Feasibility of using quantitative 1H-NMR spectroscopy and ultra-microbalances for investigation of a PET microplastic reference material

Here, we report on the feasibility of using quantitative NMR and ultra-microbalances for additional measurements of the mass of poly-ethylene terephthalate (PET) particles in a reference material (RM). The microplastic (MP) PET particles were immobilised in solid NaCl following freeze-drying of a 1-ml NaCl suspension. The particles ranged from 30 to about 200 µm (Feretmin). In a 3-day process, more than 500 such units of PET particles in the NaCl carrier were prepared and later used in a large-scale inter-laboratory comparison. The homogeneity of PET in the salt carrier over these 500 units had previously been evaluated with respect to the mass of PET using an ultra-microbalance. In addition to the original results obtained by weighing, two independent results of quantitative 1H-NMR have been obtained for further investigation of this reference material together with one additional set of weighing data. The NMR data were used for confirmation of the weighed amount of PET (as weighing is non-specific for PET). Average masses of 0.293 ± 0.04 mg and 0.286 ± 0.03 mg of PET were obtained using two different ultra-microbalances (14% RSD for n = 14 and 9% RSD for n = 4, respectively). The corresponding 1H-NMR data was 0.300 ± 0.02 mg of PET (6.7% RSD for n = 5) and 0.345 ± 0.04 mg of PET (12.5% RSD for n = 14), respectively. The average mass of PET obtained by 1H-NMR measurements was in agreement with the weighed amounts within their standard deviations. A mean value of 0.306 mg PET with an expanded uncertainty of 0.058 mg (± 19% relative) was calculated, and it is traceable to the SI system of measurements. Measurement of PET by quantitative 1H-NMR spectroscopy is also reported for a water sample. The PET contained in one RM sample was transferred to 1 L of water to mimic a drinking water sample for microplastics.Graphical abstract

Influence of PLGA End Groups on the Release Profile of Dexamethasone from Ocular Implants.

The present study deals with the development of dexamethasone (DM)-loaded implants using ester end-capped Resomer RG 502 poly(lactic acid-co-glycolic acid) (PLGA) (502), acid end-capped Resomer RG 502H PLGA (502H), and a 502H:502 mixture (3:1) via hot melt extrusion (HME). The prepared intravitreal implants (20 and 40% DM loaded in each PLGA) were thoroughly investigated to determine the effect of different end-capped PLGA and drug loading on the long-term release profile of DM. The implants were characterized for solid-state active pharmaceutical ingredient (APIs) using DSC and SWAXS, water uptake during stability study, the crystal size of API in the implant matrix using hot-stage polarized light microscopy, and in vitro release profile. The kinetics of PLGA release was thoroughly investigated using quantitative 1H NMR spectroscopy. The polymorph of DM crystal was found to remain unchanged after the extrusion and stability study. However, around 3 times reduction in API particle size was observed after the HME process. The morphology and content uniformity of the RT-stored samples were found to be comparable to the initial implant samples. Interestingly, the samples (mainly 502H) stored at 40 °C and 75% RH for 30 d demonstrated marked deformation and a change in content uniformity. The rate of DM release was higher in the case of 502H samples with a higher drug loading (40% w/w). Furthermore, a simple digital in vitro DM release profile derived for the formulation containing a 3:1 ratio of 502H and 502 was comparable with the experimental release profile of the respective polymer mixture formulation. The temporal development of pores and/or voids in the course of drug dissolution, evaluated using μCT, was found to be a precursor for the PLGA release. Overall, the release profile of DM was found to be dependent on the PLGA type (independent of subtle changes in the formulation mass and diameter). However, the extent of release was found to be dependent on DM loading. Thus, the present investigation led to a thorough understanding of the physicochemical properties of different end-capped PLGAs and the underlying formulation microstructure on the release profile of a crystalline water-insoluble drug, DM, from the PLGA-based implant.

Head-to-Head Comparison of High-Performance Liquid Chromatography versus Nuclear Magnetic Resonance for the Quantitative Analysis of Carbohydrates in Yiqi Fumai Lyophilized Injection

Carbohydrate analysis can be used as a standard analysis for quality control of industries of plants, foods and pharmaceuticals. Quantitative 1H NMR spectroscopy (qNMR) is an excellent alternative to chromatography-based mixture analysis. However, the application of qNMR in sugar analysis has rarely been reported. In this study, the performance of qNMR in sugar analysis was investigated and compared with the results from HPLC analysis. A head-to-head comparison of qNMR (internal and external standard methods) versus HPLC (PMP pre-column derivatization HPLC, HPLC-RID and HPLC-ELSD) based on quantitative analysis of four carbohydrates (fructose, glucose, sucrose and maltose) in Yiqi Fumai lyophilized injection (YQFM) is presented. Both assays showed similar performance characteristics, including linearity range, accuracy, precision and recovery, and analysis times of less than 30 min/sample. After methodological validation, both qNMR and HPLC have good accuracy, precision and stability. Indeed, the qNMR method is simple, sensitive and rapid in quantifying the four sugars. By analysis of variance (ANOVA) for sugar content with HPLC and qNMR methods, we demonstrated that the two analytical methods had no significant difference and could be used interchangeably for the quantitative analysis of carbohydrates.

Investigation of Partial Oxidation of Methane in a Cold Plasma Reactor with Detailed Product Analysis

Cold plasma is a partially ionized state of matter that unites high reactivity and mild conditions. Therefore, cold plasma reactors are intriguing for reaction engineering. In this work, a laboratory scale dielectric barrier discharge (DBD) cold plasma reactor was designed, set up, and used for studying the influence of the specific energy input (SEI) on the product spectrum of the partial oxidation of methane. In total, 23 experiments were carried out near ambient conditions with a molar reactant ratio of methane to oxygen of 2:1 at SEI between 0.3 and 6.0 J cm−3. The feed also contained argon at a mole fraction of 0.75 mol mol−1. The product stream was split into a fraction that was condensed in a cold trap and the remaining gaseous fraction. The latter was analyzed at-line in a gas chromatograph equipped with a dual column and two carrier gases. The condensed fraction was analyzed by qualitative and quantitative 1H and 13C NMR spectroscopy, Karl Fischer titration, and sodium sulfite titration. In the product stream, 16 components were identified and quantified: acetic acid, acetone, carbon dioxide, carbon monoxide, ethanol, ethane, ethene, ethylene glycol, formaldehyde, formic acid, hydrogen, methanol, methyl acetate, methyl hydroperoxide, methyl formate, and water. A univariant influence of the SEI on the conversions of methane and oxygen and the selectivities to the products was observed. The experimental results provided here are an asset for developing reaction kinetic models of the partial oxidation of methane in DBD plasma reactors.

Quantitative 1HNMR Spectroscopy: Analysis of Zinc Gluconate in Utozinc® Tablets, a Mixture of Zinc Gluconate and Vitamin C.

Zinc is an essential metal for humans and plays key roles in several biological events such as immunity, allergy, growth, and inflammation. The deficiency in zinc causes an increased infection rate with pathogens. Organo-zincates such as zinc gluconate are known for better absorption compared with their inorganic zinc salts. Its role in enhancing the immune system has driven a huge demand for organo-zinc supplements and in the treatment protocol of coronavirus disease, the causative agent of the COVID-19 pandemic. Herein, we report on a quantitative analysis of zinc gluconate in the authentic form in presence of vitamin C, and the method was applied to their dosage form (Utozinc® tablets). The method is simple, accurate, and validated according to ICH guidelines. Quantification of zinc gluconate formulated with vitamin C (Utozinc tablets) using Q-1HNMR. Maleic acid and deuterium oxide were used as internal standards and solvents, respectively. The linearity range, the limit of detection and quantification, stability, precision, and accuracy, were validated. The validation of the method within five concentration levels (from 10 to 50 mg/0.5 mL D2O) afforded a limit of detection of 4.58 mg/mL, a quantification limit of 15.27 mg/mL, and excellent linearity. The method proposed in the present study is simple, fast, nondestructive, and accurate. Zinc gluconate quantification values obtained by the Q-1HNMR method were found to show an acceptable correlation with those obtained by the thin-layer chromatographic technique. The method was successfully applied to Utozinc tablets, and the results were compared with the reported reference pharmacopeial method. The salt exchange between maleic acid (IS) and zinc gluconate was tested by noticing the change in the chemical shift of IS and zinc gluconate.

Simultaneous Determination of the Saponification Value, Acid Value, Ester Value, and Iodine Value in Commercially Available Red Fruit Oil (Pandanus conoideus, Lam.) Using 1H qNMR Spectroscopy

Red fruit oil (RFO) can be extracted from fruits of Pandanus conoideus, Lam., an endogenous plant of Papua, Indonesia. It is a commonly used essential original traditional medicine. By applying a newly developed quantitative 1H NMR (qNMR) spectroscopy method for quality assessment, a simultaneous determination of the saponification value (SV), acid value (AV), ester value (EV), and iodine value (IV) in RFO was possible. Dimethyl sulfone (DMSO2) was used as an internal standard. Optimization of NMR parameters, such as NMR pulse sequence, relaxation delay time, and receiver gain, finally established the 1H NMR-based quantification approach. Diagnostic signals of the internal standard at δ = 2.98 ppm, SV at δ = 2.37–2.20 ppm, AV at δ = 2.27–2.20 ppm, EV at δ = 2.37–2.27 ppm, and IV at δ = 5.37–5.27 ppm, respectively, were used for quantitative analysis. The method was validated concerning linearity (R2 = 0.999), precision (less than 0.83%), and repeatability in the range 99.17–101.17%. Furthermore, this method was successfully applied to crude RFO, crude RFO with palmitic and oleic acid addition, and nine commercial products. The qNMR results for the respective fat values are in accordance with the results of standard methods, as can be seen from the F- and t-test (< 1.65 and < 1.66, respectively). The fundamental advantages of qNMR, such as its rapidity and simplicity, make it a feasible and existing alternative to titration for the quality control of RFO.

1H NMR spectrometry for methanol quantification in apple wines and ciders as optimised by comparison to SIDA-HS-GC-MS

Methanol, a hepato- and neurotoxic compound, is present in apple-based beverages as a by-product of the enzymatic degradation of pectin. Stable isotope dilution assay (SIDA) employing gas chromatography-mass spectrometry (GC–MS) is the state-of-the-art method for methanol determination in beverages. Despite higher initial investment costs, quantitative 1H NMR spectroscopy (qNMR) is a simpler and faster analytical technique to quantify numerous analytes in liquid foods. Beyond targeted analyses, qNMR spectral fingerprints in the product might be used for non-targeted analytical goals, such as adulteration and contamination detections. Here, an existing 1H-qNMR method used for wine profiling was optimised for methanol quantification in apple-based products, including cross-validation against a SIDA-HS-GC–MS method and reference values from interlaboratory trials. The optimisation involved a pivotally important estimation of a correction factor by an external calibration approach, making qNMR results comparable to SIDA-HS-GC–MS. The optimised qNMR method is suggested to be an alternative for methanol quantification in beverages.

Investigation of red clover (Trifolium pratense) isoflavonoid residual complexity by off-line CCS-qHNMR

The importance of Trifolium pratense L. as a dietary supplement and its use in traditional medicine prompted the preparation of a thorough metabolite profile. This included the identification and quantitation of principal constituents as well as low abundant metabolites that constitute the residual complexity (RC) of T. pratense bioactives. The purity and RC of isoflavonoid fractions from standardized red clover extract (RCE) was determined using an off-line combination of countercurrent separation (CCS) and two orthogonal analytical methodologies: quantitative 1H NMR spectroscopy with external calibration (EC-qHNMR) and LC-MS. A single-step hydrostatic CCS methodology (Centrifugal Partition Chromatography [CPC]) was developed that fractionated the isoflavonoids with a hexanes-ethyl acetate-methanol-water (HEMWat) 5.5/4.5/5/5, v/v solvent system (SS) into 75 fractions containing 3 flavonolignans, 2 isoflavonoid glycosides, as well as 17 isoflavonoids and related compounds. All metabolites were identified and quantified by qHNMR spectroscopy. The data led to the creation of a complete isoflavonoid profile to complement the biological evaluation. For example, fraction 69 afforded 90.5% w/w biochanin A (17), with 0.33% w/w of prunetin (16), and 0.76% w/w of maackiain (15) as residual components. Fraction 27 with 89.4% w/w formononetin (13) as the major component had, in addition, a residual complexity consisting of 3.37%, 0.73%, 0.68% w/w of pseudobaptigenin (11), kaempferol (10) and pratensein (8), respectively. Despite the relatively high resolving power of CPC, and not unexpectedly, the chromatographic fractions retained varying degrees of the original metabolomic diversity. Collectively, the extent of metabolomic diversity should be recognized and used to guide the development of isolation strategies, especially when generating samples for bioactivity evaluation. The simultaneous structural and quantitative characterization enabled by qNMR, supported by LC-MS measurements, enables the evaluation of a relatively large number of individual fractions and, thereby, advances both the chemical and biological evaluation of active principles in complex natural products.

Coumarins, psoralens, and quantitative 1H-NMR spectroscopy for authentication of lemon (Citrus limon [L.] Burm.f.) and Persian lime (Citrus × latifolia [Yu.Tanaka] Tanaka) juices

Mutual adulterations of lemon and lime juices may be detected using coumarins and psoralens as markers. Poor manufacturing practices or legal but mechanically intense processing of lemons were recently suspected to lead to false accusations of deliberate adulterations with lime juices due to potentially unspecific markers. Therefore, we studied coumarin and psoralen profiles in carefully dissected flavedo, albedo, and endocarp of lime and lemon as well as in juices produced under variable mechanical stresses at laboratory and pilot plant scale. Although the marker herniarin was detectable in juices from lime and harshly extracted lemons at low levels, isopimpinellin, bergapten and the herein proposed, tentatively assigned 5-geranyloxy-8-methoxypsoralen represented unambiguously lime-specific markers. Coumarin and psoralen data also allowed differentiating juices produced at differing degrees of mechanical stress. The latter was also possible using quantitative 1H-NMR spectroscopy, which yielded best results when combined with HPLC data on coumarins and psoralens. In the future, the reported approach may be used for establishing a robust database prior to being used in industrial practice.

A Rapid 1H NMR-Based Estimation of PONA for Light and Narrow Cut Naphtha Samples of Refinery Streams toward BS-VI Gasoline

A rapid method based on quantitative 1H NMR spectroscopy for the accurate estimation of paraffins (P), olefins (O), naphthenes (N), and aromatics (A), i.e., PONA, in extremely light to middle cut n...

Characterisation of an algerian asphaltene by 1H and 13C CPMAS NMR, DEPT and ESI mass spectroscopy

Quantitative liquid 1H and solid 13C CPMAS NMR spectroscopy were used in combination with qualitative DEPT (Distortionless Enhancement by Polarization Transfer) and liquid 13C NMR to investigate the molecular structural parameters of an Algerian asphaltenes. The molecular weight of the asphaltene ionisable species determined by positive Electro Spray Ionization mass spectrometry ESI was around 390 Da. The results indicate that the representative molecular structure of the asphaltenes contains PAH (Poly-aromatic hydrocarbon) core of 4–5 rings. The combination of 13C NMR/DEPT constitutes a good route for characterizing the chemical functions of asphaltenes.

Peptide-Conjugated Ultrasmall Gold Nanoparticles (2 nm) for Selective Protein Targeting

Ultrasmall gold nanoparticles with a metallic core diameter of 2 nm were surface-conjugated with peptides that selectively target epitopes on the surface of the WW domain of the model protein hPin1 (hPin1-WW). The binding to the gold surface was accomplished via the thiol group of a terminal cysteine. The particles were analyzed by NMR spectroscopy, high-resolution transmission electron microscopy, and differential centrifugal sedimentation. The surface loading was determined by conjugating a FAM-labeled peptide, followed by UV–vis spectroscopy, and by quantitative 1H NMR spectroscopy, showing about 150 peptide molecules conjugated to each nanoparticle. The interaction between the peptide-decorated nanoparticles with hPin1-WW was probed by 1H–15N-HSQC NMR titration, fluorescence polarization spectroscopy (FP), and isothermal titration calorimetry (ITC). The particles showed a similar binding (KD = 10–20 μM) compared to the dissolved peptides (KD = 10–30 μM). Small-angle X-ray scattering (SAXS) showed that the particles were well dispersed and did not agglomerate after the addition of hPin1-WW (no cross-linking by the protein). Each nanoparticle was able to bind about 20 hPin1-WW protein molecules. An unspecific interaction with hPin1 was excluded by the attachment of a nonbinding peptide to the nanoparticle surface. The uptake by cells was studied by confocal laser scanning microscopy. The peptide-functionalized nanoparticles penetrated the cell membrane and were located in the cytosol. In contrast, the dissolved peptide did not cross the cell membrane. Peptide-functionalized nanoparticles are promising agents to target proteins inside cells.

Influence of species and processing techniques on phlorin in Citrus juices as quantified by 1H-NMR spectroscopy

Phlorin (3,5-dihydroxyphenyl-β-d-glucopyranoside) has been discussed for a long time to be useful for the authentication of Citrus juices as marker for fraudulent processing techniques. In this study, quantitative 1H-NMR spectroscopy was used to obtain extensive data on phlorin levels in 3866 authentic Citrus juice and juice concentrate samples as well as in 159 peel extracts and whole-processed fruits as obtained from worldwide manufacturers. First, significant species-related differences were found between the phlorin levels in orange, blood orange, lemon, mandarin, as well as red and white grapefruit products. Furthermore, the used juice extraction machinery types were associated with significantly different phlorin intakes. For instance, juices of orange and blood orange made with reamer-type extractors were generally characterized by lower phlorin levels (15 ± 5 and 16 ± 8 mg/L, resp.) than those produced with squeezer-type extractors (21 ± 10 and 38 ± 15 mg/L, resp.). Part of the presented data has been used earlier to support establishing maximum levels of phlorin in the Reference Guidelines of the AIJN Code of Practice for the aforementioned Citrus fruit juices.