Spectroscopic Analysis Methods Research Articles

Abraham solvation parameter model: determination of experiment-based solute descriptor values for 3,5-dimethoxybenzoic acid based on measured solubility data

ABSTRACT A spectroscopic method of chemical analysis is used to determine the mole fraction solubilities of 3,5-dimethoxybenzoic acid dissolved in propanenitrile, in eleven additional alkanol solvents (1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 2-methyl-1-propanol, 2-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 4-methyl-2-pentanol, 2-ethyl-1-hexanol, cyclopentanol) and in three additional alkyl alkanoate solvents (isopropyl acetate, tert-butyl acetate, methyl butyrate) at 298.15 K. Results from our experimental measurements, combined with published partition coefficient and solubility data, are used to calculate the Abraham model solute descriptors of the monomeric form of 3,5-dimethoxybenzoic acid. In total 62 experimental data points were used in the computation. The calculated solute descriptors describe the experimental solubility and partition coefficient data to within an overall standard deviation of 0.064 log units.

Characterization of long-term aged bitumen with FTIR spectroscopy and multivariate analysis methods

One of the most common techniques used to investigate bitumen aging from a chemical perspective is Fourier-Transformation Infrared (FTIR) spectroscopy. However, the majority of standard spectral interpretation simply covers the observation of single bands associated with carbonyls, sulfoxides or aromatic structures. A possibility for a more comprehensive post-processing of FTIR data is to apply multivariate analysis (MVA) methods. Although, MVA methods have already been used for classifying differently aged bitumen in the past, the deployed methods only included standard laboratory aging and failed to consider factors like reactive oxygen species or light. The purpose of this study was the investigation of aged bitumen samples with FTIR spectroscopy and subsequent data processing with MVA. Therefore, nine bituminous binders were subjected to aging with the Pressure Aging Vessel (PAV), Viennese Binder Aging (VBA) and light, followed by a characterization with FTIR spectroscopy and various MVA methods. It was possible to investigate the general structure of the acquired data and differentiate between the aging methods solely based on FTIR spectra. Furthermore, important wavenumbers for the classification were identified, including spectral features around the wavenumbers 3371, 3027, 2943, 1550 and 1324 and 848 cm−1 for the original spectra. This study shows the potential of MVA to maximize the information content gained by experimental analysis and for the development of fast and easy characterization methods.

Metabolomics analysis reveals the effect of removing cotyledons on salt tolerance in castor plant roots during early seedling establishment

Castor is an important oil crop with moderate salt tolerance that is grown worldwide. The early seedling stage is the most vulnerable phase during a plant’s lifecycle in a saline environment. Cotyledons play an important role in seedling establishment as the storage and first photosynthetic organ, and in the resistance of the seedling to salt stress. However, how cotyledons affect the metabolic response of castor roots in saline environments is unclear. Here, liquid chromatography-mass spectrometry analytical methods were used to examine the effects of removing cotyledons (no cotyledons removed, NR; one cotyledon removed, OR; both cotyledons removed, TR) on the metabolite changes in the roots under salt stress. The results showed that removing cotyledons inhibited the growth of TR roots much greater than NR or OR roots. Metabolomics analysis showed that the roots of TR seedlings had fewer metabolites involved in the salt stress response than NR seedlings. Moreover, the KEGG pathway enrichment analysis demonstrated that salt-induced metabolites were associated with major biological pathways related to tropane, piperidine, and pyridine alkaloid biosynthesis; amino acid biosynthesis; phenylpropanoid biosynthesis; amino sugar and nucleotide sugar metabolism, as well as arginine and proline metabolism. However, the contents of these metabolites decreased or did not change significantly in the TR roots compared to the NR roots under salt stress. Additionally, the contents of caffeyl alcohol, scopoletin, and syringin in the phenylpropanoid biosynthetic pathway increased significantly in the roots of TR seedlings under salt stress compared to the control treatment. Therefore, we concluded that castor plants with cotyledons enhanced particular metabolic pathways in the roots that protected cell membrane structure, maintained cell osmotic potential, resisted reactive oxygen species and provided energy under salt stress. In addition, the roots of castor plants without cotyledons may be resistant to salt stress by enhancing phenylpropanoid biosynthesis to maintain cell differentiation and enhance root lignification. Taken together, these findings provide new insight into the physiological functions of cotyledons in the aboveground-belowground interactions of castor plants under salt stress.

Real-Time Phytoncide Monitoring in Forests: A Comparative Study of SIFT-MS and Conventional GC-MS Methods

Conventional gas chromatography-mass spectrometry (GC-MS) analysis methods for measuring the concentration of phytoncides in forests are limited because of the need for an extended human presence in forests, the risk of errors, and contamination. To overcome these issues, this study introduces real-time measurement devices and selected ion flow tube mass spectrometry (SIFT-MS) as potential replacements. This study was conducted in the Hongneung Experimental Forest between 19 and 21 November 2019. A correlation analysis and independent samples t-test were performed to compare the GC-MS and SIFT-MS techniques. The diurnal patterns and trends in the phytoncide concentrations analyzed using the GC-MS and SIFT-MS methods were similar, suggesting the potential replacement of GC-MS with SIFT-MS. While both methods revealed similar major components in the daytime nonvolatile organic compounds (NVOCs), with pinenes comprising approximately half of the total percentage, the 24 h SIFT-MS analysis indicated reduced proportions of pinenes and benzaldehyde, along with the detection of more diverse NVOC compounds at night. Additionally, the studies indicated that GC-MS exhibited slightly higher selectivity, resulting in the detection of fewer NVOC compounds with SIFT-MS. The correlation analysis between the microclimate indicators and phytoncide measurement methods revealed differences: GC-MS with a mini pump showed positive correlations with fine dust and industrially derived VOCs, while the 24 h real-time measurements exhibited strong negative correlations. Consequently, while the GC-MS and SIFT-MS methods exhibited both similarities and differences in phytoncide concentrations, an independent samples t-test, confirming no statistically significant differences between the two methods, suggests the suitability of adopting SIFT-MS over GC-MS for phytoncide collection and analysis in forest environments. Nevertheless, this study contributes to the literature by comparing outdoor phytoncide levels using the GC-MS and SIFT-MS methodologies. These findings, which show that the methods are closely aligned, can guide future researchers in considering SIFT-MS equipment for phytoncide studies, offering a more accessible and efficient option with real-time capabilities.

Machine Learning Applied to Predict Key Petroleum Crude Oil Constituents

AbstractSulfur compounds are the most important inorganic constituents of petroleum and require to be estimated beforehand because of their corrosive nature and other processing anomalies during crude oil processing. Paraffins, naphthene, and aromatics form the bulk of crude oil. Machine learning (ML) predictions of these constituents were made by training the ML model with a diverse industrial data set of 515 oils. The XGBoost model gave an excellent R2 in the range 0.88–0.99 for the bulk compounds. R2 for sulfur was in the modest range of 0.45–0.6, which improved significantly to 0.8 for additional inputs. ML applicability was thereby found to depend on the nature of the constituent. This work furthers ML‐based predictions, with the incentive of reducing expensive spectroscopic analytical methods.

Ternifoliasaponin, a new triterpenoid saponin from the roots of Gardenia ternifolia Schumach & Thonn (Rubiaceae)

A new triterpenoid saponin, Ternifoliasaponin (1), together with four known compounds (2–5) chikusetsusaponin IVa (2), chikusetsusaponin IVa methyl ester (3), bonushenricoside B (4) and Dianoside C (5) were isolated from roots of Gardenia ternifolia Schumach. & Thonn (Rubiaceae). The structures of isolated compounds were elucidated on the basis of spectroscopic analysis and chemical methods. The antibacterial activities of compounds (3), and (4) were performed by the Muller–Hinton agar diffusion method. The antimicrobial activities of the compounds were studied on Salmonella typhi (Enterobacteriaceae), Staphylococcus aureus and Pseudomonas aeruginosa microorganisms. Compound (3) at 25 mg/mL, showed moderately sensitive effect (8.0 ˂ DIZ ˂14.0 mm) on S. typhi, S. aureus and P. aeruginosa. Compound (4) at 25 mg/mL and compound (3) at 12.5 mg/mL exhibited moderately sensitive effect on S. typhi and S. aureus. Compound (4) inhibited moderately sensitive the S. typhi and P. aeruginosa colonies at 12.5 mg/mL.

Synthesis of palladium complexes containing benzimidazole core and their catalytic activities in direct C[sbnd]H functionalization of five-membered heterocyclic bioactive motifs

Herein, we reported the synthesis of nine new PEPPSI-Pd(II)–N-heterocyclic carbene (NHC) complexes 3a-h containing benzimidazole core and examined their catalytic activities in the direct arylation of heteroaromatic species with aryl bromides and arylchlorides. Nine new Pd benzimidazole complexes were synthesized and their structures were elucidated by 1H and 13C NMR spectroscopy, FT-IR spectroscopy, and elemental analysis methods. In the 13C NMR spectra, NCHN peaks of Pd(II) complexes were observed between 159.7–161.5 ppm. Further, direct arylation reaction, using KOAc as base and DMAc as solvent under inert conditions, were carried out with the synthesized complexes in the presence of various aryl bromides and chlorides, and 2-acetyl furan, and furfuryl acetate 2-acetylthiophene and N-methylpyrrole-2- carboxaldehyde. Moreover, moderate to hig yields were obtained in the direct arylation of heteroarenes and aryl bromides under 1 mol% catalyst loading at 150 °C for 2 h loading conditions. The results showed that electron-donating and electron-withdrawing substituents on the aryl halides might produce coupling products in good yields in the presence of complexes 2a-d. Taken together, our findings are expected to contribute to the synthesis and design of palladium complexes containing benzimidazole core structures.

Classification of Vietnamese Cashew Nut (Anacardium occidentale L.) Products Using Statistical Algorithms Based on ICP/MS Data: A Study of Food Categorization

Fingerprinting techniques, which utilize the unique chemical and physical properties of food samples, have emerged as a promising approach for food authentication and traceability. Recent studies have demonstrated significant advancements in food authentication through the use of fingerprinting methods, such as multivariate statistical analysis techniques applied to trace elements and isotope ratios. However, further research is required to optimize these methods and ensure their validity and reliability in real-world applications. In this study, the inductively coupled plasma mass spectrometry (ICP-MS) analytical method was employed to determine the content of 21 elements in 300 cashew nut (Anacardium occidentale L.) samples from 5 brands. Multivariate statistical methods, such as principal components analysis (PCA), were employed to analyze the data obtained and establish the provenance of the cashew nuts. While cashew nuts are widely marketed in many countries, no universal method has been utilized to differentiate the origin of these nuts. Our study represents the initial step in identifying the geographical origin of commercial cashew nuts marketed in Vietnam. The analysis showed significant differences in the means of 21 of the 40 analyzed elements among the cashew nut samples from the 5 brands, including 7Li, 11B, 24Mg, 27Al, 44Ca, 48Ti, 51V, 52Cr, 55Mn, 57Fe, 60Ni, 63Cu, 66Zn, 93Nb, 98Mo, 111Cd, 115In, 121Sb, 138Ba, 208Pb, and 209Bi. The PCA analysis indicated that the cashew nut samples can be accurately classified according to their original locations. This research serves as a prerequisite for future studies involving the combination of elemental composition analysis with statistical classification methods for the accurate establishment of cashew nut provenance, which involves the identification of key markers for the original discrimination of cashew nuts.

Neurotrophic phenolic glycosides from the roots of Armoracia rusticana

Armoracia rusticana P. G. Gaertner. belongs to the Brassicaceae family and has aroused scientific interest for its anti-inflammatory and anticancer activities. In a continuing investigation to discover bioactive constituents from A. rusticana, we isolated 19 phenolic glycosides including three undescribed flavonol glycosides and one undescribed neolignan glycoside from MeOH extract of this plant. Their structures were elucidated based on NMR spectroscopic analysis (1H, 13C, 1H–1H COSY, HSQC, and HMBC), HRESIMS, and chemical methods. The determination of their absolute configuration was accomplished by ECD and LC-MS analysis. All the compounds were assessed for their potential neurotrophic activity through induction of nerve growth factor in C6 glioma cell lines and for their anti-neuroinflammatory activity based on the measurement of inhibition levels of nitric oxide production and pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) in lipopolysaccharide-activated microglia BV-2 cells.

Utilization of green ATR-FTIR spectroscopic method for quantitative analysis of Ibuprofen tablets

Aim: The aim of this study is to propose a green and nondestructive method using the ATR-FTIR method for the quantitative analysis of Ibuprofen tablet dosage forms. Herein, the technique has been validated as an alternative green tool that evades necessary sample preparation procedures required in traditional quantitative methods. Methods: The method depends on selecting CO of the Ibuprofen stretching band in the range 1620–1750 cm-1 to quantitatively determine Ibuprofen in original samples. The pack area (AUC) from ATR-FTIR spectral scanning of samples has been determined through the first derivative measurements. Results: The assay results indicated that no interferences between the excipients or additives and the active ingredients of the commercial tablets interferences. The linearity is excellent within the concentration range of 0.2 to 1.5 w/w % (r = 0.9994). A percentage of recoveries ranged between 99.7–10.5 which are in good agreement with the pharmacopeial percent recovery standards. The high degree of sensitivity of the technique was demonstrated by obtaining a 0.028 w/w % detection limit and a 0.1599 w/w % limit of quantification values.

Interactions of Clotrimazole with Certain d-Metal Compounds and with Organic Acids

During the interaction of aqueous-ethanol or ethanol solutions AgNO3, H[AuCl4], and CuCl2, as well as aqueous suspensions of slightly soluble copper(II) salts Cu(C6H4NO2)2·H2O, Cu(C7H5O2)2∙3H2O, and CuC7H4O3∙H2O with the ethanol solution of clotrimazole at pH of ~(5.0–5.5), the [Ag(C22H17ClN2)2]NO3·2H2O, [Au(C22H17ClN2)Cl3], [Cu(C22H17ClN2)2Cl2]·5H2O, Cu(C22H17ClN2)4(C6H4NO2)2, Cu(C22H17ClN2)4(C7H5O2)2, and Cu(C22H17ClN2)3(C7H4O3)·2H2O compounds are synthesised. They are characterised by elemental, thermal, thermogravimetric, and IR spectroscopic methods of analysis. The [Ag(C22H17ClN2)2]NO3·2H2O complex was shown to have a higher antimycotic activity against Saccharomyces cerevisiae fungi than that of AgNO3 and C22H17ClN2. Cocrystals/salts of the composition C22H17ClN2·C6H5NO2, C22H17ClN2·C7H6O2, 2C22H17ClN2·C7H6O3, and 2C22H17ClN2·C19H19O6N7·H2O are obtained from aqueous and aqueous ethanol suspensions containing nicotinic, benzoic, salicylic, and folic acids and clotrimazole (pH is 4.5–6.0). These cocrystals and salts were studied usin thermogravimetric, IR-spectroscopic methods. Diffraction patterns of the powders were obtained. The influence of the difference in the pKa components on the ability to form cocrystals/salts was assessed.

Establishment and optimization of the three-band fluorometric indices for oil species identification: Implications on the optimal excitation wavelengths and the detection band combinations

BackgroundThe oil pollutants have become a major threat to the ocean environment with the rapid development of social economy and offshore production activities. The detection and identification of petroleum pollutants is the foundation and pre-requisite for controlling offshore oil pollution. Spectroscopic analysis using the ultraviolet–induced fluorescence of oil pollutants is a potential way for oil species identification. Nevertheless, the current fluorescence spectroscopic analysis methods, such as excitation-emission matrix, has high requirements on the detection equipment. There is a great need for rapid oil species identification method when it comes to practical applications. (93) ResultsThis study established the three-band fluorometric index (TBFI) method and examined its capability on the oil species identification. The fluorescence of five different types of oil samples under various excitation wavelengths were excited using a tuneable xenon lamp, and measured using a high-resolution spectrometer. The optimal excitation wavelengths and the corresponding detection band combinations were explored through the iterations of an optimal band combination algorithm through different excitation wavelengths. According to the results, three out of the four TBFIs tested in this study are suitable for oil species identification. They share the same sets of the excitation wavelengths under 365 nm, 375 nm, and 435 nm. The detection band combinations corresponding to each TBFI under each feasible excitation wavelength were also determined in this study. (125) SignificanceThe three-band fluorometric index method proposed in this study provides a feasible way for the rapid oil species identification using fluorescence spectroscopic analysis. The optimal excitation and detection wavelengths determined in this study also provide the theoretical basis for the designs of fluorescence sensors for rapid oil spill detection and identification. (51).

Utilizing a Combination of Network Pharmacology and Experimental Validation to Unravel the Mechanism by Which Kuanxiongzhuyu Decoction Ameliorates Myocardial Infarction Damage.

Background and Objectives: With the growing incidence and disability associated with myocardial infarction (MI), there is an increasing focus on cardiac rehabilitation post-MI. Kuanxiongzhuyu decoction (KXZY), a traditional Chinese herbal formula, has been used in the rehabilitation of patients after MI. However, the chemical composition, protective effects, and underlying mechanism of KXZY remain unclear. Materials and Methods: In this study, the compounds in KXZY were identified using a high-performance liquid chromatography-mass spectrometry (HPLC-MS) analytical method. Based on the compounds identified in the KXZY, we predictively selected the potential targets of MI and then constructed a protein-protein interaction (PPI) network to identify the key targets. Furthermore, the DAVID database was used for the GO and KEGG analyses, and molecular docking was used to verify the key targets. Finally, the cardioprotective effects and mechanism of KXZY were investigated in post-MI mice. Results: A total of 193 chemical compounds of KXZY were identified by HPLC-MS. In total, 228 potential targets were obtained by the prediction analysis. The functional enrichment studies and PPI network showed that the targets were largely associated with AKT-pathway-related apoptosis. The molecular docking verified that isoguanosine and adenosine exhibited excellent binding to the AKT. In vivo, KXZY significantly alleviated cardiac dysfunction and suppressed AKT phosphorylation. Furthermore, KXZY significantly increased the expression of the antiapoptotic proteins Bcl-2 and Bcl-xl and decreased the expression of the proapoptotic protein BAD. Conclusions: In conclusion, the network pharmacological and experimental evidence suggests that KXZY manifests anti-cardiac dysfunction behavior by alleviating cardiomyocyte apoptosis via the AKT pathway in MI and, thus, holds promising therapeutic potential.

A-327 Pre-analytical and Analytical Modifications to Improve the Quality of Pediatric Lead Tests

Abstract Background The recent decrease in the CDC Blood Lead Reference Value (BLRV) to 3.5 ug/dL has highlighted the need for systematic changes that some clinicians and laboratories need to implement to assure the quality of reported pediatric lead results. Building upon the recommendations of the Blood Lead Reference Value Workgroup, APHL has developed specific recommendations and resources designed to assist medical professionals and laboratory scientists in minimizing the likelihood of contamination when collecting and testing blood specimens. Also included are analytical practices that can improve precision, accuracy, and sensitivity of pediatric lead assays. Methods Evaluation of existing protocols and practices to identify potential sources of contamination and opportunities for process improvement. Results APHL will provide practical recommendations to systematically improve point of care and fixed laboratory testing to improve confidence in reported pediatric lead concentrations. These improved measurements will improve environmental health surveillance and inform public health intervention and patient care. Conclusion Rigorous pre-analytical practices including education, updated specimen collection guidance, pre-screening of collection materials, improvement of point of care technology and refinement of spectroscopic analytical methods result in less ambient contamination, improved sensitivity, precision and accuracy of pediatric lead testing. Clinician office practices, public health and commercial clinical laboratories will all benefit from implementing these strategies.

Aroma Comparison between Plant-based Hamburgers and Traditional Beef Hamburgers

The aim of the study was to investigate the differences in aroma in the many plant-based hamburger analogues in the market today when compared with traditional beef hamburger. For that purpose, we investigated the aroma components using headspace gas chromatography-olfactometry and gas chromatography coupled to mass spectrometry analytical methods. The lipid-derived aldehydes and Maillard reaction compounds were key contributors to the characteristic aroma of a beef hamburger. And that most of the plant-based burgers in the market today were unable to replicate that profile. The plant-based meat analogues displayed a more roasted odor character with higher pyrazine levels and then many show evidence to the addition of other flavor compounds and spices to give the perception of a flavored meat analogue. These approaches resulted in higher flavor intensity; however, the result was something very sensorially different than the sweet, juicy, buttery, and meaty character of beef hamburger. Based on these findings, replication of the characteristic aroma of beef hamburger is complex. This work provides new insights into the key aroma contributors of beef hamburgers and the challenges that exist in trying to replicate it with plant-based meat analogues.

Optical Differentiation of Brain Tumors Based on Raman Spectroscopy and Cluster Analysis Methods.

In the present study, various combinations of dimensionality reduction methods with data clustering methods for the analysis of biopsy samples of intracranial tumors were investigated. Fresh biopsies of intracranial tumors were studied in the Laboratory of Neurosurgical Anatomy and Preservation of Biological Materials of N.N. Burdenko Neurosurgery Medical Center no later than 4 h after surgery. The spectra of Protoporphyrin IX (Pp IX) fluorescence, diffuse reflectance (DR) and Raman scattering (RS) of biopsy samples were recorded. Diffuse reflectance studies were carried out using a white light source in the visible region. Raman scattering spectra were obtained using a 785 nm laser. Patients diagnosed with meningioma, glioblastoma, oligodendroglioma, and astrocytoma were studied. We used the cluster analysis method to detect natural clusters in the data sample presented in the feature space formed based on the spectrum analysis. For data analysis, four clustering algorithms with eight dimensionality reduction algorithms were considered.

Calcination and ion substitution improve physicochemical and biological properties of nanohydroxyapatite for bone tissue engineering applications

Nanohydroxyapatite (nanoHAP) is widely used in bone regeneration, but there is a need to enhance its properties to provide stimuli for cell commitment and osteoconduction. This study examines the effect of calcination at 1200 °C on the physicochemical and biological properties of nanoHAP doped with magnesium (Mg2+), strontium (Sr2+), and zinc (Zn2+). A synergistic effect of dual modification on nanoHAP biological properties was investigated. The materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), BET analysis, Fourier-transform spectroscopy, and thermal analysis methods. Furthermore, ion release tests and in vitro biological characterization, including cytocompatibility, reactive oxygen species production, osteoconductive potential and cell proliferation, were performed. The XRD results indicate that the ion substitution of nanoHAP has no effect on the apatite structure, and after calcination, β-tricalcium phosphate (β-TCP) is formed as an additional phase. SEM analysis showed that calcination induces the agglomeration of particles and changes in surface morphology. A decrease in the specific surface area and in the ion release rate was observed. Combining calcination and nanoHAP ion modification is beneficial for cell proliferation and osteoblast response and provide additional stimuli for cell commitment in bone regeneration.

Spice and Herb Frauds: Types, Incidence, and Detection: The State of the Art.

There is a necessity to protect the quality and authenticity of herbs and spices because of the increase in the fraud and adulteration incidence during the last 30 years. There are several aspects that make herbs and spices quite vulnerable to fraud and adulteration, including their positive and desirable sensorial and health-related properties, the form in which they are sold, which is mostly powdered, and their economic relevance around the world, even in developing countries. For these reasons, sensitive, rapid, and reliable techniques are needed to verify the authenticity of these agri-food products and implement effective adulteration prevention measures. This review highlights why spices and herbs are highly valued ingredients, their economic importance, and the official quality schemes to protect their quality and authenticity. In addition to this, the type of frauds that can take place with spices and herbs have been disclosed, and the fraud incidence and an overview of scientific articles related to fraud and adulteration based on the Rapid Alert System Feed and Food (RASFF) and the Web of Science databases, respectively, during the last 30 years, is carried out here. Next, the methods used to detect adulterants in spices and herbs are reviewed, with DNA-based techniques and mainly spectroscopy and image analysis methods being the most recommended. Finally, the available adulteration prevention measurements for spices and herbs are presented, and future perspectives are also discussed.

Spectroscopic Methods for Pollution Analysis─Course Development and Delivery Using the Integrated Course Design Framework

Spectroscopic Methods for Pollution Analysis─Course Development and Delivery Using the Integrated Course Design Framework

A Research Journey: Over a Decade of Denaturing and Native-MS Analyses of Hydrophobic and Membrane Proteins in Amgen Therapeutic Discovery.

Membrane proteins and associated complexes currently comprise the majority of therapeutic targets and remain among the most challenging classes of proteins for analytical characterization. Through long-term strategic collaborations forged between industrial and academic research groups, there has been tremendous progress in advancing membrane protein mass spectrometry (MS) analytical methods and their concomitant application to Amgen therapeutic project progression. Herein, I will describe a detailed and personal account of how electrospray ionization (ESI) native mass spectrometry (nMS), ion mobility-MS (IM-MS), reversed phase liquid chromatographic mass spectrometry (RPLC-MS), high-throughput solid phase extraction mass spectrometry, and matrix-assisted laser desorption ionization mass spectrometry methods were developed, optimized, and validated within Amgen Research, and importantly, how these analytical methods were applied for membrane and hydrophobic protein analyses and ultimately therapeutic project support and progression. Additionally, I will discuss all the highly important and productive collaborative efforts, both internal Amgen and external academic, which were key in generating the samples, methods, and associated data described herein. I will also describe some early and previously unpublished nano-ESI (nESI) native-MS data from Amgen Research and the highly productive University of California Los Angeles (UCLA) collaboration. I will also present previously unpublished examples of real-life Amgen biotherapeutic membrane protein projects that were supported by all the MS (and IM) analytical techniques described herein. I will start by describing the initial nESI nMS experiments performed at Amgen in 2011 on empty nanodisc molecules, using a quadrupole time-of-flight MS, and how these experiments progressed on to the 15 Tesla Fourier transform ion cyclotron resonance MS at UCLA. Then described are monomeric and multimeric membrane protein data acquired in both nESI nMS and tandem-MS modes, using multiple methods of ion activation, resulting in dramatic spectral simplification. Also described is how we investigated the far less established and less published subject, that is denaturing RPLC-MS analysis of membrane proteins, and how we developed a highly robust and reproducible RPLC-MS method capable of effective separation of membrane proteins differing in only the presence or absence of an N-terminal post translational modification. Also described is the evolution of the aforementioned RPLC-MS method into a high-throughput solid phase extraction MS method. Finally, I will give my opinion on key developments and how the area of nMS of membrane proteins needs to evolve to a state where it can be applied within the biopharmaceutical research environment for routine therapeutic project support.