Coffee quality is strongly influenced by key bioactive compounds such as trigonelline, 5-caffeoylquinic acid (5-CQA), and caffeine. This study optimized and validated a fast, accessible HPLC-UV method for their simultaneous quantification in five Coffea arabica cultivars. The method demonstrated excellent linearity, accuracy, precision, robustness, and suitable detection limits, meeting analytical validation criteria.Application of the method revealed notable differences among cultivars, including the lowest caffeine level in Laurina and variable degradation of trigonelline and 5-CQA after roasting. The Arara cultivar showed the greatest 5-CQA loss, while Laurina exhibited the highest trigonelline degradation. Due to its speed and simplicity, the method is well suited for investigating how these compounds influence sensory attributes and potential health benefits.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c05526">Optimization of Methodology for Simultaneous Quantification of Trigonelline, 5-Caffeoylquinic Acid, and Caffeine in Green and Roasted Coffee Extracts by HPLC</a></h4>Walace Breno da Silva*, Larissa Martins Rocha, Lucca Dornelas Guimarães Moura, Márcio Santos Soares, Sabrina Alves da Silva, Daniele Birck Moreira, Pedro Ivo Vieira Good God, Geraldo Humberto SilvaACS Omega 2025, 10, 35, 40304–40312<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.5c05526">https://doi.org/10.1021/acsomega.5c05526</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Various analytical techniques have been employed in the characterization of bioactive compounds and in the authentication of coffee, including ultraviolet (UV) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS). <a href="javascript:void(0);">(16)</a> Recent studies have demonstrated the feasibility of quantifying compounds such as sucrose, caffeine, and trigonelline in green coffee beans using advanced techniques such as hyperspectral imaging (HSI), although these approaches require sophisticated instrumentation and involve higher analytical complexity. <a href="javascript:void(0);">(17)</a> Simplified methodologies have been proposed for the determination of alkaloids and phenolic acids in coffee, such as the use of the QuEChERS method coupled with ultraviolet–visible (UV–vis) spectrophotometry for detecting trigonelline, caffeine, and 5-caffeoylquinic acid in green coffee bean extracts. <a href="javascript:void(0);">(18)</a> However, this approach presents limitations, as the spectral signals of caffeine and chlorogenic acids overlap in the 200–500 nm range, hindering their simultaneous quantification in simple aqueous extracts. <a href="javascript:void(0);">(19)</a> Therefore, UV–vis spectroscopy lacks adequate specificity in complex matrices, such as coffee infusions.From a chromatographic standpoint, HPLC-based methodologies have proven to be effective for the simultaneous quantification of these compounds. For instance, Santiago et al. (2020) developed a method for the simultaneous analysis of trigonelline, caffeine, and 5-caffeoylquinic acid with a total chromatographic runtime of 20 min. <a href="javascript:void(0);">(20)</a> Additionally, Mehari et al. (2015) reported the application of HPLC in the quantification of alkaloids in green coffee beans, including trigonelline, caffeine, theobromine, and theophylline. However, due to the naturally low concentrations of theobromine and theophylline in coffee beans, the methodology has limitations in simultaneously quantifying all target components in the coffee matrix. <a href="javascript:void(0);">(21)</a>Nevertheless, these methods often require elaborate sample preparation steps, such as extractions with organic solvents and filtration, which negatively impact their applicability in routine analyses and their sustainability profile. In this context, this study proposes a more straightforward and sustainable approach for the simultaneous analysis of trigonelline, 5-caffeoylquinic acid, and caffeine in extracts from both raw and roasted coffee beans. This method is based solely on the direct infusion of ground coffee beans with hot water, eliminating the use of organic solvents and reducing the sample preparation time. The analysis is performed using high-performance liquid chromatography with a diode array UV detector (HPLC-UV-DAD), with a short analysis time. Accurate determination of these compounds contributes to a better understanding of the chemical and sensory characteristics of coffee, facilitating the standardization and optimization of postharvest processes to enhance and ensure product quality, thereby adding value. Furthermore, proper method validation increases the reliability of results, enabling comparisons across different studies and laboratories. <a href="javascript:void(0);">(22)</a><h4>2. Materials and Methods</h4><h5>2.2. Equipment</h5>An <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+LC">Agilent 1260 HPLC liquid chromatograph</a> equipped with a G7111B 1260 Quat pump, a G7129A 1260 Vial sampler autosampler, and a G7117C 1260 DAD HS detector were used (Agilent, Santa Clara). The column used was a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1529">Luna Omega Polar C18 LC column</a> with an internal diameter of 4.6 mm, length of 150 mm, and particle size of 5 μm (Phenomenex, Aschaffenburg, Germany) connected to an Eclipse XDB-C18 precolumn with an internal diameter of 4.6 mm, length 12.5 mm, particle size 5 μm (Agilent, Santa Clara).<h4>3. Results and Discussion</h4><h5>3.1. Method Validation</h5>To check the quality and resolution of the chromatograms of the raw and roasted coffee extracts, the standard was added to the raw and roasted extracts, and the same concentration was added to both extracts (10 μg mL–1) in triplicate. An elution time of less than 5 min was observed for the analytes and good resolution of the chromatographic bands (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c05526#fig2">Figure 2</a>).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_35_40304_40312_Figure_2_Chromatogram_with_the_analytes_trigonelline_cade61c3cb.jpg" alt="ACS Omega 2025, 10, 35, 40304–40312: Figure 2. Chromatogram with the analytes trigonelline (band a), 5-CQA (band b), and caffeine (band c). Conditions: Luna Omega Polar C18 column (internal diameter 4.6 mm, length 150 mm, particle size 5 μm); mobile phase: water with 1% acetic acid: acetonitrile (85:15); flow rate 1 mL min–1; detection: UV 272 nm, roasted coffee (1) and raw coffee (2), k’ is the retention factor; N is the number of theoretical plates; S is the symmetry; Rs is the resolution, and Se is the selectivity." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_35_40304_40312_Figure_2_Chromatogram_with_the_analytes_trigonelline_cade61c3cb.jpg 148w," sizes="100vw" width="992" height="1051">The results for the theoretical plates were above the acceptance criteria of the Food and Drug Administration, <a href="javascript:void(0);">(28)</a> which states that they should be higher than 2000. The symmetry of the bands was above 0.65, which indicates that the characteristics of the compounds influence the symmetry. Better symmetry can be obtained by changing the chromatographic conditions, such as column pressure and mobile phase flow rate. <a href="javascript:void(0);">(29)</a> Despite the symmetry values being higher than 0.65, the evaluated accuracy and precision demonstrate that the method is reliable. Trigonelline had a k’ of less than 1, which shows its low affinity with the stationary phase. Resolution and selectivity were also affected by trigonelline’s poor interaction with the stationary phase, which was not the case with 5-CQA and caffeine.<h5>3.2. Application of the Method to Quantify Bioactive Compounds</h5>Validation of the method showed that the results were within established standards, confirming its suitability for quantification of bioactive compounds in coffee. This quantification helps us to study and understand how these compounds influence the sensory characteristics of the beverage. The method was then applied to measure three specific compounds in five varieties of Coffea arabica, each processed differently after harvest.The concentration of trigonelline in raw coffee beans varied between 13.38–15.24 mg g–1 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c05526#tbl8">Table 8</a>), while in roasted beans, it was between 10.68–12.75 mg g–1, values similar to those found by Mehari. <a href="javascript:void(0);">(21)</a> Trigonelline varies between 10 and 22 mg g–1 in raw beans and is generally 10 mg g–1 or less in roasted beans. <a href="javascript:void(0);">(32)</a> The Tukey test revealed significant variation in trigonelline content among the roasted coffee samples, with Yellow Catuai and Guesha exhibiting the highest concentrations. In the raw beans, Yellow Catuai and Arara stood out with lower trigonelline levels compared with the other cultivars.Caffeine concentrations ranged from 7.93 to 12.72 mg g–1 in raw and roasted coffee beans, similar to those found in the literature. <a href="javascript:void(0);">(21)</a> When comparing raw and roasted beans of the same variety, it was observed that there was a significant variation in concentration only in Guesha beans. The lack of significant variation between the other four varieties is because caffeine belongs to a class of compounds called methylxanthines, which is considered a natural ergogenic that is not degraded or hydrolyzed during the roasting process. <a href="javascript:void(0);">(41)</a> Degradation was significant only for the Guesha variety (15%) and may be related to the roasting and processing processes to which the beans were subjected. The method proved to be efficient in showing that the Laurina genotype has a lower caffeine content, as reported in the literature.Recent studies, such as those by Santanatoglia et al. and Farag et al., confirm the reliability of LC-MS/MS for the simultaneous determination of caffeine, trigonelline, and chlorogenic acids, serving as a comparative basis for validating more accessible methods. <a href="javascript:void(0);">(42,43)</a> The good agreement between the values obtained by the proposed methodology and the LC-MS/MS data reinforces its applicability as a viable alternative for quality control and phytochemical analysis laboratories, especially in contexts where access to highly complex techniques is limited. The proposed methodology can be compared with established reference methods, such as ISO 20481:2008 and AOAC 979.08, which establish chromatographic parameters for the quantification of caffeine by HPLC-UV in coffee matrices. <a href="javascript:void(0);">(44,45)</a> These methods utilize a mobile phase with a higher proportion of organic solvents and more elaborate sample preparation steps (such as reflux extraction). The methodology proposed in this work offers shorter analysis time and lower environmental impact, representing a cleaner, safer, and faster analytical alternative suitable for industrial quality control and laboratory studies.In conclusion, the method developed for the simultaneous analysis of trigonelline, 5-caffeoylquinic acid, and caffeine in extracts of raw and roasted coffee using HPLC-UV has demonstrated effectiveness in terms of linearity, accuracy, and precision for the concentrations tested. This method offers a rapid and efficient alternative for analyzing the compositions of these bioactive compounds in coffee samples. The concentrations of trigonelline, 5-caffeoylquinic acid, and caffeine vary significantly among different coffee samples. However, the most important variation is observed in 5-caffeoylquinic acid. This compound may be essential for understanding how the composition of chlorogenic acids affects the characteristics of different coffees.

Optimization of Methodology for Simultaneous Quantification of Trigonelline, 5-Caffeoylquinic Acid, and Caffeine in Green and Roasted Coffee Extracts by HPLC

Bacillus species produce diverse bioactive lipopeptides that contribute to their ecological and biocontrol functions, yet some families remain poorly characterized. This study focuses on kurstakins, a lipopeptide family associated with biocontrol activity, using metabolomic, structural, and multiomics approaches.Metabolomic analysis of a Bacillus cereus extract revealed approximately 50 kurstakin analogs with diverse linear and branched lipid tails. Two new analogs were isolated and fully characterized by NMR, providing the first experimental determination of absolute configuration within this family. Integration of public genomic and MS data further demonstrated the broad chemical diversity and global distribution of kurstakins, highlighting their potential ecological significance in Bacillus-based biocontrol systems.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212">Structural Characterization and Multiomics Analysis Reveal Extensive Diversity and Global Distribution of Kurstakin Lipopeptides</a></h4>Rose Campbell, and Emily Mevers*J. Nat. Prod. 2025, 88, 12, 2960–2967<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.jnatprod.5c01212">https://doi.org/10.1021/acs.jnatprod.5c01212</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Lipopeptides are a well-known family of microbial natural products that possess a range of applications, including their potential medicinal uses and as crop biocontrol agents. <a href="javascript:void(0);">(1)</a> Several lipopeptides or synthetic analogs are FDA-approved for the treatment of infectious diseases, including as antibiotics (e.g., polymyxin B, oritavancin, telavancin, and daptomycin) and antifungals (e.g., rezafungin and micafungin), with many others in various stages of clinical trials. <a href="javascript:void(0);">(2,3)</a> Their bioactive properties arise from their amphiphilic structures, which consist of a fatty acid tail (lipid) and a polar peptide. The lipopeptide antibiotics are known to bind to cell membranes, forming pores that lead to depolarization. <a href="javascript:void(0);">(4)</a> Lipopeptides are commonly produced by a diverse range of microorganisms, including bacteria, fungi, and cyanobacteria; however, Bacilli are particularly well-known for their ability to produce structurally diverse bioactive lipopeptides. This includes the widespread production of surfactins, iturins, fengycins, polypeptins, and octapeptins. <a href="javascript:void(0);">(2,5)</a> This biosynthetic capability has led to their wide use as biocontrol agents in agricultural settings, such as Bacillus thuringiensis being leveraged for insect and disease control and Bacillus velezensis for controlling plant pathogens. <a href="javascript:void(0);">(6,7)</a> The diverse lipopeptides produced by these strains have been shown to work synergistically as antifungals, biofilm formation regulators, and plant defense modulators. <a href="javascript:void(0);">(5,8)</a>Although many lipopeptides have been well characterized from Bacilli, this does not represent their full biosynthetic potential. <a href="javascript:void(0);">(9)</a> In addition, many lipopeptide families have been mentioned in the literature, but lack appropriate analytical data to confirm their planar and secondary structures and the diversity of the family. <a href="javascript:void(0);">(2)</a> This includes the kurstakins, gavaserin, and saltavalin, which have all been implicated to play a critical role in the biocontrol properties of the producing organisms. <a href="javascript:void(0);">(10)</a> The kurstakins were first discovered to be produced by B. thuringiensis, and have been considered a biomarker for Bacillus cereus group species. <a href="javascript:void(0);">(11,12)</a> However, they have since been isolated from a range of bacteria, including non-cereus group Bacillus species (e.g., B. subtilis, B. licheniformis) <a href="javascript:void(0);">(13,14)</a> and more distantly related bacteria (e.g., Enterobacter cloacae, and Citrobacter sp.). <a href="javascript:void(0);">(15,16)</a> Several of the producing strains have been studied extensively for their potential use as a biocontrol agent, due to their antimicrobial activity and ability to swarm and produce protective biofilms, <a href="javascript:void(0);">(12,17,18)</a> which has been attributed in part to the kurstakins. <a href="javascript:void(0);">(19−22)</a> Four cyclic analogs, kurstakins 1–4 (1–4), were elucidated through tandem mass spectrometry (MSMS) analyses and chemical derivatization. <a href="javascript:void(0);">(12)</a> There have been reports of many additional analogs, but these reports are primarily based on MS analyses. <a href="javascript:void(0);">(23−25)</a> The studies indicate that all analogs contain the same peptide core, but incorporate diverse lipid tails. Additionally, amino acid configurations were deduced through bioinformatic analyses alone, <a href="javascript:void(0);">(17)</a> which can only predict the configuration of the α-position. Therefore, chemical analyses are needed to unequivocally determine the amino acid configurations, particularly of the β-hydroxyl in the threonine residue. Herein, we leveraged our metabolomic data set from marine bacteria to characterize the kurstakins, including the first NMR analysis, report of two new analogs, kurstakins 5 and 6 (5–6), deduction of the absolute configuration, and analysis of the true chemical diversity and distribution of the kurstakins.<h4>Results and Discussion</h4>Analysis of a Global Natural Product Social (GNPS) molecular network representing the chemical potential of diverse marine bacteria from our internal library revealed that it contained a rather large spectral family, with masses ranging from 850 to 940 m/z (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig1">Figure 1</a>A). <a href="javascript:void(0);">(26)</a> A subset of these nodes were identified as 1–4 based on their exact mass and key fragment ions (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig1">Figure 1</a>B). A peak at 110.1 m/z, which putatively represents a decarboxylated histidine, was abundant in all MSMS spectra and became a key identifying fragment (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#notes-2">Figure S1</a>). Other nodes matched masses of reported kurstakin analogs with hypothetical structures, <a href="javascript:void(0);">(15,19,23,27)</a> as well as new analogs. Based on key MS fragments, this spectral family represents both cyclic and linear core peptide sequences that have incorporated various lipid tails (e.g., C9–C14 with and without hydroxylation; <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#notes-2">Figure S2–S3</a>).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Nat_Prod_2025_88_12_2960_2967_Figure_1_A_Molecular_family_of_kurstakins_found_in_the_marine_isolates_library_8818fa75c4.jpg" alt="J. Nat. Prod. 2025, 88, 12, 2960–2967: Figure 1. (A) Molecular family of kurstakins found in the marine isolates library, showing structural diversity in tail structure and peptide linearity. (B) Key common fragments of each core peptide used to identify the kurstakins. Fragments not including histidine were rarely observed. (C) Overlaid EICs of all kurstakin masses observed in an enriched semipure fraction, 850–996 m/z, representing a large diversity of cyclic (aqua), linear (blue), and hydroxyl-tail (red) analogs." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Nat_Prod_2025_88_12_2960_2967_Figure_1_A_Molecular_family_of_kurstakins_found_in_the_marine_isolates_library_8818fa75c4.jpg 245w," sizes="100vw" width="1600" height="889">To characterize the structural breadth of the kurstakin family, the producing strain, B. cereus EM195W, was grown in large-scale (8 L) liquid media containing hydrophobic resins (HP-20, XAD4, and XAD7) to capture the secreted small molecules. After 9 days, the resin was extracted with organic solvents to generate a crude extract. The crude extract was first subjected to reverse-phase (RP) flash chromatography to yield eight refined fractions. MS analysis revealed that the kurstakins were present in the nonpolar fractions, which were combined and processed through normal-phase (NP) flash chromatography, generating an additional 13 fractions. Fractions that were highly enriched with the kurstakins were reanalyzed by high-resolution liquid chromatography-MS (HR-LCMS), revealing the presence of many more analogs than previously detected in the original GNPS analysis. Manual interpretation of the MS data indicated that there is significantly more tail variation than previously thought, which was likely overlooked due to the isobaric nature of many of the analogs and low relative abundance of several analogs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig1">Figure 1</a>C). In total, 31 masses ranging from 850–996 m/z were detected, representing approximately 50 distinct analogs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#notes-2">Table S1, Figure S4</a>). The breadth of these analogs spanned tail lengths ranging from C9–C17, most of which appeared as all possible combinations of linear- or cyclic-peptide core with and without hydroxylated tails, and represented many previously undescribed compounds.To discover the breadth of tail diversity, we turned to gas chromatography-MS (GC-MS) analysis. A portion of one of the semipurified NP fractions was treated with HCl (aq) to liberate the free acid, which was subsequently methylated with TMS-diazomethane. The methylated fatty acids were analyzed by GC-MS, comparing the retention times and fragmentation patterns with authentic standards of linear, iso-branched, and anteiso-branched fatty acid methyl esters (LCFA, iBCFA, and aBCFA, respectively). <a href="javascript:void(0);">(34)</a> Comparing the derivatized natural material to the LCFA standards revealed the presence of linear fatty acid tails ranging in length from C9–C18 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig2">Figure 2</a>A). This agreed with the masses found on the HR-LCMS, but with the addition of the C18 LCFA.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Nat_Prod_2025_88_12_2960_2967_Figure_2_Chemical_characterization_of_the_family_of_kurstakins_405ad6222e.jpg" alt="J. Nat. Prod. 2025, 88, 12, 2960–2967: Figure 2. Chemical characterization of the family of kurstakins. (A) GC-MS traces (EIC of common 74.1 m/z fragment) of the semipure B. cereus EM195W fraction (purple) compared to the LCFA (blue) and iBCFA (aqua) standards. (B) Summary of all fatty acid tails identified in kurstakins produced by EM195W." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Nat_Prod_2025_88_12_2960_2967_Figure_2_Chemical_characterization_of_the_family_of_kurstakins_405ad6222e.jpg 245w," sizes="100vw" width="1300" height="673"><h4>Experimental Section</h4><h5>General Experimental Procedures</h5>Optical rotation data were recorded on a JASCO P-2000 polarimeter. NMR spectra were recorded in d6-DMSO with the residual solvent peak as an internal standard (δC 39.5, δH 2.50) on a Bruker Avance III 600 MHz instrument equipped with a triple resonance inverse (CP-TCI) Prodigy N2 cooled CryoProbe (1H 600 MHz; 13C 150 MHz) or a Bruker Avance II 500 MHz instrument equipped with a CPBBO Prodigy N2 cooled CryoProbe (1H 500 MHz; 13C 125 MHz). TFA fuming was monitored on a JEOL ECZ 400 MHz instrument equipped with a Royal HFX probe (1H 400 MHz). GC-MS data were obtained using an <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=5975C">Agilent 5975C VL MSD GC-MS</a>. LR-LCMS data were obtained using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1200">Agilent 1200 series HPLC system</a> equipped with a photodiode array detector and a Thermo LTQ mass spectrometer. HR-ESIMS and HR-MSMS were carried out using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6530">Agilent 6530 Q-TOF</a> equipped with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II">1290 Infinity II UPLC system</a> or a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/403">Thermo Orbitrap Exploris 120 mass spectrometer</a>. Reverse-phase flash chromatography purification was carried out with a Biotage Selekt automated flash chromatography system. HPLC purifications were carried out using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+Infinity+II">1260 Infinity II HPLC system</a> equipped with a photodiode array detector. All solvents were of HPLC quality.

Structural Characterization and Multiomics Analysis Reveal Extensive Diversity and Global Distribution of Kurstakin Lipopeptides

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 2nd February 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!<blockquote>👉 <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></blockquote><blockquote>👉 Need info about different analytical techniques? Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library">LabRulezGCMS</a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library">LabRulezICPMS</a> libraries.</blockquote>This week we bring you application notes by Agilent Technologies and Waters Corporation, technical note by Shimadzu and poster by Thermo Fisher Scientific / MSACL!<h3>1. Agilent Technologies: Quantitative Separation of THC Isomers and Metabolites from Whole Blood</h3>Using Agilent Captiva Enhanced Matrix Removal (EMR) cartridges and LC/MS/MS<ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_thc_isomers_captiva_emr_lctq_5994_8664en_agilent_5eb50dc6f9.pdf">Full PDF for download</a></li></ul>THC isomers (Δ8- and Δ9-THC, in particular) are psychoactive compounds of increasing interest in forensic toxicology, where accurate identification and quantification of these individual isomers and their respective metabolites are essential for legal and clinical assessments. However, an analytical challenge derives from their structural or empirical similarity and the presence of potential isobaric interferences.&nbsp;Isobaric compounds are molecules that are empirically identical, share the same nominal mass, but differ in structure. In the context of THC analysis, CBD and exo-THC are notable isobaric interferences. These compounds can coelute or produce overlapping mass spectral signals, complicating the interpretation of results. CBD, a nonpsychoactive cannabinoid, shares an identical molecular weight and similar fragmentation pattern with THC isomers, making it difficult to distinguish using conventional mass spectrometry alone. Exo-THC, another structural analog, further adds to the complexity for the same reasons.&nbsp;Without adequate chromatographic separation, these isobaric interferences can lead to false positives or inaccurate quantitation. This is particularly problematic in forensic settings, where precise identification of psychoactive substances is critical. Therefore, a robust analytical method that can resolve these compounds and potential interferents is&nbsp;desirable.&nbsp;This study introduces a workflow that combines enhanced matrix removal (EMR) with LC/MS/MS to achieve baseline separation of THC isomers and their metabolites from the isobaric interferences that were examined within the scope of this application note. The method enables confident separation, identification, and quantification in a single 15-minute analysis.<h4>Experimental</h4><h5>Chromatographic separation and LC/MS/MS detection&nbsp;</h5>This procedure can be accomplished using different instrument configurations. Chromatographic separation was verified using both an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+Infinity+II+LC">Agilent 1260 Infinity II LC</a> and an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC">Agilent&nbsp;1290 Infinity II LC</a>. Analyte detection and quantitation were performed via both an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6475">Agilent 6475 triple quadrupole LC/MS</a> and an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Ultivo">Agilent&nbsp;Ultivo triple quadrupole LC/MS</a>. Method&nbsp;parameters are detailed in Tables 1 and 2. Detailed MRM transition settings are outlined in Appendix A.<h4>Conclusion&nbsp;</h4>This study presents an effective, integrated workflow for the separation and quantification of THC isomers and metabolites in whole blood. Agilent&nbsp;Captiva EMR–Lipid cartridges removed sufficient matrix interferences to enable clean extraction and high analyte recovery. Chromatographic separation using an Agilent InfinityLab Poroshell 120 PFP column provided reliable resolution of isobaric compounds, including CBD and exo-THC. Coupled with Agilent LC/MS/MS systems featuring Agilent Jet Stream ionization technology, the method delivered consistent sensitivity and selectivity across a complex panel of analytes. Together, this methodology offers the basis of a streamlined workflow for the separation of Δ8-, Δ9-, and Δ10-THC isomers and their respective hydroxy and carboxy metabolites and potential interferents from whole blood.<h3>2. Shimadzu: Optimization of Supercritical Fluid Extraction Parameters for Vitamins D2, D3, and K1 from Pharmaceutical Preparations</h3><ul><li>Technical note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/c190_e333_6198eb84b9.pdf">Full PDF for download</a></li></ul>Vitamins are organic compounds, essential in small amount, crucial for the normal functioning of the human body, facilitating numerous enzymatic and metabolic functions[1]. Since the human organism cannot produce vitamins, the diet represents the main route of ingestion of these compounds. As consumer awareness grows, reliable information about the vitamin content in foods is becoming a critical issue. However, considering that vitamins could be losses during food processing and storage, the readyto-use multi-vitamin pharmaceutical preparations are becoming widely employed among consumers. These facts, together with the introduction of labelling regulations, introduced the need for ensure the quality control of these types of formulation.<h4>2. Experimental</h4><h5>2.3 Instrumentation</h5>The supercritical fluid extraction (SFE) was carried out by using a SFE pretreatment system (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+UC">Shimadzu Nexera UC</a>) equipped with: a CBM-40 controller, an SFE-30A module, an LC-30ADSF CO2 pump, an LC-40D XR modifier pump, an LC-40D make-up pump, an SFC-30A back pressure regulator, a DGU-40 on-line degasser, an FRC-40 fraction collector. A combination of static and dynamic extraction was employed (Figure 2).<h4>Conclusion&nbsp;</h4>Considering the obtained results, the vitamins extraction revealed as strictly matrix dependent, according to the formulation excipients. Therefore, detailed study needs to be performed to better understand the vitamin formulation.<h3>3. Thermo Fisher Scientific / MSACL: Simultaneous quantitation and discovery (SQUAD) of fecal bile acids and their conjugates in children with autism spectrum disorder (ASD)</h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_squad_fecal_bile_acids_children_asd_po_en_af787058d3.pdf">Full PDF for download</a></li></ul>This study presents a Simultaneous Quantitation and Discovery (SQUAD) metabolomics workflow applied to the analysis of fecal bile acids and their microbial conjugates in children with Autism Spectrum Disorder (ASD) undergoing microbiota transfer therapy (MTT). Bile acids play a key role in lipid metabolism and gut–brain signaling, and alterations in gut microbiota composition in ASD have been linked to disrupted bile acid metabolism. The SQUAD approach was designed to address the need for both targeted quantification of known bile acids and untargeted discovery of novel metabolic features within a single LC–MS injection.The workflow integrates reversed-phase liquid chromatography with advanced mass spectrometry using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/748">Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer</a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744">Thermo Scientific Vanquish Horizon UHPLC system</a>. Quantitative analysis was performed using parallel reaction monitoring (PRM) in the linear ion trap, while high-resolution MS¹ scanning in the Orbitrap enabled confident metabolite annotation. The Tribrid architecture—combining a quadrupole, linear ion trap, and Orbitrap—allowed high selectivity and sensitivity, including MSⁿ capabilities for resolving co-eluting structural isomers such as leucine- and isoleucine-conjugated bile acids.Using calibrated bile acid standards, the method achieved absolute quantitation across six orders of magnitude, with limits of quantitation below 1.8 femtomoles on column. The SQUAD workflow was applied to fecal samples collected before and after MTT, revealing distinct shifts in bile acid and lipid-related metabolite profiles following treatment. Multivariate analysis, including principal component analysis (PCA), demonstrated clear separation between pre- and post-treatment samples in the MTT group, while no comparable changes were observed in placebo controls.Overall, the results demonstrate that SQUAD metabolomics enables high-throughput, highly sensitive, and information-rich analysis by combining quantitative rigor with deep metabolic coverage in a single experiment. This integrated LC–MS workflow provides valuable insight into the biochemical effects of microbiome modulation in ASD and highlights the power of Orbitrap Tribrid–based metabolomics for studying complex host–microbiome interactions in clinical research.<h3>4. Waters Corporation: Analysis of Organic Carbonates in Lithium- Ion Battery Electrolyte by High-Performance Liquid Chromatography (HPLC) with Mass Detection (MS)</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009211_en_46cbebda90.pdf">Full PDF for download</a></li></ul><h5>Benefits&nbsp;</h5><ul><li>Sensitive method for low-level detection and quantification of carbonate solvents (0.05–1 ppm) and carbonate-based aging products (0.00075–0.0025 ppm) in lithium-ion battery electrolyte solutions using the ACQUITY QDa II Mass Detector with SIR acquisition mode.</li><li>Robust chromatographic separation using the XSelect Premier HSS PFP Column under 7 minutes analysis time, operated under reversed-phase conditions.</li></ul>The electrolyte is one of the main components in the lithium-ion battery.1,2 It is a conductive solution that transports lithium ions from the cathode to the anode during the charging process. During discharge, the lithium ions flow from the anode to the cathode. The electrolyte solution is composed of lithium salt dissolved in a mixture of organic solvents.1,2 The organic solvents are typically a mixture of cyclic and linear carbonates, including EC, PC, DMC, EMC, and DEC.2 Lithium hexafluorophosphate (LiPF₆) is commonly used as the conducting lithium salt,2 providing the necessary ionic conductivity for efficient battery operation.&nbsp;The LiPF6 salt in carbonate-based systems can exhibit chemical and thermal instability, which can impact the performance of the lithium-ion batteries.2-4 Electrolyte solutions can also degrade, forming aging products including DMDOHC and DEDOHC.2 Various techniques are employed to investigate quality and properties of the electrolytes. Techniques such as scanning calorimetry (DSC) and thermogravimetric analysis (TGA) offer a comprehensive analysis of an electrolyte’s thermal properties, while rheology measures viscosity of the conductivity solutions.1 Additionally, chromatographic methods are used to measure the quantity of carbonate solvents in electrolyte solutions.2-4 Gas chromatography mass spectrometry (GC/MS) and liquid chromatography mass spectrometry (LC/MS) are common techniques used to investigate carbonate-based electrolytes.3&nbsp;In this application note, an HPLC method with mass detection was developed for the analysis of five common carbonate solvents and two carbonate-based aging products (Table 1). The method employed an ACQUITY QDa II Mass Detector coupled with an Arc Premier System. Method performance characteristics including limit of detection (LOD), limit of quantification (LOQ), linearity and system suitability were demonstrated. Additionally, the developed method was applied for the analysis of LiPF6-based electrolyte solution.<h4>Experimental</h4><h5>Method Conditions&nbsp;</h5><ul><li>System: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/306">Arc Premier System</a>, Column Manager with active pre-heating, PDA and the ACQUITY QDa II Mass Detector&nbsp;</li><li>Column: XSelect Premier HSS PFP Column, 2.5 µm, 4.6 mm x 100 mm (Waters, p/n: 186010051)</li><li>MS system: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1543">ACQUITY QDa II Mass Detector</a></li></ul><h5>Data Management&nbsp;</h5><ul><li>Chromatography data software (CDS): <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/627">Empower™ 3.6.1</a></li></ul><h4>Results and Discussion&nbsp;</h4>Chromatographic columns with a wide range of selectivity were explored during this study to ensure separation between carbonate solvents and carbonate-based aging products. The selected columns (XSelect Premier CSH™ C18, XSelect Premier CSH Phenyl Hexyl, XSelect Premier HSS T3, XSelect Premier HSS PFP) were tested with acetonitrile and methanol solvents. The study showed that XSelect Premier HSS PFP Column with methanol provided the best retentivity and separation for all analytes (Figure 1). The method successfully separated carbonate solutions and carbonate-based aging products in less than a 7-minute gradient time. The MS total ion chromatogram (TIC) data was collected across the mass range of 35–300 Da using electrospray ionization in positive (ESI+) mode (Figure 1A). The mass spectral data facilitated quick and accurate identification of the components. The carbonate solvents formed [M + H]+ molecular ions, while the selected aging products formed sodium adducts, [M + Na]+. These ions were measured using a selected ion recording (SIR) acquisition mode, which determines intensity for a single ion of interest (Figure 1B).<h4>Conclusion&nbsp;</h4>A sensitive and reliable HPLC-MS method using the Arc Premier System coupled with the ACQUITY QDa II Mass Detector was developed for the analysis of carbonate solvents and carbonate-based aging products in lithium-ion battery electrolyte. This reversed-phase LC method successfully separated all five carbonate solvents (EC, PC, DMC, EMC, DEC) and two aging products (DMDOHC/DEDOHC) on an XSelect Premier HSS PFP Column. Low-level quantification limits were achieved using SIR acquisition mode ranging from 0.05 to 1 ppm and 0.00075–0.0025 ppm for carbonate solvents and aging products, respectively. The method was demonstrated to be applicable for the analysis of lithium-ion battery electrolyte.

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