LC Method Research Articles

Comprehensive characterization of active components in Salvia miltiorrhiza using polarity-partitioned two-dimensional liquid chromatography coupled with mass spectrometry

Salvia miltiorrhiza, a widely used traditional Chinese medicine, contains a complex matrix of hydrophobic diterpenoids and hydrophilic phenolic acids, presenting significant challenges in comprehensive analysis. In this study, an online polarity-partitioned two-dimensional liquid chromatography coupled with mass spectrometry (2D-LC-MS) method was developed for comprehensive analysis of both lipophilic and hydrophilic active components in Salvia miltiorrhiza. The method integrated hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), facilitating the efficient separation of compounds across a wide range of polarities. An online dilution strategy was implemented, minimizing sample loss and enhancing the method's utility for quality control and chemical characterization of complex herbal matrices. Compared with other LC methods, this approach significantly improved analyte coverage, resolution, and analysis efficiency. Under optimal conditions, 150 active components were successfully identified, including 33 compounds newly discovered in Salvia miltiorrhiza. Additionally, the validated online method was applied to the quantitative determination of 16 quality markers of Salvia miltiorrhiza from different sources. The results demonstrated the online method's potential as a superior alternative to existing techniques, offering broader applicability in traditional Chinese medicine research.

Considerations for method development and method translation in capillary liquid chromatography: A tutorial

HPLC remains one of the most widely used measurement techniques for chemical analysis. Capillary LC, which utilizes narrow diameter columns operated at lower flow rates than analytical-scale LC, continues to gain adoption based on its reduced mobile phase consumption and increased sensitivity when coupled to MS detection. This tutorial offers practical insights into the most critical aspects of translating analytical-scale separations to the capillary scale. The selection of pumping systems, detectors, and the potential for performance loss due to extra-column effects are examined within the context of separations using columns with inner diameters ≤ 0.3 mm. Column choices within this diameter range are also detailed, both in terms of stationary phase support options and general commercial availability. The impact of these various factors on the effective development/translation of LC methods down to flow rates under 10 µL/min is described to provide readers with a basis for implementing these strategies within their own analytical workflows.

B-268 Detection of xylazine in fentanyl-positive urine drug screen specimens using a quantitative LCMS/MS method

Abstract Background Fentanyl, a synthetic opioid used clinically for severe pain management, sedation, and as a perioperative analgesic, has been at the forefront of fatal drug-involved overdoses for almost a decade. Xylazine, an α-2 adrenergic receptor agonist, is an increasingly common adulterant in illicitly manufactured fentanyl products and analogs. Given its class, xylazine is non-responsive to naloxone, an opioid receptor antagonist used to reverse opioid overdose. Importantly, the U.S. Drug Enforcement Administration reported xylazine in 23% of seized fentanyl powders and 7% of fentanyl pills; additionally, a recent study by Kariisa et. al. identified an increase of xylazine presence in fatal fentanyl-involved overdoses (2.9% in January 2019 to 10.9% in June 2022) across twenty states.1 Collectively, these trends indicate a need for increased surveillance of xylazine in overdose-presenting patients to inform appropriate clinical responses. In this study, we developed a sensitive LC-MS/MS method for xylazine quantitation and used it to determine xylazine presence in our fentanyl positive patients. Methods An LC-MS/MS method for quantitation of xylazine from urine samples was developed using a Waters Xevo TQ-XS and reversed-phase CORTECS C8 2.7 µm column. MRMs and optimal MS tuning parameters were generated for xylazine and xylazine-d6 via IntelliStart, with transition ions of 221.18→90.04 (Quan) and 221.18→136.31 (Qual) selected for xylazine quantitation against a 227.23→89.98 xylazine-d6 transition as an internal standard. An LC method was developed using water (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) and methanol (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) gradient for a 4-minute runtime. Calibrators were prepared by addition of the pure drug to water, while QCs were prepared using healthy volunteer urine that was confirmed negative in a routine urine drug screen (UDS). Calibrators were value-assigned using a reference laboratory. A simple processing method consisting of the addition of 200 µL methanol containing 25 ng/mL xylazine-d6 to 50 µL of standards, QCs, or urine was used for sample preparation. Remnant fentanyl-positive patient samples (determined by ARK Fentanyl II Immunoassay - Abbott Architect C16000 analyzer) submitted for routine UDS evaluation were collected for xylazine analysis. Results Using custom xylazine standards, the method yielded a linear range from 2 ng/mL to 500 ng/mL with an average R2 of 0.9995 across runs (n = 12). The LLoQ for this assay was 2 ng/mL with a CV of 18.1%. The intra-assay CVs were < 5.0% and inter-assay CVs were < 15.0%. Of preliminary patient samples tested (n = 25), xylazine was detected in 32% of fentanyl-positive samples, ranging in concentration from 2.8 ng/mL to 192.8 ng/mL with an average of 64.8 ng/mL. Conclusions Here we report a simple LC-MS/MS method for quantitation of xylazine in remnant fentanyl-positive UDS specimens by LC-MS/MS. Preliminary testing revealed a xylazine positivity rate of 32% in fentanyl-positive patients. Uncomplicated sample work-up makes this method applicable to xylazine surveillance in all UDS-positive patient samples in high-throughput clinical laboratories. Reference: 1. Kariisa M, et. al. “Illicitly Manufactured Fentanyl-Involved Overdose Deaths with Detected Xylazine.” MMWR Morb Mortal Wkly Rep 2023;72:721-727.

B-159 Fast Cleanup for 19 Steroid Analytes in Serum Using Supported Liquid Extraction (SLE)

Abstract Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool that holds promise in designing the future of steroid analysis in the clinical laboratory. It is already widely used in steroid analysis in endocrinology and is being utilized increasingly in newer applications for individual steroids and panels relevant in clinical research. LC-MS/MS is gaining user base owing to greater sensitivity and specificity and gradually even replacing some of the legacy test methods. Improved sample preparation, modern chromatography methods, and sensitive, faster scanning mass spectrometers have all played a role in improving LC-MS/MS. Steroid analysis for clinical research can require very low limits of detection which demand high recoveries and very clean extracted samples. Supported Liquid Extraction (SLE) can provide very clean extracted samples with less method development and a streamlined workflow compared to solid phase extraction (SPE). Laboratories desire high-throughput methods which consolidate analytes into one panel with fast chromatographic run times. Chromatographic separation of steroid isomers with the same m/z is necessary for accurate quantitation by LCMS/MS. Meeting these criteria can be challenging in a single LC-MS/MS method. Methods Each 200 µL serum sample was pretreated with 200 µL of pH 6 Ammonium Acetate buffer (aq) before being loaded onto a Novum™ PRO SLE, MAX 96-well plate, following the standard SLE protocol. Samples were eluted using 2 × 900 µL of Dichloromethane or Ethyl Acetate. After evaporation of the elution solvent, samples were reconstituted in 100 µL of 0.5 mM Ammonium Fluoride / Methanol (60:40, v/v). A Kinetex™ 2.6 μm C18, 50 x 3.0 mm column using a gradient of 0.5 mM Ammonium Fluoride (aq) and Methanol on an Agilent® 1290 Infinity UHPLC and SCIEX® 7500 Triple Quad™ were used for quantitation. Results Here, we present an effective sample extraction method and LC-MS/MS analysis method for the quantitation of a 19-analyte steroid panel from serum. The Kinetex 2.6 μm C18 column provided a fast 8-minute chromatographic separation and good selectivity for steroid analytes in both positive ion mode and negative ion mode. Calibration curves with a linear fit and 1/x weighting, showed good linearity with R2 of >0.99 for all analytes except 17-OH-Pregnenolone and DHEAS when using Dichloromethane as the elution solvent, and R2 of >0.980 for all analytes when using Ethyl Acetate as the elution the solvent. Accuracy of samples using Dichloromethane as the elution solvent were 80-112 %, except for 17-OH-Pregnenolone and DHEAS. Accuracy of all samples using the Ethyl Acetate elution solvent were 91-118 %. Conclusions The-96 well plate extraction method was robust, reproducible, and can be automated. SLE using Novum Pro MAX combined with a fast LC method provides a simple, rapid, high-throughput workflow for identification and quantitation of 19 steroids from serum.

B-168 Development and Validation of a Quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) Method for Urine Organic Acid Analysis

Abstract Background Urine organic acid (UOA) analysis is essential for the diagnosis of inborn errors of metabolism (IEMs). Traditionally, UOA analysis is performed with gas chromatography-mass spectrometry (GC-MS) and requires time-consuming sample preparation steps including liquid-liquid extraction and derivatization. The rapid development of Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) in the past few years provides the opportunity to perform UOA analysis with a dilute-and-shoot methodology. We describe the development and validation of a quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) method for UOA analysis. Methods Urine specimens were diluted to normalize creatinine concentrations to 1 mmol/L. 20 µL of urine specimen (diluted), calibrator, or quality control (QC) material was mixed with 400 µL of mobile phase A (0.05% formic acid in water) and a mixture of isotope-labeled internal standards. After centrifugation, 10 µL of the supernatant was analyzed using a Xevo G3 QTOF mass spectrometer (Waters) with a ACQUITYTM Premier HSS T3 1.8 µm VanGuardTM FIT 2.1 x 150 mm column (Waters). Data collection was performed with negative electrospray ionization (ESI) mode using the MSE method to produce fragment ions when applicable. Repeatability, reproducibility, and carryover were assessed using the QC materials. The analytical measuring range (AMR) was assessed using synthetic urine spiked with increasing concentrations of each organic acid. Accuracy was assessed by method comparison with the UOA test performed at Mayo Clinic Laboratory and by spike-recovery study using a pooled urine specimen. Matrix effect was also evaluated with matrix dilution study. Results An optimized LC method was used to enable high-resolution separation of selected UOAs (N = 29) and isomers. Total analytical time was 20 min per injection. Both linear and quadratic regressions were used to build the calibration curves. AMR and correlation coefficients of a few representative UOAs were: orotic acid (3.4 to 214.2 mmol/mol creatinine, R^2 = 0.99, linear regression); 2-methylcitric acid (4 to 189 mmol/mol creatinine, R^2 = 0.99, linear regression); 3-methylcrotonylglycine (0.3 to 18.0 mmol/mol creatinine, R^2 = 0.99, linear regression). Repeatability and reproducibility were mostly <=10% CV and no carryover was observed. Spike-recovery study demonstrated recoveries between 80% and 120%, and method comparison study demonstrated no discrepancies with results from Mayo Clinic Laboratory. Conclusions We have developed and validated a novel UPLC-QTOF method for UOA analysis to support the diagnosis of IEMs with acceptable analytical and clinical performances. Compared with the traditional GC-MS method, the UPLC-QTOF method requires a very small specimen volume and does not require laborious and time-consuming sample preparation steps. Continued optimization of the method will be pursued to measure more UOAs to support the diagnosis of more IEMs.

Development and validation of a stability-indicating method, structural elucidation of new degradation products from misoprostol by LC-MS time-of-flight, and an ex vivo study of vaginal permeation.

Misoprostol (MSP) is commonly prescribed in obstetrics and gynecology clinical practice for labor induction, cervical ripening, first-trimester pregnancy termination, and the treatment of postpartum hemorrhage. Furthermore, there is a lack of comprehensive discussion evaluating how different commercially available formulations influence the overall efficacy of MSP, even though reports indicate issues with the quality of these formulations, particularly regarding stability and vaginal absorption processes. This study investigates the stability of MSP under acidic conditions and its in vitro permeation using swine vaginal mucosa. A forced degradation study was conducted using 0.2 M HCl, and a high-efficiency LC method was developed. Three degradation products were identified and characterized using electrospray ionization-high-resolution quadrupole-time-of-flight-MS, with respective m/z values of 391.2508, 405.2705, and 387.2259, respectively. These results suggest that the degradation mechanism involves dehydration of the β-hydroxy ketone moiety, followed by isomerization to its most resonance-stable form and de-esterification. Finally, the in vitro permeation study revealed that the esterified form of MSP was unable to permeate the mucosa and required prior degradation for any component to be detected in the receptor fluid.

DiaPASEF Enables High-Throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development.

Monitoring and quantifying host cell proteins (HCPs) in biotherapeutic production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs, such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.

Estimating LoD-s Based on the Ionization Efficiency Values for the Reporting and Harmonization of Amenable Chemical Space in Nontargeted Screening LC/ESI/HRMS.

Nontargeted LC/ESI/HRMS aims to detect and identify organic compounds present in the environment without prior knowledge; however, in practice no LC/ESI/HRMS method is capable of detecting all chemicals, and the scope depends on the instrumental conditions. Different experimental conditions, instruments, and methods used for sample preparation and nontargeted LC/ESI/HRMS as well as different workflows for data processing may lead to challenges in communicating the results and sharing data between laboratories as well as reduced reproducibility. One of the reasons is that only a fraction of method performance characteristics can be determined for a nontargeted analysis method due to the lack of prior information and analytical standards of the chemicals present in the sample. The limit of detection (LoD) is one of the most important performance characteristics in target analysis and directly describes the detectability of a chemical. Recently, the identification and quantification in nontargeted LC/ESI/HRMS (e.g., via predicting ionization efficiency, risk scores, and retention times) have significantly improved due to employing machine learning. In this work, we hypothesize that the predicted ionization efficiency could be used to estimate LoD and thereby enable evaluating the suitability of the LC/ESI/HRMS nontargeted method for the detection of suspected chemicals even if analytical standards are lacking. For this, 221 representative compounds were selected from the NORMAN SusDat list (S0), and LoD values were determined by using 4 complementary approaches. The LoD values were correlated to ionization efficiency values predicted with previously trained random forest regression. A robust regression was then used to estimate LoD values of unknown features detected in the nontargeted screening of wastewater samples. These estimated LoD values were used for prioritization of the unknown features. Furthermore, we present LoD values for the NORMAN SusDat list with a reversed-phase C18 LC method.

Atypical enantioseparation of a non-ionic form of allantoin with Cinchona alkaloid-based zwitterionic chiral stationary phases

Allantoin represents a compound widely employed in pharmaceutical and cosmetic fields. Its safety has been acknowledged by regulatory bodies such as the US Food and Drug Administration, the European Commission for Cosmetics and Consumer and Health and European Directorate for the Quality of Medicines & HealthCare. This justifies its wide use in dermatological/cosmetic formulations and allows their safe use.Allantoin possesses an asymmetric carbon atom, resulting in two enantiomers, with the (S)-enantiomer predominating in plants, although racemization may potentially occur during manufacturing processes. Notably, literature currently lacks enantioselective LC methods for allantoin analysis.In this study, two zwitterionic Cinchona alkaloid-based chiral stationary phases (CSPs), commercially known as CHIRALPAK® ZWIX(+) (CSP1) and CHIRALPAK® ZWIX(-) (CSP2), were utilized for the enantioseparation of allantoin under polar-ionic conditions. By employing a mobile phase consisting of acetonitrile/methanol/water/acetic acid (96:2:2:0.1, v/v/v/v), nearly complete baseline separation (with α=1.08) of allantoin enantiomers was achieved in less than 15 min with both CSPs. Due to the “pseudo-enantiomeric” nature of the two chiral selectors (quinine-based in CSP1 and quinidine-based in CSP2), an inversion of the enantiomer elution order was observed with the two CSPs under identical experimental conditions. Remarkably, this represents a rare instance where these CSPs demonstrate the ability to enantioseparate a non-ionic, non-ionizable species.The application of a molecular dynamics in silico protocol proved useful in elucidating the retention mechanism in depth, casting light on the central role of the H-bond formation and the involvement of the anionic moiety of the CSP 1.

Two-dimensional sequential selective comprehensive chiral×reversed-phase liquid chromatography of synthetic phosphorothioate oligonucleotide diastereomers

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.

Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.

Improving the Accuracy of Predictive 2D-LC Optimization Strategies: Incorporation of Simulated Elution Profiles to Account for Injection Band Broadening in Online Comprehensive Two-Dimensional Liquid Chromatography.

Method development in online comprehensive two-dimensional liquid chromatography (LC × LC) requires the selection of a large number of experimental parameters. The complexity of this process has led to several computer-based LC × LC optimization algorithms being developed to facilitate LC × LC method development. One particularly relevant challenge for predictive optimization software is to accurately model the effect of second dimension (2D) injection band broadening under sample solvent mismatch and/or sample volume overload conditions. We report a novel methodology that combines a chromatographic numerical simulation model capable of predicting elution profiles of analytes under conditions where peak distortion occurs with a predictive multiparameter Pareto optimization approach for online LC × LC. Preliminary method optimization is performed using a theoretical model to predict 2D injection profiles, and optimal experimental configurations obtained from the Pareto fronts are then subjected to further optimization using the simulation model. This approach drastically reduces the number of simulations and therefore the computational demand. We show that the optimal experimental conditions obtained in this manner are similar to those obtained using a complete optimization using only the simulation model. Online HILIC × RP-LC separation of phenolic compounds was used to compare experimental data to simulated two- and three-dimensional contour plots. The main advantage of the proposed approach is the ability to predict the formation of split or deformed peaks in the 2D, a significant benefit in online LC × LC method optimization, especially for separation combinations with mismatched mobile phases. A further benefit is that simulated elution profiles can be used for the visualization of predicted two-dimensional chromatograms for method selection.

Development and validation of a simple and affordable LC-UV method for identification and assay of selected antimicrobial medicines

Antimicrobials, particularly antibiotics, are among the most common classes of drugs reported as substandard and falsified (SF) in developing countries. Therefore, it is important to develop simple and affordable analytical methods for the quality control of antimicrobial medicines. In this study, a liquid chromatographic method with ultraviolet detection (LC-UV) was developed and validated for the screening and quantification of 13 antimicrobial medicines and one beta-lactamase inhibitor in pharmaceutical formulations. LC separation was carried out on a Kinetex C18 column (150 mm × 4.6 mm, 2.6 µm) with gradient elution. The mobile phase consisted of mixtures of acetonitrile-water-10 mM phosphate buffer pH 3.5 at ratios of 3:92:5, v/v/v for mobile phase A and 50:45:5, v/v/v for mobile phase B with a flow rate of 0.5 mL/min. The screening method was intended for confirmation of the identity of the actives and validated for specificity and robustness, whereas the quantification method (using only a different detection wavelength) was further validated in terms of linearity, accuracy, sensitivity and precision (repeatability, intermediate precision). For all compounds, the method was found to be linear (r2 > 0.999), precise (%RSD < 1%), accurate (% recovery of 98–102%), sensitive, specific and robust. The developed LC method was successfully applied for the identification and assay of 12 antimicrobial samples from Ethiopia. Among the 12 samples analyzed, one (8.3%) product was confirmed to be falsified.

Simultaneous analysis of cannabinoids and terpenes in Cannabis sativa inflorescence using full comprehensive two-dimensional liquid chromatography coupled to smart active modulation

Nowadays, the higher peak capacity achievable by comprehensive two-dimensional liquid chromatography (LC×LC) for the analysis of vegetal samples is well-recognized. In addition, numerous compounds may be present in very different amounts. Cannabinoids and terpenes represent the main components of Cannabis sativa inflorescence samples, whose quantities are relevant for many application purposes. The analyses of both families are performed by different methods, at least two different separation methodologies, mainly according to their chemical characteristics and concentration levels. In this work, concentration differences and sample complexity issues were addressed using an LC×LC method that incorporates an optimized modulation strategy, namely smart active modulation, for the simultaneous analysis of cannabinoids and terpenes. The system was built by interposing an active flow splitter pump between both dimensions. This set up aimed to exploit the known advantages of LC×LC. In addition, here we proposed to use the splitter pump for online control over the splitting ratio to facilitate the selective dilution of different eluted fractions containing compounds with highly different concentrations.This work represents the first application and demonstration of smart active modulation (SAM) in LC×LC to simultaneously determine analytes with significant differences in concentration levels present in complex samples. The proposed method was tested with eight different strains, from which fingerprints were taken, and numerous cannabinoids and terpenes were identified in these samples. With this strategy, between 49 and 54 peaks were obtained in the LC×LC chromatograms corresponding to different strains. THCA-A was the main component in six strains, while CBDA was the main component in the other two strains. The main terpenes found were myrcene (in five strains), limonene (in two strains), and humulene (in one strain). Additionally, numerous other cannabinoids and terpenes were identified in these samples, providing valuable compositional information for growers, as well as medical and recreational users. The SAM strategy here proposed is simple and it can be extended to other complex matrices.

Structure Based Machine Learning Prediction of Retention Times for LC Method Development of Pharmaceuticals.

Significant resources are spent on developing robust liquid chromatography (LC) methods with optimum conditions for all project in the pipeline. Although, data-driven computer assisted modelling has been implemented to shorten the method development timelines, these modelling approaches require project-specific screening data to model retention time (RT) as function of method parameters. Sometimes method re-development is required, leading to additional investments and redundant laboratory work. Cheminformatics techniques have been successfully used to predict the RT of metabolites & other component mixtures for similar use cases. Here we will show that these techniques can be used to model structurally diverse molecules and predictions of these models trained on multiple LC conditions can be used for downstream data-driven modelling. The Molecular Operating Environment(MOE) was used to calculate over 800 descriptors using the strucutres of the analytes. These descriptors were used to model the RT of the analytes under four chromatographic conditions. These models were then used to create data-driven models using LC-SIM. A structural-based Random Forest (RF) model outperformed other techniques in cross-validation studies and predicted the RTs of a randomized test set with a median percentage error less than 4% for all LC conditions. RTs predicted by this structure-based model were used to fit a data-driven model that identifies optimum LC conditions without any additional experimental work. These results show that small training sets yield pharmaceutically relevant models when used in a combination of structure-based and data-driven model.

Development of novel methods based on GC-HRMS and LC-HRMS for the determination of non-phthalate plasticizers in soil

Non-phthalate plasticizers (NPPs) are a suitable alternative to phthalates, which are harmful compounds for human, animal health, and the environment. In this study, 28 commercial non-phthalate plasticizers (NPPs) from different families, including adipates, citrates, phosphates, sebacates, trimellitates, benzoates and cyclohexanoates, were determined. Two novel methods for determining these alternative compounds in soil were developed using gas chromatography coupled to high-resolution mass spectrometry (GC-HRMS-Q-Orbitrap) and liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS-Q-Orbitrap). Solid-liquid extraction (SLE) with ethyl acetate or acetonitrile, along with water as extraction solvents, were employed. In most cases, the GC method exhibited recoveries ranging from 84.9 % to 110.8 % at 20, 40 and 200 μg/kg, while the LC method achieved recoveries between 73.1 % and 115.4 % at 10, 20, 40 and 200 μg/kg. Most of the relative standard deviation (RSD) values were below 20 % for both methods. The validated methods were then applied to analyse soil samples collected from four different areas in Almeria. The results indicated that the compounds detected most frequently at high concentrations were 1-hydroxycyclohexyl phenyl ketone (HCPK) using GC, in the range 29.1–67.4 μg/kg and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB) using LC, in the range 39.9–51.5 μg/kg. Additionally, suspect and unknown analysis were carried out, and other plasticizers as phthalates, were also detected, in addition to other substances present in the analysed samples. All the soils exhibited the presence of a few plasticizers, either phthalic and/or non-phthalic.

Comprehensive two-dimensional liquid chromatography with high resolution mass spectrometry to investigate the photoelectrochemical degradation of environmentally relevant pharmaceuticals and their degradation products in water

The widespread presence of organic micropollutants in the environment reflects the inability of traditional wastewater treatment plants to remove them. In this context, advanced oxidation processes (AOPs) have emerged as promising quaternary wastewater treatment technologies since they efficiently degrade recalcitrant components by generating highly reactive free radicals. Nonetheless, the chemical characterization of potentially harmful byproducts is essential to avoid the contamination of natural water bodies with hazardous substances. Given the complexity of wastewater matrices, the implementation of comprehensive analytical methodologies is required. In this work, the simultaneous photoelectrochemical degradation of seven environmentally relevant pharmaceuticals and one metabolite from the EU Watch List 2020/1161 was examined in ultrapure water and simulated wastewater, achieving excellent removal efficiencies (overall >95%) after 180 min treatment. The reactor unit was linked to an online LC sample manager, allowing for automated sampling every 15 min and near real-time process monitoring. Online comprehensive two-dimensional liquid chromatography (LC × LC) coupled with high resolution mass spectrometry (HRMS) was subsequently used to tentatively identify degradation products after photoelectrochemical degradation. Two reversed-phase liquid chromatography (RPLC) columns were used: an SB-C18 column operated with 5 mM ammonium formate at pH 5.8 (1A) and methanol (1B) as the mobile phases in the first dimension and an SB-Aq column using acidified water at pH 3.1 (2A) and acetonitrile (2B) as the mobile phases in the second dimension. This resulted in a five-fold increase in peak capacity compared to one-dimensional LC while maintaining the same total analysis time of 50 min. The LC x LC method allowed the tentative identification of 12 venlafaxine, 7 trimethoprim and 10 ciprofloxacin intermediates. Subsequent toxicity predictions suggested that some of these byproducts were potentially harmful.This study presents an effective hybrid technology for the simultaneous removal of pharmaceuticals from contaminated wastewater matrices and demonstrates how multidimensional liquid chromatography techniques can be applied to better understand the degradation mechanisms after the treatment of micropollutants with AOPs.

Development and validation of a stability indicating lc method for the analysis of chlordiazepoxide and trifluoperazine hydrochloride in the presence of their degradation products

Development and validation of a stability indicating lc method for the analysis of chlordiazepoxide and trifluoperazine hydrochloride in the presence of their degradation products

The Gradient Delay Volume, Part I: Theory

The gradient delay volume is arguably one of the most important, yet least appreciated, parameters that affect how gradient elution separations in LC work. This has implications both for method development and for method transfer during the lifecycle of a LC method. In this installment, I will review the concept of gradient delay volume, its physical connection to the LC instrument, and how it can impact method development and separation quality.