Total Run Time Research Articles

Eco-friendly RP-HPLC approach for simultaneously estimating the promising combination of pentoxifylline and simvastatin in therapeutic potential for breast cancer: Appraisal of greenness, whiteness, and Box–Behnken design

Abstract Breast cancer affects millions of women worldwide. This study explores the potential of combining pentoxifylline (PTX) and simvastatin (SIM) as a treatment for breast cancer. We aimed to develop six sustainability tools using green and white metrics to evaluate the environmental impact of reversed-phase high-performance liquid chromatography (RP-HPLC) methods for analyzing and separating PTX and SIM in their pure forms. The tools include analytical GREEnness, green analytical procedure index, Complexgreen analytical procedure index, analytical greenness metric for sample preparation, blue applicability grade index, and the RGB 12 algorithm. For the separation, we used a Novapack C8 column (15 × 0.46 cm, 5 µm) at 25°C. The injection volume was 5.0 µL, the wavelength was set to 210 nm, and the total runtime was 5 min. We identified optimal chromatographic conditions efficiently using the Box–Behnken design with minimal trials. We investigated the effects of three factors on retention time and resolution: acetonitrile ratio, pH, and flow rate. We used overlay plots with a 60:40 ratio (v/v) of acetonitrile and bi-distilled water to forecast the most effective mobile phase. The calibration curves for PTX and SIM showed a correlation value of over 0.999 within the range of 5–60 µg·mL−1. The recovery rates ranged from 99.9% to 100.2%, indicating high accuracy. Our RP-HPLC technique proves to be reliable and efficient for the simultaneous estimation of multiple anticancer drugs. We evaluated the environmental sustainability of this approach using green and white metrics, and the recommended method has been thoroughly validated according to International Council for Harmonisation guidelines, making it highly reliable for analyzing new formulations.

Performance of ITER pressure gauges during deuterium operation in the Large Helical Device

Abstract During deuterium campaigns on the heliotron large helical device (LHD), ITER pressure gauges with different cathode materials were used to measure the neutral pressure in the sub-divertor region. Throughout these campaigns, it was observed that the performance of the LaB6 cathodes was unsatisfactory, credited to the generation of neutrons impacting the gauges. Conversely, measurements taken with pressure gauges with a ZrC cathode performed well throughout the deuterium pulses. The ITER pressure gauge with the ZrC cathode could be operated with a very high electron current of 800 µA, thus improving the lower detection limit of the neutral pressure in LHD. With this design it was also possible to avoid jumps in the ion current within strong magnetic fields, improving the accuracy of the measurement from 15% uncertainty to 5%. These features allowed very precise neutral pressure measurements to be made in a fusion device with magnetic confinement. The total running time in the magnetic field was around 60 hours, less than the cathode life time of 350 hours.

Trace-level quantification of NDMA in levosulpuride active pharmaceutical ingredient and tablet formulation Using UFLC-MS/MS

Nitrosamine impurities identified in several pharmaceuticals during recent times has raised concerns leading to product recalls worldwide and necessitating sensitive liquid and gas chromatographic methods for trace level detection of nitrosamine impurities. This study developed and validated a ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the quantification of NDMA in Levosulpuride drug substance and tablet formulation. Current method utilizes a triple quadrupole analyzer, atmospheric pressure chemical ionization (APCI) ionization source and multiple reaction monitoring (MRM) scan mode for the analysis. Chromatographic separation was achieved on a Gemini NX-C18 column (150 × 4.6 mm, 3 µm) maintained at 40 °C. The mobile phase consisted of a binary gradient of solvent A (0.1 % formic acid in water) and solvent B (methanol), with a total run time of 18 minutes. Current method achieved excellent linearity, recovery, precision, and sensitivity. Greenness of the developed method was evaluated using the GAPI, AGREE, and AES metrics. Current method is sensitive and selective for NDMA in levosulpuride drug substance and tablet formulations and can be employed for routine quality control analysis in pharmaceutical industry.

Novel Application of Quantum Computing for Routing and Spectrum Assignment in Flexi-Grid Optical Networks

Flexi-grid technology has revolutionized optical networking by enabling Elastic Optical Networks (EONs) that offer greater flexibility and dynamism compared to traditional fixed-grid systems. As data traffic continues to grow exponentially, the need for efficient and scalable solutions to the routing and spectrum assignment (RSA) problem in EONs becomes increasingly critical. The RSA problem, being NP-Hard, requires solutions that can simultaneously address both spatial routing and spectrum allocation. This paper proposes a novel quantum-based approach to solving the RSA problem. By formulating the problem as a Quadratic Unconstrained Binary Optimization (QUBO) model, we employ the Quantum Approximate Optimization Algorithm (QAOA) to effectively solve it. Our approach is specifically designed to minimize end-to-end delay while satisfying the continuity and contiguity constraints of frequency slots. Simulations conducted using the Qiskit framework and IBM-QASM simulator validate the effectiveness of our method. We applied the QAOA-based RSA approach to small network topology, where the number of nodes and frequency slots was constrained by the limited qubit count on current quantum simulator. In this small network, the algorithm successfully converged to an optimal solution in less than 30 iterations, with a total runtime of approximately 10.7 s with an accuracy of 78.8%. Additionally, we conducted a comparative analysis between QAOA, integer linear programming, and deep reinforcement learning methods to evaluate the performance of the quantum-based approach relative to classical techniques. This work lays the foundation for future exploration of quantum computing in solving large-scale RSA problems in EONs, with the prospect of achieving quantum advantage as quantum technology continues to advance.

Two-Dimensional Liquid Chromatography Tandem Mass Spectrometry Untangles the Deep Metabolome of Marine Dissolved Organic Matter.

Dissolved organic matter (DOM) is an ultracomplex mixture that plays a central role in global biogeochemical cycles. Despite its importance, DOM remains poorly understood at the molecular level. Over the last decades, significant efforts have been made to decipher the chemical composition of DOM by high-resolution mass spectrometry (HR-MS) and liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Yet, the complexity and high degree of nonresolved isomers still hamper the full structural analysis of DOM. To address this challenge, we developed an offline two-dimensional (2D) LC approach using two reversed-phase dimensions with orthogonal pH levels, followed by MS/MS data acquisition and molecular networking. 2D-LC-MS/MS reduced the complexity of DOM, enhancing the quality of MS/MS spectra and increasing spectral annotation rates. Applying our approach to analyze coastal-surface DOM from Southern California (USA) and open-ocean DOM from the central North Pacific (Hawaii), we annotated in total more than 600 structures via MS/MS spectrum matching, which was up to 90% more than that in iterative 1D LC-MS/MS analysis with the same total run time. Our data offer unprecedented insights into the molecular composition of marine DOM and highlight the potential of 2D-LC-MS/MS approaches to decipher the chemical composition of ultracomplex samples.

The effects of inflation, shortages, and partial backlogs on products that deteriorate over time in response to varied demand

This study aims to determine an optimal policy to deal with a situation where a retailer should have enough stock to meet customer demands and prevent supply loss and deterioration. In this study, we demonstrate the impact of partial backlogging, shortages, and inflation on decaying items to provide the most relevant solutions to this problem. The model uses a two-parameter Weibull deterioration distribution with an exponential demand rate. Numerical results and sensitivity analysis are combined with a graphical demonstration, and the proposed approach is reliable and accurate, providing a new explanation for a different type of system. This paper determines the total cost and optimal production run time by developing an inventory model. Sensitivity analysis is carried out to demonstrate the proposed model.

A rapid and efficient approach for quantifying metformin in human plasma using Ultra-High performance liquid chromatography with UV-Detection: Relevance in Non-Diabetes clinical studies

Metformin, a first-line drug to treat type-2 diabetes, has gained attention in various non-diabetes studies, such as anti-aging, reduced cancer incidence and mortality, and reduced symptomatic viral infection due to its immunomodulatory properties. This study aims to develop and validate a simple and cost-effective ultra-high-performance liquid chromatography method with UV detection (uHPLC-UV) for quantifying metformin in human plasma amidst operational challenges and restricted accessibility to mass spectrometry. Plasma samples were protein precipitated with acetonitrile, and metformin was separated from endogenous plasma components using a Waters Acquity UPLC BEH Amide column, 1.8 µm, (2.1 x 100 mm) with gradient elution using 0.1 M potassium phosphate adjusted with phosphoric acid to pH 5.8, and acetonitrile as mobile phases and detected at 240 nm. Method validation was performed according to U.S. FDA guidelines. Total run time achieved was 3 min and linearity was demonstrated over the range of 0.1–40 mg/L based on linear regression using 1/x weighting. The lowest quantitation limit of 0.1 mg/L was achieved. Excellent recoveries (90.3–98.4 %) and negligible matrix factors (94.6–108.1 %) were observed in the sample preparation, negating the need for using any internal standard. Finally, the developed assay was utilised to measure metformin plasma levels from healthy volunteers recruited in a randomised, double-blind, placebo-controlled study investigating potential immunomodulatory effects of metformin influence on yellow fever viremia.

RP-HPLC Method for Simultaneous Quantification of Withanolides in <i>Withania somnifera</i>

Background: Withanolides are the primary bioactive components of Withania somnifera (L.) Dunal. Among them, withanolide A, and 12-deoxy-withastramonolide are the herb’s most physiologically active ingredients. The steroidal lactones are isomers, which makes them difficult to separate. Aim: In the current research, a simple, precise, accurate, and decent RP-HPLC method has been developed, optimised and validated for simultaneous quantification and separation. Methods: Resolution was conducted on Gemini, Phenominex C18 (250mm X 4.6mm, 5 μ) column at 40oC utilising binary mobile phases of acetonitrile and ammonium acetate (10 mM) in gradient mode with 1.0 mL/min flow rate and observed at 230 nm. The total run time was twenty-five minutes. The procedure complied with ICH guidelines, covering the limit of detection, specificity, linearity, quantification, precision, accuracy, and robustness. Results: The standard curves of both relevant analytes displayed a linear pattern with regression values > 0.999 in the 1–10 μg/mL range. For both herbal markers, 1 μg/mL was the lowest limit of quantification. The method’s accuracy was 100.38-100.93 % and 99.13-100.75 % for with withanolide A, and 12-deoxy-withastramonolide respectively. The precision of within and between days was found in the tolerable limit of >2% for both herbal markers. Conclusion: The suggested method worked well for simultaneously analyzing the extracted samples and identifying withanolides.

More quantum chemistry with fewer qubits

Quantum computation is one of the most promising new paradigms for the simulation of physical systems composed of electrons and atomic nuclei, with applications in chemistry, solid-state physics, materials science, and molecular biology. This requires a truncated representation of the electronic structure Hamiltonian using a finite number of orbitals. While it is, in principle, obvious how to improve on the representation by including more orbitals, this is usually unfeasible in practice (e.g., because of the limited number of qubits available) and severely compromises the accuracy of the obtained results. Here, we propose a quantum algorithm that improves on the representation of the physical problem by virtue of second-order perturbation theory. In particular, our quantum algorithm evaluates the second-order energy correction through a series of time-evolution steps under the unperturbed Hamiltonian. An important application is to go beyond the active-space approximation, allowing to include corrections of virtual orbitals, known as multireference perturbation theory. Here, we exploit the fact that the unperturbed Hamiltonian is diagonal for virtual orbitals and show that the number of qubits is independent of the number of virtual orbitals. This gives rise to more accurate energy estimates without increasing the number of qubits. Moreover, we demonstrate numerically for realistic chemical systems that the total runtime has highly favorable scaling in the number of virtual orbitals compared to previous studies. Numerical calculations confirm the necessity of the multireference perturbation theory energy corrections to reach accurate ground-state energy estimates. Our perturbation theory quantum algorithm can also be applied to symmetry-adapted perturbation theory. As such, we demonstrate that perturbation theory can help to reduce the quantum hardware requirements for quantum chemistry. Published by the American Physical Society 2024

B-231 Evaluation of the KingFisher Flex for DNA Isolation From FFPE Tissue Utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

B-192 Covid on the PCR Pro: a consumer-friendly PCR test

Abstract Background The emergence of COVID-19 highlighted the need for rapid and accurate diagnostic solutions that can be deployed on a broad scale. The complexity and long turn-around-time of PCR testing typically performed in molecular laboratories is not compatible with community-wide screening. To overcome these challenges, we present the Co-Dx PCR Pro™, a rapid and simple test platform developed for self-collection and at-home use. The instrument is controlled from the user’s smartphone or tablet and performs sample treatment, RT-PCR, and DNA melting analysis. An automatic calling algorithm provides a simple, “Detected,” or, “Not detected,” result. We demonstrate the utility of this platform using a test for the detection of SARS-COV-2. Methods A self-collected anterior nasal swab sample and 2 mLs of water are added to the collection cup. Closing the lid of the cup initiates rehydrating a dried Proteinase K mixture to digest proteins in the sample. After a brief Proteinase K heat-kill step, the instrument mixes the sample with a dried PCR master mix. Primers target two SARS-COV-2 specific genes (RdRp and E gene) and the human RNase P gene as a control. The sample undergoes an RT step followed by 45 cycles of PCR and DNA melting analysis. Detection is by an automatic calling algorithm that identifies a SARS-COV-2 amplicon melting peak at 82°C and RNase P peak at 87°C. The total runtime is less than 30 minutes. The limit of detection for the assay was assessed by contriving intact virus in negative sample matrix, which was then applied to dry swabs. In addition, frozen and banked clinical samples were tested using the Logix Smart®™ Covid-19 test as a comparator assay. A third party performed a usability assessment by having (1) naïve users follow the instructions and self-administer the test (2) expert trained users perform an independent assessment of the assay. Participants were then asked to complete a usability assessment. Results The 95% limit of detection was 2,100 copies/swab using contrived nasal swabs. Clinical samples were blinded to the operator and run on the platform. All samples resulted in a diagnosis concordant with the comparator assay. The usability assessment was performed by six naïve users and resulted in a systems usability scale score of “Best” (84.2 ± 14.3, out of 100). The two expert users determined an 80% limit of detection of 700 genome equivalents and “ease of use,” and “satisfaction,” scores of 4.5/5.0 and 5.0/5.0, respectively. Conclusions Performance of the PCR Pro platform is on par with laboratory-based PCR testing. The benefits of our assay are its speed and the low complexity platform accessible to the lay. This platform could improve access to molecular diagnostic testing, including use at home. Future expansion to include common respiratory pathogens (Flu, RSV, Strep) will increase utility. Additionally, anonymized results can be aggregated and provide a wealth of epidemiological information at the community level to better inform public health initiatives and health outcomes.

A-320 Determination of Guanidinoacetate Methyltransferase Enzyme Biomarkers in Newborn Dried Blood Spot by Tandem Mass Spectrometry-A Two Tier Approach

Abstract Background Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Following a recommendation by the Recommended Uniform Screening Panel and in line with California State statute, all California newborns will be screened for GAMT deficiency beginning July 2024. Here, we developed and validated a quantitative two-tier tandem mass spectrometry (MS/MS) assay for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). Methods This method punches a 3.2 mm disc of controls and newborn patient DBS into a 96-well plate. 100 μL of extraction solution, containing internal standards of GAMT biomarkers, is added to each well and incubated at 40°C for 45 minutes. The extract is transferred, evaporated, and derivatized into a 3N-butanol-HCl solution at 60°C for 30 minutes. The excess HCl is evaporated to dryness. The conversion of GUAC and creatine to their butyl esters has improved their detection sensitivity by MS/MS. The butyl esters are reconstituted into the mobile phase and shaken for 10 minutes at 27°C. The final 10 μL extract was analyzed by flow injection analysis (FIA)-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. The presumptive positive samples from initial analysis are prepared following the same method, and a 5 μL extract is resolved through an ultra-performance liquid chromatography (UPLC) column and analyzed by UPLC-MS/MS in MRM positive ion mode with a total run time of 3.5 minutes per sample. The validation was performed by assessing the linearity, accuracy, imprecision, carryover, limit of detection (LOD) and lower limit of quantitation (LLOQ), matrix effect, analytical measurement range (AMR), percent total allowable error (TEa), and reference range. Results This method is linear for GUAC, creatine and creatinine over a measured range of 1.17-40.00, 121.20-857.36 and 41.34-387.46 µmol/L respectively. The R2 values=1.000, 0.997, 0.9998; slope=0.999, 0.9945, 0.9982; intercept=0.0398, 4.881, 1.7027; mean recoveries=99-104%, 100-104%, 100-104%; intra-assay precision (n=20) =4.05-5.95%, 4.62-5.31%, 3.27-5.19%, inter-assay precision (n=40)=3.7-12.6%, 4.3-7.6%, 3.8-7.4%; TEa=6-21%, 7-17%, 6-17%; LOD=0.12, 0.54, 1.11 µmol/L; AMR=0.33-1300, 1.11-2700, 3.50-4500 µmol/L; median patient results observed from 2293 normal patient=1.48, 494.02, 89.97 μmol/L for GUAC, creatine, and creatinine respectively. The tier-1 and tier-2 assay screening cutoff for GUAC at the population 95th percentile is 2.53 and 4.55 μmol/L respectively. Conclusions The tier-1 and tier-2 methods has been developed and validated to determine GAMT biomarkers in newborn DBS, making it suitable for routine screening of ∼2000 newborn specimens per day by tier-1 and followed by more specific tier-2 to separate GAMT biomarkers from potential isobaric interfering compounds. This two-tier approach greatly improved the test performance and significantly reduced false positive rate.

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100×3mm; 1.7μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8min including column equilibration, which enables the analysis of 160 samples per day.

B-178 A Sensitive LC-MS/MS Assay for Total Testosterone Quantification

Abstract Background Total testosterone immunoassay is a commonly ordered test for the diagnosis of gonadal disorders. However, immunoassays suffer from low specificity and sensitivity, which leads to inaccuracy especially in the low concentration range (< 100 ng/dL). As a result, commercial testosterone immunoassays have difficulties to accurately measure total testosterone levels for women, children, and hypogonadal men. LC-MS/MS has emerged as the method of choice for the accurate quantitation of steroid hormones. We presented here a sensitive laboratory-developed total testosterone assay, which was validated on two LC-MS/MS instruments. Methods An internal standard containing [2,3,4-13C3]-testosterone was added to each sample. The spiked serum sample was loaded on a supported liquid extraction (SLE) cartridge and eluted using methylene chloride. The elute was dried down under nitrogen and reconstituted in sample reconstitution buffer. Chromatographic separation was achieved using a Restek Ultra C18, 50 x 2.1 mm, 3µm analytical column and a total run time of each sample was 5 minutes. The assay was validated on two triple quadrupole mass spectrometers, API 6500 and API 4500. Results were compared with those of a reference laboratory. Results The assay was linear in the range of 1 to 1200 ng/dL for total testosterone and the AMR was set at 2-1200 ng/dL. The interday and intraday imprecision were less than 8%. Split sample comparison to a reference laboratory demonstrated acceptable accuracy with the slope of 1.053 and correlation coefficient (R2) of 0.991. The limit of detection (LOD) was 0.2 ng/dL and the limit of quantitation (LOQ) was 0.5 ng/dL. Results obtained from two instruments agreed well with the slope of 0.996 and the R2 of 0.999. No carryover or interferences was observed for this assay. Conclusions A LC-MS/MS based method was developed and validated for measuring total testosterone levels for female and children. This sensitive and specific assay will improve the diagnosis of hypogonadism and monitoring of testosterone-suppressing treatment.

High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids.

Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1-12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1-500μM is achieved for most SCFAs, while it is 0.05-100μM for MCFAs. Validation of the procedure depicts good linearity (R2>0.98) and repeatability (CV≤20%). The lower limit of detection (LLOD) is 10-30nM. The lower limit of quantification (LLOQ) is 50-100nM for most analytes, while it is 0.5μM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150×2.1mm; 1.7μm) for the separation of lipids from 23 subclasses with a total run time of 25min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

A quality by design paradigm, novel, and fast LC-MS/MS method to quantify vortioxetine in rat brain homogenate and its assessment of greenness, whiteness, and blueness tools

Vortioxetine (VOR) is an antidepressant drug with multimodal mechanism of action. In this work a sensitive and simple bioanalytical LC-MS/MS method for analysis of VOR in spiked rat brain tissue was developed and validated. Chromatographic analyses were performed in isocratic mode, and Citalopram (CT) was found as a suitable internal standard. Supelco Ascentis® Express Phenyl-Hexyl column was used as stationary phase, and the mobile phase was 0.15 % formic acid in acetonitrile:water (55:45, v/v). A Box-Behnken Design − based method optimization was applied to explore the impact of analytical conditions on chromatographic separation of VOR and CT. Detection was realized via a triple quadrupole LC-MS/MS system; the mass transition ion-pair has been followed as m/z 299.05 → 149.90 for VOR, m/z 325.20 → 109.05 for CT. Analytical method validation was performed according to International Council for Harmonisation (ICH) guideline M10 on bioanalytical method validation. The calibration curve was within the range of 30 to 450 ng/mL. LOD and LLOQ were calculated at 10 and 30 ng/mL, respectively. Recovery was 90 % for the lower limit of quantification and between 98 % to 105 % for the other concentration levels. Total run time was 5 min, retention times of citalopram and vortioxetine were 2.4 and 3.1 min, respectively, and a good chromatographic separation was obtained under optimal conditions. The solvents used to make the method greener were evaluated with the Green Solvent Selection Tool and Green Index Spider chart. The AGREE score for the greenness of the developed method was calculated as 0.61 and was also evaluated with the ComplexGAPI pictogram. The BAGI score for blueness was calculated as 72.5, and the RGBfast score for whiteness was calculated as 50. The method was determined to be most green, white, and blue.

A quality by design assisted RP-HPLC method utilizing central composite design for the assay of eliglustat and its organic impurities in drug

This study aimed to develop a stability-indicating RP-HPLC assay method for eliglustat and its organic impurities using a central composite design approach. The chromatographic separation was achieved with a 10 mM phosphate buffer at pH 4.0 on a Thermo Accucore C18 column (150 x 4.6 mm, 2.6 μm) at a wavelength of 210 nm. Gradient elution was performed using a mobile phase-A consisting of buffer and acetonitrile in a 90:10 v/v ratio and a mobile phase-B composed of water and acetonitrile in a 35:65 v/v ratio, at a flow rate of 1.1 mL/min at 45°C for a total run time of 55 minutes. Based on preliminary experiments, central composite design was utilized to evaluate the effects of independent variables such as flow rate, acetonitrile ratio and column oven temperature on the response factors. Eliglustat and its impurities (EGS-5A, EGS-Diastereomers, and EGS-N-Oxide) had retention time of 3.5 minutes, 21.5 minutes, 23.2 minutes and 25.9 minutes, respectively. The limits of detection and quantitation for eliglustat and its impurities were established in relation to the test concentration, achieving a desirability of 1. The developed method was validated as per regulatory guidelines and successfully applied in bulk drugs and formulation.

Method Development and Validation for the Simultaneous Estimation of Lobeglitazone Sulphate and Glimepiride by HPLC Method.

A simple, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of lobeglitazone sulfate and glimepiride in bulk and tablet dosage forms. The method involved selecting various chromatographic parameters. Specifically, a new method was developed using a 250 mm × 4.6 mm (HSS) reverse-phase C18 column with 5 µm particle size (Shim-pack C18, 250 × 4.6 mm; 5 µm). The mobile phase consisted of 40 volumes of phosphate buffer (pH 3) and 60 volumes of acetonitrile, with methanol as the diluent, running as an isocratic elution. The flow rate was set at 1.0 mL/min, and UV detection was performed at 254 nm. The injection volume was 2 µL, and the total runtime was 10 minutes. The recoveries for lobeglitazone sulfate were found to be in the range of 101% to 100.6%, while those for glimepiride were in the range of 101.5% to 100%. The method’s accuracy is evident from these results. The inter-day and intra-day precision of the new method were both below the maximum allowable limit (RSD% ≤ 2.0), as per International Council on Harmonisation (Q2 R1) guidelines. The method exhibited a linear response, with correlation coefficient (r2) values of 0.9972% for pioglitazone and 0.998 for glimepiride. The validation parameters, such as accuracy, precision, linearity, specificity, stability in the analytical solution and robustness, met the acceptance criteria. Hence, this method can be effectively used for the simultaneous estimation of lobeglitazone sulfate and glimepiride in both bulk and pharmaceutical dosage forms.

Simultaneous multianalyte trace-level quantification of eight genotoxic nitrosamine impurities in valsartan Active Pharmaceutical Ingredient and tablet formulation using UFLC-MS/MS and greenness assessment

Valsartan, a widely prescribed antihypertensive drug, was identified for the presence of carcinogenic nitrosamine impurities in 2018, leading to product recalls worldwide and necessitating sensitive liquid and gas chromatographic methods for trace level detection of nitrosamine impurities. This study developed and validated an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the quantification of eight genotoxic nitrosamines (NDMA, NDEA, NDPA, NEIPA, NDIPA, NMBA, NDBA, and NMPA) in valsartan drug substance and tablet formulations. The method employed a triple quadrupole analyzer, atmospheric pressure chemical ionization (APCI) ionization source and multiple reaction monitoring (MRM) scan mode for the analysis. Chromatographic separation was achieved on a Phenomenex biphenyl column (150 × 4.6 mm, 3 µm) maintained at 55 °C. The mobile phase consisted of a binary gradient of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in methanol), with a total run time of 20 min for the sensitive and specific detection of the analytes. Method validation demonstrated excellent linearity, recovery, precision, and sensitivity. The greenness of the developed analytical method was critically assessed through comprehensive evaluations using the GAPI, AGREE, and AES metrics and the results obtained are compared with the reported methods in the literature. This novel UFLC-MS/MS method offers a reliable and sensitive approach for routine quality control analysis of nitrosamine impurities in Valsartan drug substance and tablet formulations.