Liquid Chromatography Instrument Research Articles

B-155 Analysis of Monoclonal Antibodies Using a Quantification LC-MS/MS Kit - Spotlight on Infliximab

Abstract Background The use of immunoassays for measuring mAbs is well established but has some limitations, including poor performance, lack of standardization, a high cost when processing a limited number of samples, limited dynamic range, and the potential for cross-reactivity. Using LC-MS/MS associated with the ready-to-use commercial mAbXmise Kit is a simple way to implement mAbs measurement for infliximab, providing high analytical performance, ease of use, and flexibility. Methods The Promise Proteomics Inflammation mAbXmise Kit contains all the calibrators, QCs, internal standard and sample preparation consumables to run the LC-MS/MS method for infliximab. Briefly, 20µL samples are dispensed into the mAbXmise plates containing lyophilized stable labelled infliximab. Samples are transferred to the PuriXmise plate to perform the immunocapture of infliximab and allow washing of the samples. Samples are eluted into a collection plate, evaporated to dryness, resuspended, and the protease (CutXmise) is added to the sample to digest infliximab overnight. The digestion is quenched, with the samples immediately ready for analysis. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ Premier HSS T3 Column, eluted using a water/acetonitrile/formic acid gradient profile and quantified with both a Waters Xevo™ TQ-XS and Xevo TQ-S micro Mass Spectrometer. Results The method was shown to be linear from 2 - 100 µg/mL. Analytical sensitivity at lowest calibrator of 2µg/mL provided S/N (PtP) ≥15:1 for all tryptic peptides on both mass spectrometers. Coefficients of variation (CV) at 4 and 25µg/mL QC concentrations were all ≤ 10.0% (n = 5). The relative concentrations of the tryptic peptides were compared across 29 anonymized samples and good agreement was observed for GLE, SAV, DIL and ASA peptides. Conclusions We have demonstrated a clinical research method for quantifying Infliximab in plasma using a commercially available kit for sample preparation followed by LC-MS/MS analysis. The Promise Proteomics mAbXmise Kit aids in the day-to-day reproducibility of the immunocapture and tryptic digestion of IFX for targeted LC-MS/MS analysis with the correct peptide selection. Compared to a direct digest surrogate peptide workflow, the process used in the kit reduces matrix interference, improves analytical sensitivity, and enables the use of lower sample volumes. The method can be run on both the Xevo TQ-XS and Xevo TQ-S micro Mass Spectrometers which provide the dynamic range to quantify IFX across the expected range, and the selectivity and analytical sensitivity to obtain low-level quantification of the IFX surrogate peptide in plasma samples. The data presented combines the use of a kit dedicated to the preparation of samples and the use of liquid chromatography and mass spectrometry instrumentation to perform the quantitative analyses. The mAbXmise Kit described has not been cleared by any regulatory entity for diagnostic purposes outside of Europe. Analytical performance depends on the instrument characteristics and its settings. The end user is responsible for completion of the method development and validation. Proteomics mAbXmise Kits are not available for sale in all countries. For Research Use Only. Not for use in diagnostic procedures.

Determination of Aloin A, Aloin B, and Aloe-Emodin in Raw Materials and Finished Products Using HPLC Multi-Laboratory Validation Study, AOAC 2016.09, Final Action Status.

A multi-laboratory validation study was performed on AOAC Official Method of Analysis (OMA) 2016.09, for final action. Eight different laboratories throughout the world participated in the study tested the same set of 6 different laboratory samples of raw materials (commercial) and formulated products (commercial), and all the laboratories successfully generated results within the acceptance criteria. The intention of the study was to evaluate specificity, precision (variation), linearity/range, system suitability, limit of detection (LOD), and limit of quantitation (LOQ) by multiple laboratories to satisfy the requirement of moving the OMA 2016.09 to the final action status. Accuracy and ruggedness were already validated in earlier work of single laboratory validation (1), and it was not necessary to include these validation parameters in the multi-laboratory validation study. Laboratory samples containing Aloe were sent out to participating laboratories. Each lab followed AOAC 2016.09 (First Action status) to analyze contents of aloin A, aloin B, and aloe emodin using high performance liquid chromatography (HPLC) instrument. The results generated by each laboratory were collected and evaluated. The specificity results show that blank and matrix chromatograms do not contain major interfering peaks on the retention time of aloin A, aloin B, and aloe-emodin. The precision (variation) results of duplicated preparations are not more than 0.05 parts per million (ppm) different. The linearity/range results from six standards (10 parts per billion (ppb), 20 ppb, 40 ppb, 80 ppb, 160 ppb, and 500 ppb) have correlation coefficient (R) value of ≥ 0.999. The system suitability results meet the acceptance criteria to show the instrument validity. The limit of detection (LOD) results show that the signal-to-noise (S/N) ratio of 10 ppb standards for all three components are about 3. The limit of quantitation (LOQ) results show that the S/N ratio of 20 ppb standards for all three components are about 10. The validation parameters (specificity, precision (variation), linearity/range, system suitability, LOD, and LOQ) have been successfully analyzed, and it shows that the test method is suitable for its intended use. The test method has been successfully validated by the eight different laboratories around the world (US, UK, Germany, and Canada). Each of the laboratory is managed independently by the site lab management team. This multi-lab study validated the method of Determination of Aloin A, Aloin B, and Aloe-Emodin in Raw Materials and Finished Products Using high performance liquid chromatography (HPLC), and the method fits for its intended purpose.

Automated multicolumn screening workflow in ultra-high pressure hydrophilic interaction chromatography for streamlined method development of polar analytes

The pharmaceutical industry is rapidly advancing toward new drug modalities, necessitating the development of advanced analytical strategies for effective, meaningful, and reliable assays. Hydrophilic Interaction Chromatography (HILIC) is a powerful technique for the analysis of polar analytes. Despite being a well-established technique, HILIC method development can be laborious owing to the multiple factors that affect the separation mechanism, such as the selection of stationary phase chemistry, mobile phase eluents, and optimization of column equilibration time. Herein, we introduce a new automated multicolumn and multi-eluent screening workflow that streamlines the development of new HILIC assays, circumventing the existing tedious ‘hit-or-miss’ approach. A total of 12 complementary columns packed with sub-2 µm fully porous and 2.7 µm superficially porous particles operated on readily available ultra-high pressure liquid chromatography (UHPLC) instrumentation across a diverse set of commercially available polar stationary phases were investigated. Different mobile phases with pH ranging from pH 3 to 9 were evaluated using different organic modifiers. The gradient and column re-equilibration were judiciously set to ensure a reliable assay screening framework that indicates promising conditions for subsequent method optimization to achieve resolution of challenging mixtures. This UHPLC screening system is coupled with a diode array and charged aerosol detectors (DAD, CAD and mass spectrometry) to ensure versatile detection for a variety of compounds. This fast-screening platform lays the foundation for a convenient generic workflow, accelerating the pace of HILIC method development and transfer across both academic and industrial sectors.

Review on Recent Advance in High Performance Liquid Chromatography and Ultra-Performance Liquid Chromatography Instrumentation.

Review on Recent Advance in High Performance Liquid Chromatography and Ultra-Performance Liquid Chromatography Instrumentation.

Isomerization of E-Cinnamamides into Z-Cinnamamides Using a Recycling Photoreactor.

The photocatalytic synthesis of thermodynamically less-stable Z-alkenes has received considerable research attention in recent years. In this study, a recycling photoreactor was applied to the photoisomerization of E-alkenes (cinnamamide and Weinreb amide derivatives) to produce Z-alkenes. The closed-loop recycling system comprises an immobilized photosensitizer to achieve rapid photoisomerization and a high-performance liquid chromatography instrument for separation of the Z/E diastereomers. After 4-10 cycles, the desired pure Z-alkenes were obtained efficiently. In the photoreactor system, a photosensitizer (thioxanthone) was covalently immobilized on silica gel via amide bonding, which led to an enhanced photocatalytic activity compared to the parent thioxanthone. This recycling photoreactor shows promise as an alternative system for the production of Z-alkenes.

Exploring a reversible adaptation of conventional HPLC for capillary-scale operation

This study introduces a feasible approach for utilizing a conventional High-Performance Liquid Chromatography (HPLC) instrument at the capillary scale (1 - 10 µL/min). The development of an active flow splitter and an adapted UV-visible (UV–vis) detection cell are described. The system employs an Arduino Uno board to monitor a flow sensor and control a stepper motor that automates a split valve to achieve capillary-scale flow rates from a conventional pump. A capillary UV–vis cell compatible with conventional detectors, featuring an optical path length with a volume of 14 nL, was developed to address the detection challenges at this scale and minimize extra column band broadening. The system performance was assessed by a lab-packed LC capillary column with 0.25 mm x 15 cm dimensions packed with 3.0 µm C18 particles. Model compounds, particularly polycyclic aromatic hydrocarbons (PAHs), were employed to assess the functionality of all developed components in terms of theoretical plates, resolution, and band broadening. The proposed system is a profitable, reliable, and cost-effective tool for miniaturized liquid chromatography.

Evolutions in Particle, Surface Chemistry, and Hardware Designs: New Liquid Chromatography (LC) Columns and Accessories for 2024

This article covers liquid chromatography (LC) columns and accessories commercially released after Pittcon 2023 through the 2024 conference. As in the past, LCGC International sent out a survey in late 2023 and early 2024 asking vendors to supply information on products launched over the course of the year. Note that new products for gas chromatography (GC), LC instrumentation and software, and sample preparation are covered elsewhere. Information for this article is obtained over the course of many months, and thus, it is possible that some information could have been missed or misinterpreted. The reader is encouraged to check with specific vendor sites for additional product releases, as well as more detailed information on product usage and attributes. Links to vendor sites are provided where applicable.

An HPLC-SEC-based rapid quantification method for vesicular stomatitis virus particles to facilitate process development

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size exclusion chromatography (SEC) separation based on high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method's performance and applicability to in-process samples were characterized. The assay’s repeatability variation was < 1% and its intermediate precision variation was < 3%. The linear range of the method spans from 7.08×108 to 1.72×1011 VP/mL, with a limit of detection (LOD) of 7.72×107 VP/mL and a lower limit of quantification (LLOQ) of 4.20×108 VP/mL. The method, characterized by its high precision, requires minimal hands-on time and provides same-day results, making it efficient for process development.

Enhanced Light Absorption and Elevated Viscosity of Atmospheric Brown Carbon through Evaporation of Volatile Components.

Samples of brown carbon (BrC) material were collected from smoke emissions originating from wood pyrolysis experiments, serving as a proxy for BrC representative of biomass burning emissions. The acquired samples, referred to as "pyrolysis oil (PO1)," underwent subsequent processing by thermal evaporation of their volatile compounds, resulting in a set of three additional samples with volume reduction factors of 1.33, 2, and 3, denoted as PO1.33, PO2, and PO3. The chemical compositions of these POx samples and their BrC chromophore features were analyzed using a high-performance liquid chromatography instrument coupled with a photodiode array detector and a high-resolution mass spectrometer. The investigation revealed a noteworthy twofold enhancement of BrC light absorption observed for the progression of PO1 to PO3 samples, assessed across the spectral range of 300-500 nm. Concurrently, a decrease in the absorption Ångstrom exponent (AAE) from 11 to 7 was observed, indicating a weaker spectral dependence. The relative enhancement of BrC absorption at longer wavelengths was more significant, as exemplified by the increased mass absorption coefficient (MAC) measured at 405 nm from 0.1 to 0.5 m2/g. Molecular characterization further supports this darkening trend, manifesting as a depletion of small oxygenated, less absorbing monoaromatic compounds and the retention of relatively large, less polar, more absorbing constituents. Noteworthy alterations of the PO1 to PO3 mixtures included a reduction in the saturation vapor pressure of their components and an increase in viscosity. These changes were quantified by the mean values shifting from approximately 1.8 × 103 μg/m3 to 2.3 μg/m3 and from ∼103 Pa·s to ∼106 Pa·s, respectively. These results provide quantitative insights into the extent of BrC aerosol darkening during atmospheric aging through nonreactive evaporation. This new understanding will inform the refinement of atmospheric and chemical transport models.

ChromatoShiny: an interactive R/Shiny App for plotting chromatography profiles

Background Unicorn™ software on Äkta liquid chromatography instruments outputs chromatography profiles of purified biological macromolecules. While the plots generated by the instrument software are very helpful to inspect basic chromatogram properties, they lack a range of useful annotation, customization and export options. Methods We use the R Shiny framework to build an interactive app that facilitates the interpretation of chromatograms and the generation of figures for publications. Results The app allows users to fit a baseline, to highlight selected fractions and elution volumes inside or under the plot (e.g. those used for downstream biochemical/biophysical/structural analysis) and to zoom into the plot. The app is freely available at https://ChromatoShiny.bio.ed.ac.uk. Conclusions It requires no programming experience, so we anticipate that it will enable chromatography users to create informative, annotated chromatogram plots quickly and simply.

Effect of Hydrochar Modification on the Adsorption of Methylene Blue from Aqueous Solution: An Experimental Study Followed by Intelligent Modeling

Wheat straw, which is a carbon-rich precursor and a common agriculture waste in Sanandaj, was modified to produce hydrochar with high adsorption capacity by the hydrothermal carbonization (HTC) method. The hydrochars were tested as adsorbents to remove methylene blue (MB) from aqueous solution, and the effects of various interfering parameters, including pH, MB concentration, and adsorbent dosage, were investigated using artificial neural networks (ANNs) on adsorption modeling. Adsorption isotherms and kinetics were studied to explain the MB adsorption process. The prepared hydrochars were characterized using Brunauer–Emmett–Teller (BET), scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDAX), and high-performance liquid chromatography (HPLC) instruments. The maximum MB removal efficiency achieved by hydrochar modified by KOH (0.1 M) and adsorption data fitted well with the Langmuir isotherm and pseudo-second-order kinetics. In terms of elemental composition, the hydrochar sample contained 52.19% carbon (C), 3.37% hydrogen (H), 0.1% nitrogen (N), 0.15% sulfur (S), and 35.66% oxygen (O). The ash content in the sample was 8.50%. The recorded hydrogen-to-carbon ratio (H/C) and oxygen-to-carbon ratio (O/C) indicated a shift towards humification, implying the influence of KOH addition during the hydrochar production process. Additionally, the specific surface area of the hydrochar, as measured by the BET method, was found to be 11.54 m²/g. Among the aromatics, a significant presence of hydroxymethylfurfural (HMF) was detected, with a concentration of 4.70 g/kg DM. The modeling results demonstrated that the concentration of MB had the most substantial impact on the predicted removal, followed by pH, adsorbent dosage, and contact time.

Identification of Anthocyanidins in Anthocyanin Extract of Purple Sweet Potato (Ipomoea batatas l), black rice (Oryza sativa l), and Black Glutinous rice (Oryza sativa v. glutinosa)

Anthocyanins originating from different commodities have different constituent components and anthocyanidin levels. This will undoubtedly affect the anthocyanin content of total in foodstuffs. Local items such as; Purple sweet potato, black rice, and black glutinous rice have different structural components and levels of anthocyanidins. The research objective was to measure the total anthocyanin content and identify anthocyanidins qualitatively and quantitatively from anthocyanin extracts of purple sweet potato, black rice, and black glutinous rice. The research method used was an experimental method , qualitative and quantitative identification analyses were carried out using the UHPLC-PDA (Ultra high Performance Liquid Chromatographic) instrument system. Based on the study with pH-difference measurements, the total anthocyanin extract in purple sweet potato is 0.6 mg / g of material, much lower than black rice (5.50 mg / g of material) and black glutinous rice (6.00 mg / g of material). g material). Based on the identification of the purple sweet potato anthocyanin extract, the main components of anthocyanidins were Delfinidin, Cyanidin, and Malvidin with concentrations of 0.12 mg, 0.12 mg, and 1.20mg / 100g of ingredients, respectively. Meanwhile, black rice and black glutinous rice were identified with cyanidine and malvidin with concentrations of 0.73 and 13.17 mg / 100g and 1.07 mg and 17.6 mg / 100g of ingredients.

ChromatoShiny: an interactive R/Shiny App for plotting chromatography profiles.

Unicorn™ software on Äkta liquid chromatography instruments outputs chromatography profiles of purified biological macromolecules. While the plots generated by the instrument software are very helpful to inspect basic chromatogram properties, they lack a range of useful annotation, customization and export options. We use the R Shiny framework to build an interactive app that facilitates the interpretation of chromatograms and the generation of figures for publications. The app allows users to fit a baseline, to highlight selected fractions and elution volumes inside or under the plot (e.g. those used for downstream biochemical/biophysical/structural analysis) and to zoom into the plot. The app is freely available at https://ChromatoShiny.bio.ed.ac.uk. It requires no programming experience, so we anticipate that it will enable chromatography users to create informative, annotated chromatogram plots quickly and simply.

Strongly Adsorbing Analytes: What, Why, and How to Fix It

In many applications of liquid chromatography (LC), excellent peak shapes are observed with simple, easy-to-use mobile phases and operating conditions. However, some specific combinations of analyte and LC column chemistry can lead to terrible peak shapes and even total adsorption of analytes to components of the LC system such that no peaks are observed at all. In this installment of “LC Troubleshooting”, I discuss two specific cases where an understanding of specific interactions between particular analyte functional groups and LC instruments or column materials is critical to achieving successful separations.

IDSL.CSA: Composite Spectra Analysis for Chemical Annotation of Untargeted Metabolomics Datasets.

Poor chemical annotation of high-resolution mass spectrometry data limits applications of untargeted metabolomics datasets. Our new software, the Integrated Data Science Laboratory for Metabolomics and Exposomics─Composite Spectra Analysis (IDSL.CSA) R package, generates composite mass spectra libraries from MS1-only data, enabling the chemical annotation of high-resolution mass spectrometry coupled with liquid chromatography peaks regardless of the availability of MS2 fragmentation spectra. We demonstrate comparable annotation rates for commonly detected endogenous metabolites in human blood samples using IDSL.CSA libraries versus MS/MS libraries in validation tests. IDSL.CSA can create and search composite spectra libraries from any untargeted metabolomics dataset generated using high-resolution mass spectrometry coupled to liquid or gas chromatography instruments. The cross-applicability of these libraries across independent studies may provide access to new biological insights that may be missed due to the lack of MS2 fragmentation data. The IDSL.CSA package is available in the R-CRAN repository at https://cran.r-project.org/package=IDSL.CSA. Detailed documentation and tutorials are provided at https://github.com/idslme/IDSL.CSA.

Capacitively coupled contactless conductivity detection to account for system-induced gradient deformation in liquid chromatography

The time required for method development in gradient-elution liquid chromatography (LC) may be reduced by using an empirical modelling approach to describe and predict analyte retention and peak width. However, prediction accuracy is impaired by system-induced gradient deformation, which can be especially prominent for steep gradients. As the deformation is unique to each LC instrument, it needs to be corrected for if retention modelling for optimization and method transfer is to become generally applicable. Such a correction requires knowledge of the actual gradient profile. The latter has been measured using capacitively coupled “contactless” conductivity detection (C4D), featuring a low detection volume (approximately 0.05 μL) and compatibility with very high pressures (80 MPa or more). Several different solvent gradients, from water to acetonitrile, water to methanol, and acetonitrile to tetrahydrofuran, could be measured directly without the addition of a tracer component to the mobile phase, exemplifying the universal nature of the approach. Gradient profiles were found to be unique for each solvent combination, flowrate, and gradient duration. The profiles could be described by convoluting the programmed gradient with a weighted sum of two distribution functions. Knowledge of the exact profiles was used to improve the inter-system transferability of retention models for toluene, anthracene, phenol, emodin, sudan-I and several polystyrene standards.

Ion-Pair Chromatography for the Determination of Capreomycin Sulfate Components and Related Substances

Chromatographic methods for the analysis of antibiotic degradation products are widely used to evaluate the quality of medicines. Natural multicomponent antibiotics, such as capreomycin, are the most challenging compounds in terms of developing analytical procedures for related substances. Capreomycin sulfate monographs of the leading pharmacopoeias do not contain specifications for related substances. The key requirement concerns the sum of the main components of capreomycin calculated by normalising the peak areas in the test solution chromatogram. Therefore, it is important to develop an analytical procedure for determining not only the main components but also related substances of capreomycin.The aim of the study was to develop an analytical procedure for determining both the main components (IA, IB, IIA, and IIB) and related substances of capreomycin by ion-pair ultra-high-performance liquid chromatography (UHPLC).Materials and methods. This study examined capreomycin sulfate powder, an active pharmaceutical ingredient (API). Capreomycin sulfate solutions were analysed after artificial degradation (alkaline or acid hydrolysis) to demonstrate the resolution, selectivity, and efficiency of the experimental chromatographic system. The authors used an Agilent 1100 liquid chromatography instrument (Agilent Technologies) and chromatographic columns: Kinetex C18, YMC-Triart С18, ACQUITY UPLC BEH C18, ACQUITY UPLC BEH C8, ACQUITY UPLC BEH Phenyl, and ACQUITY UPLC CSH C18 (experimental procedure) or Acclaim C18, Zorbax SB-C18, and XBridge BEH130 C18 (The International Pharmacopoeia procedure).Results. In contrast to pharmacopoeial procedures, which evaluate only the component composition, the experimental procedure under the selected chromatography conditions can determine both the component composition and related substances of capreomycin. This advantage results from substituting a column packed with 1.7 µm particles for a 5 µm column required for pharmacopoeial procedures. The experimental procedure remains suitable for liquid chromatography instruments with a pressure limit of no more than 400 bar in the gradient elution mode with two mobile phases. According to the efficiency and selectivity evaluation, ACQUITY UPLC BEH C18 columns (150 × 2.1 mm, 1.7 µm) provide optimal peak resolution for capreomycin isoforms and related substances after artificial degradation of capreomycin.Conclusions. This experimental procedure based on ion-pair UHPLC may be used in the production and stability testing of capreomycin medicines to evaluate the API quality by the content of its main components and related substances.

Exploring Biopharmaceutical Analysis with Compact Capillary Liquid Chromatography Instrumentation.

A recent trend in the design of liquid chromatography (LC) instrumentation is the move towards miniaturized and portable systems. These smaller platforms provide wider flexibility in operation, with the opportunity for conducting analysis directly at the point of sample collection rather than transporting the sample to a centralized laboratory facility. For the manufacturing of pharmaceutical and biopharmaceutical products, these platforms can be implemented for process monitoring and product characterization directly in manufacturing environments. This article describes a portable, miniaturized LC instrument coupled to a mass spectrometer (MS) for characterization of a biopharmaceutical monoclonal antibody (mAb).

Evaluation of PGC Stationary Phases Under High-Temperature LC Conditions for the Analysis of Parabens in Food Samples

Porous graphitic carbon (PGC) columns for liquid chromatography (LC) represent an alternative to octadecyl‑bonded silica columns for the separation of both polar and nonpolar molecules. This is accomplished by exploiting the polarizability of the stationary phase interacting with the functional groups of the analytes. However, the elution of nonpolar compounds requires a high percentage of organic solvent, losing the intrinsic advantage of reversed‑phase aqueous separations. In this article, we aimed to exploit an additional advantage of such columns, viz. the resistance at high temperatures. Superheated water was employed as the mobile phase, taking advantage of the decrease in water dielectric constant by increasing the temperature. In this context, our goal was to minimize the percentage of organic solvent utilizing high temperatures (up to 250 °C) to achieve fast and “green” separations. The new developed high-temperature LC instrument was applied to the analysis of parabens in food samples.

Isolation of Glyphosate-Resistant Bacterial Strains to Improve the Growth of Maize and Degrade Glyphosate under Axenic Condition

Glyphosate is a non-selective herbicide that is used to control perennial weeds in agriculture. However, its vast application may result in glyphosate residues in the food chain. Due to its toxicity to non-target organisms, glyphosate-contaminated soils needed to be remediated, and bioremediation is a conventional remedial method. The success of this depends on the isolation of bacteria with the ability to degrade glyphosate. The goal of this study was to isolate glyphosate-degrading bacteria from the rhizosphere of maize and wheat with a repeated application history of glyphosate for 5–10 years and test their roles in promoting the growth of maize (Zea mays) and glyphosate degradation in vitro. Eleven isolated bacteria were inoculated, and their role in plant growth was compared at different levels (100 and 200 mg/kg) of glyphosate. The results revealed that E. ludwigii improved the highest shoot length by 26% and the root length by 34% compared to the control at 100 mg/kg. The relative water contents in leaves significantly improved by 58% using P. aeruginosa at 100 mg/kg. The maximum electrolyte leakage from leaves significantly reduced by 73% using E. ludwigii at 100 mg/kg compared to the control (uninoculated). A high-pressure liquid chromatography instrument was used to assess the glyphosate concentrations. The highest degradation of glyphosate was observed in treatments inoculated with E. ludwigii (99 and 40%), P. aeruginosa (95 and 39%), K. variicola, (91 and 38%) E. cloacae (92 and 38%), and S. liquefaciens (87 and 36%), respectively, at 100 and 200 mg/kg within 28 days. These five strains demonstrated a great potential for degrading glyphosate and promoting the growth of maize in vitro, and they will be further exploited for the biodegradation of glyphosate and the growth promotion of broader crop species in situ in the near future.