Peak Tailing Research Articles

Comparative Studies on Simultaneous Estimation of Metformin, Empagliflozin, Remogliflozin, Glimepride and Gliclazide by using RP-HPLC and Diol Technique

Retaining extremely polar metformin and its combination with other non-polar antidiabetic drugs can be difficult and challenging by using the most common reverse-phase high-performance liquid chromatography (RP-HPLC). The retention time of metformin is comparatively short when separated, utilizing C18-based RPC. Peak fronting and tailing effects are commonly observed with metformin (MET), its combination with highly polar antidiabetic medicines, and early elution with void volume. However, normal phase chromatography may occasionally be rendered ineffective due to the low solubility of polar analytes in organic solvents. Therefore, a different and complementary strategy is required for the separation of such a highly polar drug. A more advantageous platform is mixed-mode chromatography. The use of this newer technique of separations has been increased because of its advantages over C18-based RPC for its efficiency, selectivity, sensitivity, specificity and reproducibility towards simultaneous quantification of selected drugs. In this study, we compared two established chromatographic methods for simultaneous quantification of metformin and other moderately polar antidiabetic medications, including empagliflozin, remogliflozin, glimepride, and gliclazide, both in their pure form and in combination tablet dosage. An isocratic elution based on solvent a 15 mM ammonium acetate-Methanol: acetonitrile (10:90 v/v) was used as the mobile phase in first method, which is reversed-phase HPLC with a Zodiac C18 column and chromatographic separation on a 150 × 4.6 mm acclaimed mix mode HILIC-1 column with a particle size of 5 μm. In accordance with ICH standards, both approaches underwent exhaustive validation for linearity, accuracy, precision, selectivity, and resilience. Metformin, empagliflozin, ramogliflozin, glimepride, and gliclazide have not yet been reported to be separated using any method.

An Ultra-Throughput Boost Method for Gamma-Ray Spectrometers

(1) Background: Generally, in nuclear medicine and nuclear power plants, energy spectrum measurements and radioactive nuclide identification are required for evaluation of strong radiation fields to ensure nuclear safety and security; thereby, damage is prevented to nuclear facilities caused by natural disasters or the criminal smuggling of nuclear materials. High count rates can lead to signal accumulation, negatively affecting the performance of gamma spectrometers, and in severe cases, even damaging the detectors. Higher pulse throughput with better energy resolution is the ultimate goal of a gamma-ray spectrometer. Traditionally, pileup pulses, which cause dead time and affect throughput, are rejected to maintain good energy resolution. (2) Method: In this paper, an ultra-throughput boost (UTB) off-line processing method was used to improve the throughput and reduce the pileup effect of the spectrometer. Firstly, by fitting the impulse signal of the detector, the response matrix was built by the functional model of a dual exponential tail convolved with the Gaussian kernel; then, a quadratic programming method based on a non-negative least squares (NNLS) algorithm was adopted to solve the constrained optimization problem for the inversion. (3) Results: Both the simulated and experimental results of the UTB method show that most of the impulses in the pulse sequence from the scintillator detector were restored to δ-like pulses, and the throughput of the UTB method for the NaI(Tl) spectrometer reached 207 kcps with a resolution of 7.71% @661.7 keV. A reduction was also seen in the high energy pileup phenomenon. (4) Conclusions: We conclude that the UTB method can restore individual and piled-up pulses to δ-like sequences, effectively boosting pulse throughput and suppressing high-energy tailing and sum peaks caused by the pileup effect at the cost of a slight loss in energy resolution.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

Highly efficient GPCR immobilization with enhanced fouling resistance, salt tolerance, and chromatographic performance

The feasibility of immobilized protein-based biodetection relies critically on the activity of the immobilized proteins as well as the biocompatibility of the protein surface. Although many protein immobilization strategies have been developed with satisfied detection readout signals. Non-specific interactions caused by the protein-coating surface are still of great concern since they often interfere with or affect the reliability of detection. Herein, we developed a highly efficient G protein-coupled receptor (GPCR) immobilization method by the combination of polyethylene glycol (PEG) with a self-labeling enzyme-catalyzed reaction. The immobilization relies on the covalent interaction between the fusion tag of a target GPCR (kinase domain of epidermal growth factor receptor, EGFR) and its covalent inhibitor ibrutinib, which is modified on PEGylated silica gels. Two types of GPCRs, N-methyl-D-aspartate 2 A receptor (NMDAR2A) and endothelin A receptor (ETAR), were used as examples to realize protein immobilization. The GPCR modified gels and the affinity columns packed with them have been extensively characterized, in terms of non-specific adsorptions, retention factor (k′), half peak width (W1/2), tailing factor (Tf), theoretical plates (N), and association and dissociation constants of the ligands with the receptors. The immobilized GPCRs with reduced non-specific interactions and enhanced fouling resistance, salt tolerance, and chromatographic performance were clearly observed. We believe it is the first work to introduce PEGylation in GPCR immobilization and provide comprehensive proof-of-concept studies to illustrate the improved antifouling property, salt tolerance, and chromatographic performance. This method could be generally applicable in other immobilized protein-based technology for reliable biodetection.

Physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns.

The analysis of mixtures containing monoclonal antibody (mAb) (approximately 150 kDa molecular weight) and sub-unit impurities (approximately 100 kDa) is challenging, even when adopting the latest ultra-high-pressure liquid chromatography (UHPLC) columns (4.6 mm [Formula: see text] 150mm coated hardware, 1.7 [Formula: see text]m 250 BEH[Formula: see text] Surface-modified Particles) and systems (ACQUITY[Formula: see text] UPLC[Formula: see text] I-class Bio Plus). The main issue still encountered is a persistent tail of the mAb peak. Here, the physical origin(s) of such peak tailing in size-exclusion chromatography (SEC) are investigated from both fundamental and practical approaches. Up to five relevant physical origins are analyzed: sample heterogeneity (glycoforms), UHPLC system dispersion, strong residual binding of the mAb to the SEC particles (via hydrophobic and/or electrostatic interactions) and to the stainless steel column/system hardware, slow escape kinetics of the mAb from the SEC particles, and flow heterogeneity caused by the non-ideal slurry packing of SEC columns. Experiments (testing sample heterogeneity, system dispersion, and strong residual interactions) and calculations (predicting the transient absorption/escape kinetics in a single SEC particle and the two-dimensional peak concentration profiles) altogether unambiguously demonstrate that the observed mAb peak tailing is caused primarily by the long-range velocity biases across the SEC column combined with the slow transverse dispersion of mAbs. Therefore, improvement in the resolution between mAb and sub-unit fragment impurities can only be achieved by increasing the column length, e.g., by applying recycling chromatography at acceptable pressures.

Accumulation of Large Ion Populations with High Ion Densities and Effects Due to Space Charge in Traveling Wave-Based Structures for Lossless Ion Manipulations (SLIM) IMS-MS.

The accumulation of very large ion populations in traveling wave (TW)-based Structures for Lossless ion Manipulations (SLIM) has been studied to better understand aspects of "in-SLIM" ion accumulation, and particularly its use in conjunction with ion mobility spectrometry (IMS). A linear SLIM ion path was implemented that had a "gate" for blocking and accumulating ions for arbitrary time periods. Removing the gate potential caused ions to exit, and the spatial distributions of accumulated ions examined. The ion populations for a set of peptides increased approximately linearly with increased accumulation times until space change effects became significant, after which the peptide precursor ion populations decreased due to growing space charge-related ion activation, reactions, and losses. Ion activation increased with added storage times and the TW amplitude. Lower amplitude TWs in the accumulation/storage region prevented or minimized ion losses or ion heating effects that can also lead to fragmentation. Our results supported the use of an accumulation region close to the SLIM entrance for speeding accumulation, minimizing ion heating, and avoiding ion population profiles that result in IMS peak tailing. Importantly, space charge-driven separations were observed for large populations of accumulated species and attributed to the opposing effects of space charge and the TW. In these separations, ion species form distributions or peaks, sometimes moving against the TW, and are ordered in the SLIM based on their mobilities. Only the highest mobility ions located closest to the gate in the trapped ion population (and where the highest ion densities were achieved) were significantly activated. The observed separations may offer utility for ion prefractionation of ions and increasing the dynamic range measurements, increasing the resolving power of IMS separations by decreasing peak widths for accumulated ion populations, and other purposes benefiting from separations of extremely large ion populations.

Interpretation of ion cyclotron emission from sub-Alfvénic beam-injected ions heated plasmas soon after L-H mode transition in EAST

Ion cyclotron emission (ICE) at deuterium ion cyclotron harmonics, driven by sub-Alfvénic beam-injected deuterium ions, has been observed by the high-frequency B-dot probe in the EAST tokamak. The origin of ICE shifts from the plasma core to the plasma edge soon after an L-H mode transition, where the beam-injected deuterium ions have a relatively peak bump-on tail structure in the energy direction and a very intense pitch angle anisotropy. Based on the fast ion distribution function obtained from the TRANSP/NUBEAM code, together with a linear analysis theory of magnetoacoustic cyclotron instability (MCI), the growth rates of MCI could be calculated. It is shown that MCI, resulting in the generation of obliquely propagating fast Alfvén waves at deuterium ion cyclotron harmonics, can occur under such conditions. And the temporal evolution of the MCI growth rate closely follows that of the observed ICE amplitude in the EAST.

Accurate Estimation of Some Pharmaceutical Compounds Using HPLC Technology

For the assessment of different chemical components in pharmaceutical and biological samples, High Performance Liquid Chromatography (HPLC) can be defined as a commonly utilized analytical method in pharmaceutical samples. HPLC offers high sensitivity, specificity, and accuracy, making it a preferred method for the analysis of complex samples. In this technique, the sample is prepared by extracting, purifying, and concentrating the target analyte from the matrix. Mobile and stationary phases are after that used in chromatographic separation of the prepared sample. Several detectors, including fluorescence, UV-vis, and mass spectrometry, are used to identify and measure the eluted compounds. HPLC is capable of estimating a wide range of elements, including small molecules, peptides, and proteins, making it an essential tool in pharmaceutical research and development. The theoretical plates, peak tailing, and % assay have not been significantly impacted by the deliberate alterations made to the method. This implies that the existing technique is robust. The low values of LOQ and LOD show how sensitive the suggested approach is. The current research used a standard drug solution with six replicates to examine the system suitability parameters. It was discovered that the computed parameters fell under the acceptable criteria. The theoretical plate count, tailing factor, and HETP parameters are all within acceptable ranges. The results of this work are compared with those of earlier research. Upon analyzing the chart, it can be inferred that the proposed method presents certain benefits. The investigation employed a Kromasil C18 column. The obtained retention time is comparatively lower than that of all other methodologies. The maximum quantifiable threshold is 600 μg/mL, surpassing or equating all of the aforementioned techniques. The duration of the execution is also minimal. Therefore, the author draws the conclusion that the HPLC method proposed exhibits sensitivity and reproducibility in analysis of etodolac and thiocolchicoside in combined tablet dosage form, while also requiring a short analysis time.

Abstract 13049: Electrophysiological Mechanism of Potassium Channel KCNQ1 p.V162M Mutation Induced Atrial Tachycardia and Epilepsy

Objective: Arrhythmia and epilepsy are the causes of syncope and sudden death. Both of them are associated with abnormal function of potassium ion channels. Delayed rectified potassium current (IKs) is generated by combining the α subunit of potassium ion channel encoded by KCNQ1 gene with the β subunit encoded by KCNE1 gene. It plays an important role in electrical excitation and K+ circulation of nerve cells. In this study, a patient with atrial arrhythmia and epilepsy with a history of syncope was identified by genetic testing as having a mutation in the KCNQ1 p.V162M gene. The purpose of this study was to investigate the effect of KCNQ1 p.V162M mutation on the structure and function of potassium ion channels. Methods: The mutant plasmid KCNQ1-p.V162M was constructed for cell transfection. The expression of protein and mRNA were analyzed by western blotting and real-time quantitative PCR. The IKs of the two groups were detected by whole-cell patch clamp technique. Results: Whole exon gene sequencing revealed that the patient carried the KCNQ1 p.V162M mutation. The results of molecular biology experiment showed that mRNA, KCNQ1 total protein and membrane protein expressions in mutant group were significantly lower than those in wild group. Whole-cell patch clamp results showed that the peak current density and tail current of IKs in the mutant group were significantly decreased compared with those in the wild group, indicating that the mutation of KCNQ1 p.V162M could lead to decreased function of potassium ion channels. Conclusions: KCNQ1 p.V162M mutation can lead to decreased expression of KCNQ1 protein and decreased function of potassium ion channel, which may be the potential mechanism of atrial tachycardia and epilepsy induced by this mutation.

The Evolution of LC Troubleshooting: Strategies for Improving Peak Tailing

As chromatography technologies change, our approaches to using them in ways that value reliability, as well as the ways we should approach troubleshooting and fixing problems, should also change. In this installment, we continue the discussion started earlier this year about how some specific troubleshooting topics have evolved over time, this time focusing on chemical causes of peak tailing, and strategies that we can use to overcome them. Understanding how knowledge and technologies related to peak tailing have evolved is helpful for troubleshooting problems with peak shape, designing reliability into new methods, and when considering updates to legacy methods that have been in use for decades.

Addressing Acid-Catalyzed Deamidation and the Solubility of Hydrophobic Peptides in Multi-Attribute Method Workflows.

Recently, we introduced an optimized and automated Multi-Attribute Method (MAM) workflow, which (a) significantly reduces the number of missed cleavages using an automated two-step digestion procedure and (b) dramatically reduces chromatographic peak tailing and carryover of hydrophobic peptides by implementing less retentive reversed-phase column chemistries. Here, further insights are provided on the impact of postdigest acidification and the importance of maintaining hydrophobic peptides in solution using strong chaotropic agents after digestion. We demonstrate how oxidation can significantly increase the solubility of hydrophobic peptides, a fact that can have a profound impact on quantitation of oxidation levels if care is not taken in MAM workflows. We conclude that (a) postdigestion acidification can result in significant acid-catalyzed deamidation during storage in an autosampler at 5 °C and (b) a strong chaotropic agent, such as guanidine hydrochloride, is critical for preventing loss of hydrophobic peptides through adsorption, which can result in (sometimes extreme) biases in quantitation of tryptophan oxidation levels. An optimized method is presented, which effectively addressed acid-catalyzed deamidation and solubility of hydrophobic peptides in MAM workflows.

Challenges with retention and recovery of impurities containing acidic moieties during analytical UHPLC method development and validation for gefapixant freebase

Gefapixant citrate is a P2X3 purinergic receptor antagonist developed for the treatment of chronic cough. Gefapixant freebase is the penultimate intermediate in the commercial manufacturing route for gefapixant citrate and contains a complex impurity profile consisting of acidic and basic analytes. A UHPLC method was developed for assay and purity determination of gefapixant freebase utilizing a Waters Acquity Charged Surface Hybrid (CSH) C18 column (2.1 mm I.D. x 10 cm length, 1.7 µm particle size) with 0.1 % phosphoric acid and acetonitrile as the mobile phases. Method optimization was performed using ACD Labs LC Simulator to achieve baseline separation of all impurities and the method was successfully validated. During routine use and method transfer for gefapixant freebase, an increase in retention time for impurities containing strongly acidic functional groups, and poor recovery of a sulfinic acid impurity were observed. Subsequent investigation determined that the CSH column aging resulting from exposure of the column packing to the acidic mobile phase was the root cause for these behaviors. The mechanism of peak-shifting was further investigated using model compounds and determined to be due to an increase in ionic interactions with the CSH stationary phase with routine column use. The increase in ionic interactions was demonstrated to correlate with the charge state of the analyte. Poor recovery for the sulfinic acid impurity was attributed to increased peak tailing for this single impurity on older columns. This knowledge was leveraged to establish additional system suitability requirements to monitor column performance for the lifecycle of the analytical procedure.

Prolonged Exposure to Remdesivir Inhibits the Human Ether-A-Go-Go-Related Gene Potassium Current.

Remdesivir, approved for the treatment of COVID-19, has been associated with heart-rate corrected QT interval (QTc) prolongation and torsade de pointes in case reports. However, data are conflicting regarding the ability of remdesivir to inhibit the human ether-a-go-go-related gene (hERG) -related current. The objective of this study was to investigate the effects remdesivir and its primary metabolite, GS-441524, on hERG-related currents. Human embryonic kidney 293 cells stably expressing hERG were treated with various concentrations of remdesivir and GS-441524. The effects of acute and prolonged exposure on hERG-related current were assessed using whole-cell configuration of voltage-clamp protocols. Acute exposure to remdesivir and GS-441524 had no effect on hERG currents and the half-activation voltage (V 1/2 ). Prolonged treatment with 100 nM and 1 µM remdesivir significantly reduced peak tail currents and hERG current density. The propensity for remdesivir to prolong QTc intervals and induce torsade de pointes in predisposed patients warrants further investigation.

An Empirical Study on the Logarithmic Return of Securities

Objective: Previous studies have shown that the distribution of security returns has the characteristics of peak and fat tail, and it is not realistic to use logarithmic normal distribution to describe security returns. This paper mainly studies the specific form of security returns distribution and conducts empirical research. Methods: We first assume that the security returns follow a logarithmic t-distribution. Then, the maximum likelihood estimation and moment estimation are used to explore the degree of freedom of the distribution. Finally, the goodness of fit test is used to conduct an empirical analysis about Shanghai Securities Composite Index. Results: The specific expression formulas for the estimator of the degree of freedom are obtained. Empirical testing shows that the security returns follow a logarithmic t-distribution. Conclusion: The probability distribution of security returns is unrealistic to assume that the security returns follow the logarithmic normal distribution. It is more practical to use the logarithmic t-distribution to characterize the distribution of security returns. Our results provide theoretical assistance for exploring the pricing of financial derivatives.

Stagnant pore-blocked supports for improved extraction chromatography

Prior work has demonstrated that the complex pore structure of the polymeric supports employed for the preparation of commercial extraction chromatographic resins is a major contributor to their poor column efficiency and susceptibility to pronounced peak tailing. Particularly problematic is the presence of a number of small, relatively inaccessible (“stagnant”) pores. By blocking these pores with an appropriate inert filler, the extractant can be confined to the more readily accessible intermediate and large pores. The result is improved column efficiency (i.e., up to a 40% increase in N), reduced peak tailing and asymmetry (i.e., up to a 34% reduction in As), and in some instances, higher metal ion uptake capacity (up to a 64% increase) versus a commercial resin. The utility of the improved chromatographic performance arising from this “stagnant pore blocking” is illustrated in the separation of selected trivalent lanthanide ions, specifically Nd(III) and Eu(III), whose resolution has been reported to be unsatisfactory using conventional extraction chromatographic materials.

Hydro-organic mobile phase and factorial design application to attain green HPLC method for simultaneous assay of paracetamol and dantrolene sodium in combined capsules

The greenness of any analytical method has become a very important aspect of a good analytical method. However, most chromatographic methods depend on the usage of relatively large amounts of lethal and un-decaying chemicals and solvents. So, a green approach based on the full factorial design was employed to develop a simple and rapid HPLC technique for concurrent determination of paracetamol and dantrolene sodium in their combined capsules. Both drugs are highly recommended to be administered together in patients with severe musculoskeletal disorders. Avoiding the routine methodology and resorting to the modern technology represented in the usage of experimental design allows rapid determination of the studied drugs using the optimum quantity of chemicals to avoid any waste of resources. Simultaneous separation of a binary mixture of paracetamol and dantrolene sodium was accomplished using a reversed phase Hypersil C18 column using an eco-friendly isocratic eluent. The used mobile phase consisted simply of ethanol: water (40:60, v/v). Orthophosphoric acid was used to adjust the pH of the mobile phase to 4.5. Triethanolamine (0.2%) was added aiming to reduce the peak tailing. The assay was completed within less than 6 min adopting 0.8 mL/min as a flow rate. The detection was carried out using a UV-detector at 290 nm. The suggested technique shows a linear correlation over concentration ranges of 1.0–200 and 1.0–40 µg/mL for paracetamol and dantrolene sodium, respectively. The suggested technique allowed the simultaneous analysis of the two co-formulated drugs in their synthetic mixture and combined capsule. The suggested technique is considered a greener substitute for the other reported HPLC techniques through the usage of safer solvents and chemicals, along with decreasing both waste output and analysis time. The method is accurate with recoveries between 97.85 and 101.27%, precise, as %RSD for the intraday and interday precision were between 0.39 and 1.72% and very sensitive with limits of detection (LOD)’s 0.15 and 0.18 µg/ml and limits of quantification (LOQ)’s 0.48 and 0.61 µg/ml for paracetamol and dantrolene sodium, respectively. The method greenness was ensured through its assessment by four greenness metrics. It is also validated following the International Conference on Harmonization Guidelines. The recommended technique could be a good alternative to traditional methods in the routine quality control analysis of the studied drugs due to its minimum harm to the planet or human beings.

Continuous monitoring of a monoclonal antibody by size exclusion chromatography reveals a correlation between system suitability parameters and column aging

Size exclusion chromatography (SEC) is a foundational analytical method to assess product purity of biological molecules. To ensure accurate and reproducible data that meet regulatory agency standards, it is critical to monitor the chromatographic column with efficient and continuous approaches. In this study, 19 SEC columns (Waters Acquity BEH200) were evaluated using an in-house monoclonal antibody made at Regeneron. System suitability parameters (SSPs) were used to monitor the performance of the SEC assay, including USP resolution, USP plate count, USP tailing factor, asymmetry factor, elution time, peak width, and peak height. A general linear model was built and revealed that elution time, peak width, asymmetry factor, and tailing factor increased with injection number, while peak height, resolution, and plate count decreased. After 1000 injections, tailing factor and peak width increased by more than 10%, while resolution and plate count decreased by more than 10% from their respective starting values.

Interlaboratory development and proposition for a new quality control sample for chemical forensics analysis of chemical warfare agents

A new quality control (QC) test sample for gas chromatography–mass spectrometry (GC–MS) was created and analysed to test the comparability and repeatability of chemical forensics results within the Organisation for the Prohibition of Chemical Weapons (OPCW)–designated laboratories. The QC test sample was designed in collaboration between four laboratories and consists of 27 compounds which evaluate the performance of GC–MS instruments. This solution was analysed with GC–MS(EI) in 11 laboratories, seven of which were OPCW designated. The participating laboratories analysed the sample multiple times on consecutive days, as well as after the analysis of a set of complex matrix samples. Retention times, retention indices, peak areas, peak tailing values, signal-to-noise ratios, and isotope ratios were extracted from the GC–MS data, and statistical multivariate analyses with principal component analysis and Hotelling's T2-tests were conducted. The results from these analyses indicate that differences between GC–MS analyses by multiple laboratories were not statistically significant at the 5% level, as the approximate p-value for the null hypothesis of “no differences between the runs” was 0.69. However, similar data processing methods and data normalisation are essential for enabling the reliable comparison of chemical fingerprints between laboratories. A composition for the QC sample and criteria for acceptable GC–MS performance for chemical forensics are proposed. The composition and criteria differ from the currently used chemical weapons verification analysis QC sample by e.g. broadening the range for retention index calculations by addition of new alkane compounds, including new chemicals with concentrations close to the limit of detection (10–100 ng/ml), and including compounds with higher polarity to emulate real-life forensic samples. The proposed criteria include monitoring of retention indices, isotope ratios, peak tailing, signal-to-noise ratios, peak height, mass spectra, and sensitivity of the instrument. The new compounds and criteria will be the subject of future confidence building exercises to validate their relevancy on a large scale.

Resolution limits of size exclusion chromatography columns identified from flow reversal and overcome by recycling liquid chromatography to improve the characterization of manufactured monoclonal antibodies

The flow reversal (FR) technique consists of reversing the flow direction along a chromatographic column. It is used to reveal the origin (such as poor column packing, active sites, or slow absorption/escape kinetics) for the resolution limit of 4.6 mm × 150 mm long columns packed with 1.7 μm 200 Å Bridge-Ethylene-Hybrid (BEHTM) Particles. These columns are used to separate manufactured monoclonal antibodies (mAb, ∼ 150 kDa) from their close impurities (or IdeS fragments, ∼ 100 kDa) by size exclusion chromatography (SEC). FR unambiguously demonstrates that the resolution limit of these SEC columns is primarily due to long-range flow velocity biases covering distances of at least 500 μm across the column diameter. This confirms the existence of center-to-wall flow heterogeneities which cause undesirable tailing for the mAb peak. Because the transverse dispersion coefficient (Dt=1.1 × 10−6 cm2/s) of mAbs across the column diameter is intrinsically low, the bandspreading of the mAb in a single flow direction is in part reversible upon reversing the flow direction. For the very same residence time in the column, the column efficiency is found to increase by +85% relative to that observed under conventional elution mode. The observed peak tailing of the mAb and its sub-units is not caused by active surface sites or by slow absorption/escape from the BEH Particles. Therefore, the most critical mAb impurities (hydrolytic degradation Fab/c and IdeS F(ab′)2 fragments) can only be successfully separated and quantified with acceptable accuracy by adopting alternate pumping recycling liquid chromatography (APRLC). APRLC enables the full baseline separation of the mAb and 100 kDa mAb fragments and partial separation of Fab/c and F(ab′)2 fragments after increasing the number of cycles to ten. It was made possible to accurately measure the relative abundances of the mAb (99.0 ± 0.1%), F(ab′)2 fragment (0.88 ± 0.03%), and Fab/c immunogenic fragment (0.13 ± 0.02%) in less than 45 min for a total mAb sample load of only 5 μg. Still, further improvements are needed to increase the sensitivity of the APRLC method and to reduce the solvent consumption by adopting narrow-bore 2.1 mm i.d. SEC columns.

Simultaneous determination of femtogram levels of 237Np, 239Pu, and 240Pu in environmental solid samples using extraction chromatography and ICP-MS/MS

An analytical method based on triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS/MS) was developed for simultaneous determination of femtogram levels of 237Np, 239Pu, and 240Pu in environmental samples. By carefully controlling the valence states of Np and Pu in the entire separation procedure using a simple single extraction chromatographic column (TK200), the consistent behavior of Np and Pu was achieved, allowing for the reliable application of 242Pu as the chemical yield tracer for 237Np, 239Pu, and 240Pu. A high decontamination factor of 3.2 × 107 for the most critical interfering element, uranium, was achieved in the chemical separation step. The interferences of 238U1H+ and peak tailing of 238U+ during the measurement of plutonium isotopes were effectively eliminated by utilizing 7.5 mL/min He–1.1 mL/min CO2 as reaction gases in the octupole collision/reaction cell and employing sequential quadrupole mode for mass separation in ICP-MS/MS. Specifically, the interference of 238U1H+ was reduced to 10−6 and the peak tailing of 238U+ to 10−10, surpassing the performance of measurement method without reaction gases by 3 orders of magnitude. The developed method enables the accurate determination of femtogram levels of 237Np, 239Pu, and 240Pu in the samples with U/Np and U/Pu atom ratios of up to 1017 and 1012, respectively. The developed method was validated by analyzing standard reference materials and spiked soil samples.