Online ligand screening for cytochrome C from herbal medicines through "four-in-one" measurement.

Affinity-oriented online ligand screening with LC coupled to different detectors is widely popular to capture active compounds from herbal medicines (HMs). However, false-positive extensively occurs because insufficient information is recorded for the existence and stability of ligand-protein complex. Here, efforts were made to advance the hit confidences via configuring post-column infusion-LC-energy-resolved-affinity MS (PCI-LC-ER-AMS) to achieve "four-in-one" monitoring of: 1) response decrement of potential ligands; 2) response decrement of protein; 3) ions relating to ligand-protein complexes; and 4) ligand-protein binding strength. Ligand fishing for Cyt C from HMs was conducted as a proof-of-concept. For utility justification, a mimic sample containing twelve well-defined ligands and two negative controls underwent LC separation and met Cyt C prior to Qtof-MS measurements. Compared to Cyt C- or ligand-free assay, twelve ligands instead of negative controls showed response decrements that were consistent with twelve negative peaks observed at retention times corresponding to the ligands in Cyt C ion current chromatogram. Serial ions correlating to each ligand-Cyt C complex were observed. After recording breakdown graphs, optimal collision energy (OCE) corresponding to the non-covalent bond dissociation was positively correlated with binding strength. Two HMs including Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata were investigated. Consequently, 24 compounds were merely fished from SR, and particularly, flavonoid glycosides exhibited greater OCEs and also binding strengths over aglycones. Affinity assays and cellular evaluations consolidated the significant interactions between each captured compound and Cyt C. Overall, PCI-LC-ER-AMS is eligible for confidence-enhanced online ligand screening for Cyt C from HMs through "four-in-one" measurement.

Standardization via Post Column Infusion-A Novel and Convenient Quantification Approach for LC-MS/MS.

Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach-quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques.

Developments in on-line, post separation sample manipulation in the last 22 years: Pharmaceutical and biomedical applications.

On-line post separation sample manipulation is a powerful approach increasing the sensitivity and selectivity in chemical analysis. Post separation sample manipulation includes the treatment of the analytes after their separation through a suitable separation technique, mainly liquid chromatography and capillary electrophoresis. Typically, post separation approaches include either the addition of a reagent/solvent to derivatize the analyte/enhance the sensitivity, pH change, or the conversion of the analyte through a photochemical/electrochemical system (reagent-free systems). This review focuses on the developed methods using post-column manipulation of sample with pharmaceuticals and biomedical applications, covering the period from 2000 to midle-2023. Chemistries combined with fluorescence, UV-vis and mass spectrometric detection are discussed employing both liquid chromatography and electrophoretic techniques for separation. Noteworthy instrumental modifications are also discussed.

Influence of Zhuanggu Guanjie Pill on Seven Cytochrome P450 Enzymes Based on Probe Cocktail and Pharmacokinetics Approaches.

BACKGROUND: The use of herbal medicines has tremendously increased over the past few decades. Case reports and controlled clinical investigations of herbal-drug interactions have been reported. Since Cytochrome P450 (CYP) enzymes play an important role in drug interactions. The evaluation of the influence of herbal medicines on the activities of CYPs is beneficial to promote scientific and rational clinical use of herbal medicines. OBJECTIVE: Herein, we aimed to develop and validate a method to simultaneously quantify seven CYP cocktail probe drugs consisting of phenacetin (PNC), bupropion (BPP), losartan potassium (LK), omeprazole (OMP), dextromethorphan (DM), chlorzoxazone (CZZ) and midazolam (MDZ) and their respective metabolites in a single acquisition run and use this method to evaluate the influence of Zhuanggu Guanjie Pill (ZGGJP) on seven CYPs. METHODS: A cost-effective and simple UHPLC-(±)ESI-MS/MS method for simultaneous determination of seven probe drugs and metabolites in rat plasma was developed and validated. Male and female rats were randomly divided into three groups and treated with 1.2 g/kg/d ZGGJP, 5 g/kg/d ZGGJP and 0.5% CMC-Na for 14 consecutive days. After 24 h of the last administration, all rats were administrated orally with probe drugs. The influence of ZGGJP on the CYPs was carried out by comparing the metabolic ratio (Cmax, AUC0-t) of metabolites/probe drugs in rats. RESULTS: The calibration curves were linear, with correlation coefficient > 0.99 for seven probe drugs and their corresponding metabolites. Intra- and inter-day precisions were not greater than 15% RSD and the accuracies were within ±15% of nominal concentrations. The ZGGJP showed significant inductive effect on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats. CONCLUSION: ZGGJP had inductive effects on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats.

Determination of eight neonicotinoid insecticides in Chinese cabbage using a modified QuEChERS method combined with ultra performance liquid chromatography-tandem mass spectrometry.

A modified QuEChERS method was developed and combined with ultra performance liquid chromatography-tandem mass spectrometry to analyze eight neonicotinoid insecticides in Chinese cabbage. Herein, acetonitrile served as extraction solvent. Anhydrous sodium sulfate replaced the traditional anhydrous magnesium sulfate in the phase partition process to eliminate the influence of salt caking and heat release. Primary secondary amine and graphitized carbon black were selected as dispersive-solid phase extraction sorbents according to the clean-up efficiency evaluated by post-column infusion. The method showed good sensitivity with the limits of quantification below 1.0 µg/kg for eight analytes. The average recoveries ranged from 79.0% to 108.4% for three fortification levels, and the matrix effect could be ignored. For the tested samples, six neonicotinoids were detected, while cycloxaprid and imidaclothiz were not. The developed analytical method provided a powerful tool for monitoring neonicotinoid insecticides residues in Chinese cabbage.

Development and validation of an ultra performance liquid chromatography-tandem mass spectrometry method for twelve bisphenol compounds in animal feed.

Bisphenol compounds (BPs) are a group of environmental contaminants with endocrine-disrupting effects both for humans and animals. The present work developed a sensitive analytical method for the detection of multiple BPs in the animal feed based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with post-column ammonium hydroxide (NH4OH) infusion. A modified QuEChERS method was incorporated into the extraction and purification processes. The limit of detection (LODs) and quantification (LOQs) for the target BPs were in the ranges of 0.02-0.75 µg kg-1 and 0.04-0.95 µg kg-1, respectively. Average recoveries were ranged between 82.6% and 112%. The proposed method was successfully applied to determine the concentrations of BPs in 20 actual feed samples, and the preliminary profiles of BPs in products from local feed factories were obtained. Each sample was simultaneously contaminated with at least 2 to 4 BPs, and bisphenol A (BPA) was the dominant analog of BPs found in animal feed.

Quantification of vitamin D3 and 25-hydroxyvitamin D3 in food - The impact of eluent additives and labelled internal standards on matrix effects in LC-MS/MS analysis.

Deuterated vitamin D standards are used commonly as internal standards in LC-MS/MS analysis of vitamin D3 and 25-hydroxyvitamin D3 in food. However, the use of various eluent additives, such as methylamine, formic acid and ammonium formate, also contributes to matrix effects and the performance of analysis by affecting accuracy and robustness. For the first time, continuous post-column infusion experiments of isotopically labelled vitamin D3-[d6] were performed to evaluate ion-suppression in a wide variety of food (salmon, cheese, pork fat, pork meat, and egg yolk). Furthermore, results collected using five analytical methods, employing DAD/UV and MS/MS-detectors, were evaluated with in-house and standardised reference materials. The matrix effect was significant when analysing vitamin D3 in most food matrices using the deuterium labelled internal standard. Even though the use of the 13C5-labelled internal standard reduced matrix effects, a standardised method is needed to agree on the true value of vitamin D in food.

Matrix effects in the analysis of polar organic water contaminants with HILIC-ESI-MS.

Matrix effects have been shown to be very pronounced and highly variable in the analysis of mobile chemicals, which may severely exacerbate accurate quantification. These matrix effects, however, are still scarcely studied in combination with hydrophilic interaction liquid chromatography (HILIC) and for very polar chemicals. In this study, the matrix effects of 26 polar model analytes were investigated in enriched drinking water, wastewater treatment plant effluent and solutions of inorganic salts, utilizing post-column infusion of the analytes into a HILIC-electrospray ionisation (ESI)-high-resolution mass spectrometry system. These experiments revealed the occurrence of structure-specific and unspecific matrix effects. The unspecific matrix effects were mainly observed in positive ESI polarity and predominantly coincided with a high ion count, resulting in ion suppression of all analytes. Thus, the excess charge is hypothesized to be the limiting factor in ion formation. Structure-specific matrix effects were more pronounced in negative ESI polarity and even structurally similar compounds were observed to react entirely differently: perfluoroalkyl carboxylic acids were suppressed, while perfluoroalkane sulfonic acids were simultaneously enhanced. These matrix effects were traced back to inorganic anions and cations, which eluted over a significant fraction of the chromatographic run time with this setup. Hence, it was concluded that inorganic ions are a main cause for matrix effects in the analysis of mobile chemicals utilizing HILIC. Graphical abstract.

Development of a general method for quantifying IgG-based therapeutic monoclonal antibodies in human plasma using protein G purification coupled with a two internal standard calibration strategy using LC-MS/MS.

Monoclonal antibody (mAb) drugs have generated much interest in recent years for treating various diseases. Immunoglobulin G (IgG) represents a high percentage of mAb drugs that have been approved by the Food and Drug Administration (FDA). To facilitate therapeutic drug monitoring and pharmacokinetic/pharmacodynamic studies, we developed a general liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the concentration of IgG-based mAbs in human plasma. Three IgG-based drugs (bevacizumab, nivolumab and pembrolizumab) were selected to demonstrate our method. Protein G beads were used for sample pretreatment due to their universal ability to trap IgG-based drugs. Surrogate peptides that were obtained after trypsin digestion were quantified by using LC-MS/MS. To calibrate sample preparation errors and matrix effects that occur during LC-MS/MS analysis, we used two internal standards (IS) method that include the IgG-based drug-IS tocilizumab and post-column infused IS. Using two internal standards was found to effectively improve quantification accuracy, which was within 15% for all mAb drugs that were tested at three different concentrations. This general method was validated in term of its precision, accuracy, linearity and sensitivity for 3 demonstration mAb drugs. The successful application of the method to clinical samples demonstrated its' applicability in clinical analysis. It is anticipated that this general method could be applied to other mAb-based drugs for use in precision medicine and clinical studies.

Post-column infusion of internal standard quantification for liquid chromatography-electrospray ionization-tandem mass spectrometry analysis - Pharmaceuticals in urine as example approach.

Liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) technique is gaining more and more attraction as the method of choice for multi-sample analysis. However, it is strongly susceptible to the influence of matrix components. Matrix effects are the main source of substantial losses in detection sensitivity and have to be compensated via complex quantification methods In this work, we introduce a sophisticated quantification method for the LC-ESI-MS/MS analysis of 16 substances in urine samples using a single continuously post-column infused internal standard (PCI-IS) for matrix effect correction. The performance of the introduced technique was proven by the simultaneous quantification using internal standards. Our results demonstrate that a single post-column infused internal standard suffices to analyze multiple target analytes. The introduced method is a new approach to analyze complex matrices and represents a powerful alternative to the classic internal standard methodology. The proposed technique significantly reduces the required steps for sample preparation, costs of additional stable isotopically-labeled internal standards, and self-induced matrix effects.

An alternative approach for assessment of liquid chromatography-mass spectrometry matrix effects using auto-sampler programmed co-injection.

We report the use of auto-sampler programmable functions to co-inject analyte standard solution and matrix extract to assess ion enhancement and suppression (matrix effects) in LC-MS. This is effectively an automated post-extraction addition (APEA) procedure, emulating the manual post-extraction addition (PEA) approach widely adopted for assessment of matrix effects. To verify that APEA was comparable to the conventional PEA approach, matrix effects were determined using both methods for a selection of 31 illicit and pharmaceutical drugs in 10 different human urine extracts. Matrix effects measured using APEA were statistically indistinguishable from manual PEA methodology for 27 of the 31 drugs. Of the four drugs that showed significant differences using the two methods, three differed by less than 2 %, which is within the expected accuracy limits required for matrix effect determinations. The remaining analyte, trimeprazine, was found to degrade in the spiked PEA matrix extract, accounting for the difference between matrix effects measured by the PEA and APEA approaches. APEA enables a single matrix extract to be assessed at multiple analyte concentrations, resulting in a considerable reduction in sample preparation time. In addition, APEA can reduce the quantity of analyte-free sample matrix required for matrix effect assessment, which is an important consideration in certain analytical and bioanalytical fields. This work shows that APEA may be considered as an acceptable alternative to PEA for the assessment of matrix effects in LC-MS method validation and may be applicable to a variety of matrices such as environmental samples.

Evaluation of the matrix effect of different sample matrices for 33 pharmaceuticals by post-column infusion.

Matrix effects that occur during quantitative measurement by liquid chromatography mass spectrometry specifically when using electrospray ionization are a widely recognized phenomenon. Sample matrix compounds affect the ionization process of the target analytes, lead to a low signal response, and flawed analytical results. How these matrix compounds directly influence the ionization process has not yet been completely understood. In the present study, we determined the matrix effect for 33 pharmaceutical substances in sample extracts of urine, plasma and wastewater. Most of the investigated substances were subject to a signal suppression effect. Only for a small subset of the compounds we detected a signal enhancement effect. We investigated the matrix effect profiles in detail to disentangle the influence of different matrices and to correlate the impact of specific components and groups of the analyzed extract in suppressing or enhancing effects in the profile. Most signal suppression effects were detected in the first half of the chromatographic run-time for the matrix extracts of urine and wastewater. The observed effects are caused by high mass flow of salts and other diverse matrix components that were contained in high concentrations in those biological matrices. We also found signal suppression in the matrix effect profile of plasma samples over a wide time range during the chromatographic separation that were associated with a high content of triglycerides of diverse carbohydrate chain lengths. Here, we provide a broader picture of how 33 substances were influenced during analysis. Our results imply that a high number of the investigated substances had comparable effects of matrix compounds, despite differences in their chemical structure.

Gradient elution mode for the troubleshooting of matrix effect on the determination of G004 in different tissues by LC-MS/MS.

A steep gradient elution mode was applied to reduce the risk of matrix effect (ME) for the determination of G004, a novel sulfonylurea hypoglycemic drug, in a tissue distribution study by LC-MS/MS. The mass spectra of the total-ion-current chromatograms combined with the post-column infusion traces enabled the 'unseen' interfering species to be directly detected, and ensured that the chromatography conditions and sample preparation method were adequate to overcome the ME. According to this, a steep gradient elution mode was designed to overcome the intense ME from different tissues. The analysis was performed by monitoring the transitions m/z 558.1 → 419.0 for G004 and m/z 489.3 → 364.1 for glimepiride used as the internal standard. Calibration curves recovered over a range from 0.1 to 10000 ng/mL for seven different tissues. Sex-related difference was found in the tissue distribution. The drug levels in the tissues of female rats were about two to three times higher than those in male counterparts. The highest level was observed in liver, then in kidney, heart, pancreas, lung and spleen, but no G004 was detected in brain. G004 was slowly eliminated from female rats compared with male rats. There was no long-term accumulation of G004 in male or female rat tissues.