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Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: Necessity or not?
Abstract
It appears to be a general belief that stable isotopically labeled (SIL) internal standards yield better assay performance results for quantitative bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays than does any other internal standard. In this article we describe our experiences with structural analogues and SIL internal standards and their merits and demerits. SIL internal standards are the first choice, but deuterium-labeled compounds may demonstrate unexpected behavior, such as different retention times or recoveries, than the analyte. In addition, a SIL internal standard with identical chemical properties as the analyte may cover up assay problems with stability, recovery, and ion suppression. Since SIL internal standards are not always available or are very expensive, structural analogues can be used, however, with consideration of several issues, which are usually displayed during method validation.
... Although there are several analytical methods intended to organic contaminants analysis in biosolids, those involving antimicrobial analysis have only been developed in recent years. In those methods, liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) is the most used analytical technique for antimicrobials' quantification [18,19], but different sample treatments are used. Commonly, antimicrobials are extracted by ultrasoundassisted solvent extraction (USE) [20], focused ultrasound solid-liquid extraction (FUSLE), pressurized liquid extraction (PLE) [21,22] or microwave-assisted extraction (MAE), among others [18]. ...
... Regarding the peak picking criteria, only features with a minimum peak area of 500,000 and a Lorentzian peak shape were manually picked for further annotation. Only exact masses with an error lower than ± 5 ppm were considered using nine different mass lists, being five of them metabolic transformation products obtained by BioTransformer 3.0 software (19,483 compounds) and the other four including antimicrobials from different families as well as TPs (2795 compounds), summing a total of 22,278 compounds. The molecular formulas suggested by Compound Discoverer were considered for annotation if MS1 spectra was satisfactorily matched (SFit > 30% and isotopic profile > 80%), and a minimum peak to consider of 5e 5 units was established. ...
... (1) Matrix effect (ME%) = (A∕B − 1) × 100 (15) 47 (19) 124 (8) 39 (8) 70 (3) 97 (5) 50 (4) 86 (2) 103 ( (12) 120 (3) 29 (4) 100 (4) 112 (4) 26 (2) 101 (12) 100 (2) [ 2 ...
The present work aimed to develop an accurate analytical method for the simultaneous analysis of twenty-four antimicrobials in soil:compost and animal manure samples by means of ultra-high performance liquid chromatography coupled to a triple-quadrupole mass spectrometer (UHPLC-QqQ). For this purpose, the effectiveness of two extraction techniques (i.e. focused ultrasound solid–liquid extraction (FUSLE) and QuEChERS (quick, easy, cheap, effective, rugged and safe)) was evaluated, and the clean-up step using solid-phase extraction (SPE) was also thoroughly studied. The method was successfully validated at 10 μg·kg⁻¹, 25 μg·kg⁻¹, and 50 μg·kg⁻¹ showing adequate trueness (70–130%) and repeatability (RSD < 30%), with few exceptions. Procedural limits of quantification (LOQPRO) were determined for soil:compost (0.45 to 7.50 μg·kg⁻¹) and manure (0.31 to 5.53 μg·kg⁻¹) samples. Pefloxacin could not be validated at the lowest level since LOQPRO ≥ 10 μg·kg⁻¹. Sulfamethazine (7.9 ± 0.8 µg·kg⁻¹), danofloxacin (27.1 ± 1.4 µg·kg⁻¹) and trimethoprim (4.9 ± 0.5 µg·kg⁻¹) were detected in soil samples; and tetracycline (56.8 ± 2.8 µg·kg⁻¹), among other antimicrobials, in the plants grown on the surface of the studied soil samples. Similarly, sulfonamides (SAs), tetracyclines (TCs) and fluoroquinolones (FQs) were detected in sheep manure in a range of 1.7 ± 0.3 to 93.3 ± 6.8 µg·kg⁻¹. Soil and manure samples were also analysed through UHPLC coupled to a high-resolution mass-spectrometer (UHPLC-qOrbitrap) in order to extend the multitarget method to suspect screening of more than 22,281 suspects. A specific transformation product (TP) of sulfamethazine (formyl-sulfamethazine) was annotated at 2a level in manure samples, among others. This work contributes to the efforts that have been made in the last decade to develop analytical methods that allow multitarget analysis of a wide variety of antimicrobials, including TPs, which is a complex task due to the diverse physicochemical properties of the antimicrobials.
Supplementary information
The online version contains supplementary material available at 10.1007/s00216-023-04905-2.
... The matrix effects of different freshwater lakes are quite different, which will significantly affect the accuracy of the analytical method used for analysis. However, the introduction of SIL-IS can effectively eliminate the errors during sample pretreatment and analysis (Stokvis et al., 2005;Tran et al., 2020). Deuterium, 13 C and 15 N labelling are the commonly used methods for isotopic internal standards, where the Deuterium-labelled IS has the potential for H/D exchange, thus affecting the accuracy of the analytical results (Stokvis et al., 2005). ...
... However, the introduction of SIL-IS can effectively eliminate the errors during sample pretreatment and analysis (Stokvis et al., 2005;Tran et al., 2020). Deuterium, 13 C and 15 N labelling are the commonly used methods for isotopic internal standards, where the Deuterium-labelled IS has the potential for H/D exchange, thus affecting the accuracy of the analytical results (Stokvis et al., 2005). Therefore, 13 C and 15 N-labelled IS are preferred for MCs (Stokvis et al., 2005). ...
... Deuterium, 13 C and 15 N labelling are the commonly used methods for isotopic internal standards, where the Deuterium-labelled IS has the potential for H/D exchange, thus affecting the accuracy of the analytical results (Stokvis et al., 2005). Therefore, 13 C and 15 N-labelled IS are preferred for MCs (Stokvis et al., 2005). The natural isotopic peaks of MCs are mainly derived from elemental C (1.1 % for 12 C/ 13 C), while other elements contribute less to the isotope. ...
Precise quantification of microcystins (MCs) in freshwater is crucial for environmental monitoring and human health. However, the preparation of traditional multi-sample external calibration curve (MSCC) is time consuming and prone to error. Here, a novel one-point calibration strategy including one sample multi-point calibration curve (OSCC) and in sample calibration curve (ISCC) is proposed for the quantitation of eight MCs in freshwater lakes using liquid chromatography tandem mass spectrometry (LC-MS/MS). The multiple isotopologue reaction monitoring (MIRM) of MCs and its ¹⁵N-labelled internal standards were used for OSCC and ISCC, respectively. The isotopic abundance of each MIRM channel could be calculated and measured accurately. Additionally, this strategy was comprehensively validated and showed good performance in selectivity, sensitivity, accuracy and precision as the traditional MSCC. Interestingly, OSCC could realize sample dilution by monitoring the less abundant MIRM transitions, while ISCC remove blank matrixes and generate calibration curve in each study samples. Furthermore, the proposed methodology was successfully applied to analyze several freshwater lake samples contaminated by MCs. Considering the advantages of excluding the MSCC preparation, simplified workflows and improved throughput, OSCC and ISCC will be favored for MCs monitoring and as an emerging approach in environmental pollutant control and prevention.
... According to the concentration-response relationship within the specified range for targeted analyst, the suitable mathematical model is adopted to fit the regression, and the sample concentrations are back-calculated. In order to improve the trueness and stability of analytical methods, stable isotope internal standards (IS) are usually added to calibration curve samples and samples to be tested, thereby reducing the influence of matrix effects and measurement errors (Stokvis et al., 2005;Trufelli et al., 2011). It is worth noting that for the analysis of targets in different matrix samples, corresponding calibration curves need to be optimized (Trufelli et al., 2011). ...
... Considering 13 C and Deuterium-labeled (D labeled) compounds display identical or almost identical chromatographic and ionization behaviors comparing with the un-labeled compound. Herein, the 13 C labeled compounds instead of D labeled form was selected, since it's relatively stable with high purity, and no exchange of Hydrogen/Deuterium (H/D) during analysis (Stokvis et al., 2005). As known, the natural occurrence of stable isotopes can lead to cross-talk and isotopic overlap in MS analysis. ...
Preparing of calibration curves are critical steps for accurate quantitative LC-MS bioanalysis. Traditional multi-sample external calibration curve (MSCC) is labor-intensive and prone to error. In this study, a novel strategy of one sample multi-point calibration curve (OSCC) using multiple isotopologue reaction monitoring (MIRM) was proposed and validated using LC-MS for the quantitation of six aflatoxins in milk and oat-based milk samples. The developed MIRM-OSCC methodology is comprehensively validated and the results indicated that the established method exhibits good performance in selectivity, sensitivity, accuracy and precision. Furthermore, the OSCC could realize sample dilution by monitoring the MIRM channel with less intensity for samples beyond the upper limit of quantification, without the need of sample dilution, which improves the assay throughput. Considering the advantages of excluding the MSCC preparation and sample dilution in OSCC, this strategy can be widely applied in various fields such as drugs, food safety and environmental analysis.
... In addition, MQL varied for different substances in different matrices, ranging from 22 ng/kg to 105 ng/kg. Matrix components are notorious for reducing analyte signals by what is known as ion suppression (Stokvis et al., 2005). The matrix effect (ME) for the three different matrices is shown in Table 2. ...
... Additionally, the degree of ion suppression caused by impurities in the matrix was also closely related to the chemical structure of the analytes. The chemical structure of the isotope-labelled internal standard was very similar to that of the analytes, thus reducing the effect of ion suppression on quantification (Stokvis et al., 2005). ...
Erythromycin fermentation residue (EFR) is a solid waste generated from the fermentation process of erythromycin A production. Some byproducts are produced during the fermentation process of erythromycin A production, and erythromycin A can also undergo hydrolysis and biodegradation reactions in the environment with the formation of transformation products. Herein, an accurate analytical method was established and validated to quantify erythromycin A, two byproducts and five hydrolysis or biodegradation products, in solid or semi-solid media of waste EFR and the amended soil. The method mainly included ultrasonic solvent extraction, solid phase extraction, and ultra-performance liquid chromatography-tandem mass spectrometry quantification. All analytes could be effectively extracted in a single process, and the recoveries ranged from 76% to 122% for different matrices. Low matrix effects and excellent precision were achieved by optimizing the mass spectrometry parameters, extraction solution, number of extractions and eluent. This method was applied to evaluate the residual analytes in EFR, treated EFR after industrial-scale hydrothermal treatment, and the subsequent soil application. Seven analytes were detected in the EFR, while six were found in the treated EFR and amended soils. The concentration of erythromycin A in EFR was 1629 ± 100 mg/kg·TS, and the removal efficiency of hydrothermal treatment (180 °C, 60 min) was about 99.6%. Three hydrolysis products were the main residuals in treated EFR, with anhydroerythromycin A showing the highest concentration. The concentrations of the analytes in soil ranged from 2.17 ± 1.04 to 92.33 ± 20.70 μg/kg·TS, and anhydroerythromycin A contributed 65%–77% of the total concentration. Erythromycin B, a byproduct, was still detected in soil. This work provides an accurate analytical method which would be useful to evaluate the potential risk of byproducts and transformation products of erythromycin A in environment.
... Quantification was performed via RRF (ESM Table S1), which was shown to be time-saving since no calibration curves were needed. Furthermore, a stable isotope-labeled IS with identical chemical properties as the targeted analyte may compensate for issues with stability, MF, RE, and ion suppression [31,32]. However, the purchase of isotope-labeled corresponding IS for each analyte is costly. ...
Oral endocrine therapies (OET) for breast cancer treatment need to be taken over a long period of time and are associated with considerable side effects. Therefore, adherence to OET is an important issue and of high clinical significance for breast cancer patients’ caregivers. We hypothesized that a new bioanalytical strategy based on liquid chromatography and high-resolution mass spectrometry might be suitable for unbiased adherence monitoring (AM) of OET. Four different biomatrices (plasma, urine, finger prick blood by volumetric absorptive microsampling (VAMS), oral fluid (OF)) were evaluated regarding their suitability for AM of the OET abemaciclib, anastrozole, exemestane, letrozole, palbociclib, ribociclib, tamoxifen, and endoxifen. An analytical method was developed and validated according to international recommendations. The analytical procedures were successfully validated in all sample matrices for most analytes, even meeting requirements for therapeutic drug monitoring. Chromatographic separation of analytes was achieved in less than 10 min and limits of quantification ranged from 1 to 1000 ng/mL. The analysis of 25 matching patient samples showed that AM of OET is possible using all four matrices with the exception of, e.g., letrozole and exemestane in OF. We were able to show that unbiased bioanalytical AM of OET was possible using different biomatrices with distinct restrictions. Sample collection of VAMS was difficult in most cases due to circulatory restraints and peripheral neuropathy in fingers and OF sampling was hampered by dry mouth syndrome in some cases. Although parent compounds could be detected in most of the urine samples, metabolites should be included when analyzing urine or OF. Plasma is currently the most suitable matrix due to available reference concentrations.
Graphical Abstract
... [16][17][18][19][20][21][22] The use of stable isotopically labelled internal standards for all compounds, either deuterated or labelled with 13 C or 15 N, makes it possible to compensate for matrix effects or incomplete recoveries. 23 Furthermore, the chosen calibration range makes the assay very suitable for TDM, since low and high concentrations can be present in (critically ill) patients. ...
Objective
To develop and validate an UPLC–MS/MS assay for simultaneous determination of the total concentration of ceftazidime, ciprofloxacin, flucloxacillin, piperacillin, tazobactam, sulfamethoxazole, N-acetyl sulfamethoxazole and trimethoprim, and the protein-unbound concentration of flucloxacillin, in human plasma to be used for research and clinical practice.
Methods
Sample pretreatment included protein precipitation with methanol. For the measurement of protein-unbound flucloxacillin, ultrafiltration was performed at physiological temperature. For all compounds, a stable isotopically labelled internal standard was used. Reliability of the results was assessed by participation in an international quality control programme.
Results
The assay was successfully validated according to the EMA guidelines over a concentration range of 0.5–100 mg/L for ceftazidime, 0.05–10 mg/L for ciprofloxacin, 0.4–125 mg/L for flucloxacillin, 0.2–60 mg/L for piperacillin, 0.15–30 mg/L for tazobactam, 1–200 mg/L for sulfamethoxazole and N-acetyl sulfamethoxazole, 0.05–10 mg/L for trimethoprim and 0.10–50 mg/L for unbound flucloxacillin. For measurement of total concentrations, the within- and between-day accuracy ranged from 90.0% to 109%, and 93.4% to 108%, respectively. Within- and between-day precision (variation coefficients, CVs) ranged from 1.70% to 11.2%, and 0.290% to 5.30%, respectively. For unbound flucloxacillin, within-day accuracy ranged from 103% to 106% and between-day accuracy from 102% to 105%. The within- and between-day CVs ranged from 1.92% to 7.11%. Results of the international quality control programme showed that the assay is reliable.
Conclusions
The method provided reliable, precise and accurate measurement of seven commonly prescribed antibiotics, including the unbound concentration of flucloxacillin. This method is now routinely applied in research and clinical practice.
... The SANTE indications were taken into consideration for validation. Usually, quantitative analyses by MS are performed using internal standards to monitor errors that may occur due to ion suppression caused by matrix components (Stokvis et al., 2005). Deuterated compounds are widely used as internal standards, but they can be very expensive or unavailable. ...
... For quantification of biomarkers, their signal is compared to stable isotope labeled standards (SIS) that are added to each sample during sample preparation. SIS are compounds in which several atoms in the analyte are replaced by their stable isotopes, such as 2H (D, deuterium), 13C, 15N, or 17/18O [30,31]. They have the same (or highly similar) physicochemical properties than their endogenous (light) counterpart which means that they Becher et al. [24] Shibata et al. [25] show the same behaviour during sample preparation, LC separation, and electrospray ionization (ESI), but they can be distinguished in MS due to their larger molecular mass. ...
Routine measurement of cancer biomarkers is performed for early detection, risk classification, and treatment monitoring, among other applications, and has substantially contributed to better clinical outcomes for patients. However, there remains an unmet need for clinically validated assays of cancer protein biomarkers. Protein tumor markers are of particular interest since proteins carry out the majority of biological processes and thus dynamically reflect changes in cancer pathophysiology. Mass spectrometry-based targeted proteomics is a powerful tool for absolute peptide and protein quantification in biological matrices with numerous advantages that make it attractive for clinical applications in oncology. The use of liquid chromatography-tandem mass spectrometry (LC–MS/MS) based methodologies has allowed laboratories to overcome challenges associated with immunoassays that are more widely used for tumor marker measurements. Yet, clinical implementation of targeted proteomics methodologies has so far been limited to a few cancer markers. This is due to numerous challenges associated with paucity of robust validation studies of new biomarkers and the labor-intensive and operationally complex nature of LC–MS/MS workflows. The purpose of this review is to provide an overview of targeted proteomics applications in cancer, workflows used in targeted proteomics, and requirements for clinical validation and implementation of targeted proteomics assays. We will also discuss advantages and challenges of targeted MS-based proteomics assays for clinical cancer biomarker analysis and highlight some recent developments that will positively contribute to the implementation of this technique into clinical laboratories.
... Therefore, immunochemical methods are basically used for screening, LC-MS/MS methods are used for confirmation. The LC-MS/ MS methods have some disadvantages such as ion suppression/enhancement due to matrix effect, potential sources of inaccuracy due to ionization techniques, absence of stable isotope labeled internal standards, instrument cost, expertise, training, quality assurance, and lack of standardization and harmonization [42,43]. ...
Objectives
The aim of this study was to compare the analytical performance of the KIMS (kinetic interaction of microparticles in solution) immunochemical method with a validated in-house and a commercial LC-MS/MS method.
Methods
The urine samples of the 100 subjects were included in the present study. The urine samples were analysed with Roche DAT immunochemical method based on KIMS method. In-house LC-MS/MS method was validated for 58 parameters according to the CLSI C62-A recommendations with the following parameters: matrix effect, lower limit of quantification (LLOQ), linearity, intra-day and inter-day precision and accuracy. Eureka Lab Division Drugs of Abuse kit was used as the commercial LC-MS/MS method.
Results
The immunochemical method had a satisfactory performance with specificity, sensitivity and accuracy values above 80 % and met the DRUID recommendation except benzodiazepines. The sensitivity and specificity of the immunochemical method were between 97–100 % and 84–100 %, respectively (except for benzodiazepines). The bias obtained for THC-COOH, morphine and codeine parameters were −17.5, 24.6 and 43.6 between two LC-MS/MS methods. The commercial method had a tendency to have a negative bias except for cannabinoids.
Conclusions
The analytical performance of the KIMS-based urine immunochemical method was found to be satisfactory for the intended use, except for benzodiazepines. The validated urine in-house LC-MS/MS method was found to be a good alternative for confirmation of substance abuse.
... One drug may be used as an IS as long as this drug is not a part of the patient's therapeutic treatment. Guidelines are available for the correct application of the IS in the determination of drugs in biological samples (Stokvis et al., 2005). ...
... One drug may be used as an IS as long as this drug is not a part of the patient's therapeutic treatment. Guidelines are available for the correct application of the IS in the determination of drugs in biological samples (Stokvis et al., 2005). ...
... However, mass overlap between the natural and labelled compounds at their reference masses will result in nonlinear calibration curves, therefore, for organic compounds with 7 to 10 Carbon atoms, mass differences of at least 3 mass units between the labelled and natural compounds is required. Unfortunately, increasing the degree of labeling of the internal standard could involve differences in physical chemical properties and/or changes in chromatographic retention times that could modify the behavior of the added standard in comparison with the target compound, mainly when using deuterium derivatives [45] . Alternatively, isotope pattern deconvolution (IPD) pro-vides a good alternative to the conventional isotope dilution quantification [46] . ...
... These deuterated compounds have many potential applications [13], especially in the food and pharmaceutical industries. For example, in the food industry, deuterated compounds can be used as an internal standard [14,15] in the analysis of bioactive compounds. In this context, compared to other deuteration methodologies [16,17], the Cp2TiCl/D2O system [18] is a sustainable and inexpensive reagent, very useful for the preparation of deuterated phenols that can be used as an internal standard in the analysis of some of the bioactive phenolic compounds present in olive oil [15]. ...
Significant efforts have been made toward developing sustainable reduction reactions of organic and bioorganic compounds. In these studies, the selection of reagents and solvents has played a very important role in the development of environment-friendly methodologies. In this context, the reducing agent Cp2TiCl/H2O has been introduced as a safe, efficient, selective, and low-cost reagent, and thus as a sustainable alternative for the reduction of organic compounds. To facilitate understanding of the reductions mediated by this system, in this study we focus on describing the intermediates, mechanisms, and representative examples. Finally, a reflection is made on the future perspectives of this reducing agent, including its analog Cp2TiCl/D2O as a powerful tool for the preparation of deuterated phenols, which can be successfully used as an internal standard for analyzing bioactive phenols in olive oil.
... The most commonly used isotopes are non-exchangeable 2 H, 13 C and 15 N, respectively. Widely practiced is the introduction of three to eight 2 H or 13 C atoms or a combination of both to separate the isotopologs of the isotopically enriched material from that of the unenriched material on a mass spectrometer [16]. While apparently simple, some requirements have to be considered regarding the implementation and the choice of the internal standard, as described in more detail in the literature [14,15] and the guideline of IDMS [17]. ...
The methyl substitution along and among the polymer chains of methyl cellulose (MC) is commonly analyzed by ESI-MS after perdeuteromethylation of the free-OH groups and partial hydrolysis to cello-oligosaccharides (COS). This method requires a correct quantification of the molar ratios of the constituents belonging to a particular degree of polymerization (DP). However, isotopic effects are most pronounced for H/D since their mass difference is 100%. Therefore, we investigated whether more precise and accurate results could be obtained for the methyl distribution of MC by MS of ¹³ CH 3 instead of CD 3 -etherified O -Me-COS. Internal isotope labeling with ¹³ CH 3 makes the COS of each DP chemically and physically much more similar, reducing mass fractionation effects, but at the same time requires more complex isotopic correction for evaluation. Results from syringe pump infusion ESI-TOF-MS with ¹³ CH 3 and CD 3 as isotope label were equal. However, in the case of LC-MS with a gradient system, ¹³ CH 3 was superior to CD 3 . In the case of CD 3 , the occurrence of a partial separation of the isotopologs of a particular DP resulted in slight distortion of the methyl distribution since the signal response is significantly dependent on the solvent composition. Isocratic LC levels this problem, but one particular eluent-composition is not sufficient for a series of oligosaccharides with increasing DP due to peak broadening. In summary, ¹³ CH 3 is more robust to determine the methyl distribution of MCs. Both syringe pump and gradient-LC-MS measurements are possible, and the more complex isotope correction is not a disadvantage.
Graphical abstract
... This method was performed using deuterated analogues of each analyte, except for the steroid EPIPRAG, which was quantified using the deuterated analogue of its isomer, PRAG (PRAG-d4). Each deuterated IS was differing from its analogues by a minimum of 3 mass units to avoid isotopic overlap during MS analysis [47]. The number and position of the deuterium atoms of the ISs are listed in Table 1. ...
The emergence of metabolomics and quantification approaches is revealing new biomarkers applied to drug discovery. In this context, tandem mass spectrometry is the method of choice, requiring a specific validation process for preclinical and clinical applications. Research on the two classes of lipid mediators, steroids and cannabinoids, has revealed a potential interaction in cannabis addiction and metabolism-related disorders. Here we present the development of GC-MS/MS and LC-MS/MS methods for routine quantification of targeted steroids and cannabinoids, respectively. The methods were developed using an isotopic approach, including validation for linearity, selectivity, LLOQ determination, matrix effect, carryover, between- and within-run accuracy and precision, and stability tests to measure 11 steroids and seven cannabinoids in human plasma. These methods were satisfactory for most validity conditions, although not all met the acceptance criteria for all analytes. A comparison of calibration curves in biological and surrogate matrices and in methanol showed that the latter condition was more applicable for our quantification of endogenous compounds. In conclusion, the validation of our methods met the criteria for GLP-qualified rather than GLP-validated methods, which can be used for routine analytical studies for dedicated preclinical and clinical purposes, by combining appropriate system suitability testing, including quality controls in the biological matrix.
... Isotopically-labelled analogues of PBTZ169, its metabolites amino, 3-oxo and 4-oxo were available and therefore were used as internal standards. No labelled compounds were available for the other metabolites (OH and H 2 PBTZ), thus necessitating the identification of analogues suitable for quantification [45]. To this end, several molecules from the new-chemical-entities' library of the Global Health Institute (EPFL, Lausanne, Switzerland) have been selected for their structural analogies with PBTZ169 and were injected with the optimized analytical method. ...
Tuberculosis, and especially multidrug-resistant tuberculosis (MDR-TB), is a major global health threat which emphasizes the need to develop new agents to improve and shorten treatment of this difficult-to-manage infectious disease. Among the new agents, macozinone (PBTZ169) is one of the most promising candidates, showing extraordinary potency in vitro and in murine models against drug-susceptible and drug-resistant Mycobacterium tuberculosis. A previous analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed by our group to support phase I clinical trials of PBTZ169. These plasma sample analyses revealed the presence of several additional metabolites among which the most prominent was H2PBTZ, a reduced species obtained by dearomatization of macozinone, one of the first examples of Meisenheimer Complex (MC) metabolites identified in mammals.
Identification of these new metabolites required the optimization of our original method for enhancing the selectivity between isobaric metabolites as well as for ensuring optimal stability for H2PBTZ analyses. Sample preparation methods were also developed for plasma and urine, followed by extensive quantitative validation in accordance with international bioanalytical method recommendations, which include selectivity, linearity, qualitative and quantitative matrix effect, trueness, precision and the establishment of accuracy profiles using β-expectation tolerance intervals for known and newer analytes.
The newly optimized methods have been applied in a subsequent Phase Ib clinical trial conducted in our University Hospital with healthy subjects. H2PBTZ was found to be the most abundant species circulating in plasma, underscoring the importance of measuring accurately and precisely this unprecedented metabolite. Low concentrations were found in urine for all monitored analytes, suggesting extensive metabolism before renal excretion.
... However, it is necessary to have a crosstalk or isotopic overlap during MS analysis to perform the deconvolution, which is possible when SIL chemical purity and/or isotopic enrichment is less than 100% or when there is only a small mass-unit difference between the isotope labeled standard and its analogous compound [151]. Thus, Castillo and colleagues used a SIL (diclofenac-d 4 ) with only 92.8% isotopic enrichment to increase the crosstalk effect on the natural isotopic distribution of the analyte. ...
Over the last two decades, liquid chromatography coupled to mass-spectrometry (LC‒MS) has become the gold standard to perform qualitative and quantitative analyses of small molecules. When quantitative analysis is developed, an analyst usually refers to international guidelines for analytical method validation. In this context, the design of calibration curves plays a key role in providing accurate results. During recent years and along with instrumental advances, strategies to build calibration curves have dramatically evolved, introducing innovative approaches to improve quantitative precision and throughput. For example, when a labeled standard is available to be spiked directly into the study sample, the concentration of the unlabeled analog can be easily determined using the isotopic pattern deconvolution or the internal calibration approach, eliminating the need for multipoint calibration curves. This tutorial aims to synthetize the advances in LC‒MS quantitative analysis for small molecules in complex matrices, going from fundamental aspects in calibration to modern methodologies and applications. Different work schemes for calibration depending on the sample characteristics (analyte and matrix nature) are distinguished and discussed. Finally, this tutorial outlines the importance of having international guidelines for analytical method validation that agree with the advances in calibration strategies and analytical instrumentation.
... All labeled compounds were obtained with high specific activities ([ 3 H]3a-[ 3 H]3d, 41.6-99.7 Ci mmol À 1 ). 3 H NMR spectroscopy confirmed that tritiation occurred selectively at the benzylic methylene and methyl groups and at the aromatic CÀ H bonds in orthopositions to the alkoxy substituents. ...
Hydrogen isotope exchange (HIE) represents one of the most attractive labeling methods to synthesize deuterium‐ and tritium‐labeled compounds. Catalytic HIE methods that enable site‐selective C−H bond activation and exchange labeling with gaseous isotopes D2 and T2 are of vital importance, in particular for high‐specific‐activity tritiation of pharmaceuticals. As part of our interest in exploring s‐block metals for catalytic transformations, we found CsN(SiMe3)2 to be an efficient catalyst for selective HIE of benzylic C−H bonds with D2 gas. The reaction proceeds through a kinetic deprotonative equilibrium that establishes an exchange pathway between C−H bonds and D2 gas. By virtue of multiple C−H bonds activation and high activity (isotope enrichment up to 99 %), the simple cesium amide catalyst provided a very powerful and practically convenient labeling protocol for synthesis of highly deuterated compounds and high‐specific‐activity tritiation of pharmaceuticals.
... Using an aminoglycoside chemical analogue, kanamycin B, as an internal standard provides sufficient correction for sample preparation and detection of paromomycin because they are chemically structural and have similar properties. A SILIS is recommended in the use of LC-MS/MS; however, the internal standard recovery of the analyte should be assessed (Stokvis et al., 2005). A multiple deuterated internal standard was used for the quantification of paromomycin, which could introduce interference resulting in a bias in the quantification (Roseboom, Thijssen, et al., 2020). ...
Bioanalytical method development and validation for the quantification of antileishmanial drugs are pivotal to support clinical trials and provide the data necessary to conduct pharmacokinetic analysis. This review provides a comprehensive overview of published validated bioanalytical assays for the quantification of antileishmanial drugs amphotericin B, miltefosine, paromomycin, pentamidine, and pentavalent antimonials in human matrices. Each antileishmanial drug was discussed regarding their applicability of the assay for leishmaniasis clinical trials as well as providing their relevance for PK studies with emphasis on the choice of matrix, calibration range, sample volume, sample preparation, choice of internal standards, separation, and detection. Given that no published bioanalytical methods included multiple antileishmanial drugs in a single assay while antileishmanial shortened combination regimens currently were under investigation, it was recommended to combine various drugs in a single bioanalytical method. Furthermore, bioanalytical method development regarding target site matrix as well as applying microsampling strategies was recommended to optimize future clinical pharmacokinetic studies in leishmaniasis.
... Bioanalysis is an essential science in many fields of study, such as developing new medicines, forensic testing, doping controls, and the identification of biomarkers for many diseases. Due to the sophistication of the sample matrix, Bioanalysis is challenging [2][3][4][5][6][7][8][9][10][11]. There is a well-known need for intense sample preparation before being used in analytical instruments to use complex matrices such as blood, plasma, and urine. ...
A protocol is used to detect and measure biomolecules and metabolites in human and animal tissues using bimolecular methods. The biosanalinity method is effective at determining the number of drugs and metabolites in a biological system. New methods, the validation of existing procedures, and the analysis of samples are one of the prominent tasks for bioanalysis. Above all, a compound can be measured using several methods and identified by different methods of analysis. Drugs may be tested by several extraction techniques, including liquid extraction, solid-phase extraction, and protein precipitation in complex plasma and biological samples. To determine how the environment, matrix, or procedures impact the matrix estimation to the time of the analysis, all steps in the process must be investigated. The more detailed study of drug products can be performed with higher-pressure analytical techniques, such as high-extraction (HPLC), liquid chromatography coupled with double-mass spectrometry (LCMS/MS), and ultra-performance Liquid chromatography (UPLC). Both of them have flaws and strengths. At present, HPLC and GC usually perform biolysis. The parameters are linearity, repeatability, accuracy, selectivity, and continuity. We are proposing the development and validation of bioanalytical systems to assist in the quality assurance of drugs.
... Since a compound and its stable isotopic labelled analogue will theoretically co-elute, it is important that the mass difference between the compounds is at least 3 mass units, in order to be able to separate them in the mass analyser. 249 The IS is added to the sample to be analysed, so that both experience the same environmental conditions when passing through the LC-MS/MS. The Rt of the component is divided by the Rt of the IS to achieve the relative Rt. ...
... Internal standards are usually added to a sample before the extraction, after tissue homogenization, thus also allowing to account for possible losses during extraction. Compounds used as internal standards could be an isotope-labeled target PSM to allow for differentiation from the native PSM (Stokvis et al., 2005), or a chemically similar compound that is not present in the sample [e.g., borneol as internal standard of alpha-pinene (Sorensen et al., 2004), 4hydroxybenzyl benzyl glucosinolate, allyl glucosinolate, as internal standard of other glucosinolates (Martin and Müller, 2007;Abdalsamee et al., 2014;Beran et al., 2018;Sporer et al., 2021), heliotrine as internal standard of senecionine alkaloids (Narberhaus et al., 2004)]. When selecting an internal standard, it is necessary to confirm that the ionization and detection of this compound is comparable with that of the target compounds. ...
Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions.
... Internal standards are usually added to a sample before the extraction, after tissue homogenization, thus also allowing to account for possible losses during extraction. Compounds used as internal standards could be an isotope-labeled target PSM to allow for differentiation from the native PSM (Stokvis et al., 2005), or a chemically similar compound that is not present in the sample [e.g., borneol as internal standard of alpha-pinene (Sorensen et al., 2004), 4hydroxybenzyl benzyl glucosinolate, allyl glucosinolate, as internal standard of other glucosinolates (Martin and Müller, 2007;Abdalsamee et al., 2014;Beran et al., 2018;Sporer et al., 2021), heliotrine as internal standard of senecionine alkaloids (Narberhaus et al., 2004)]. When selecting an internal standard, it is necessary to confirm that the ionization and detection of this compound is comparable with that of the target compounds. ...
Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions.
... The use of appropriate ILIS for correcting ME, as well as sample treatment recoveries, when added in the first step of the analysis (i.e., as surrogates) has evident advantages. Nevertheless, it should be kept in mind that isotopically-labelled compounds may produce "cross-talk" if the mass difference between natural and labelled compound is less than 3 Da, as the isotope peaks of the unlabeled analyte may interfere with the signal of the ILIS (Stokvis et al., 2005). This was the case for one of the pesticide metabolites included in this work, for which the requirement of > 3Da was not met, causing challenges. ...
Wastewater-based epidemiology (WBE) relies on the assessment and interpretation of levels of biomarkers in wastewater originating from a well-defined community. It has provided unique information on spatial and temporal trends of licit and illicit drug consumption, and has also the potential to give complementary information on human exposure to chemicals. Here, we focus on the accurate quantification of pesticide biomarkers (i.e., predominantly urinary metabolites) in influent wastewater at the ng L⁻¹ level to be used for WBE. In the present study, an advanced analytical methodology has been developed based on ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), for the simultaneous determination of 11 specific human biomarkers of triazines, urea herbicides, pyrethroids and organophosphates in urban wastewater. The sample treatment consisted of solid-phase extraction using Oasis HLB cartridges. Direct injection of the samples was also tested for all compounds, as a simple and rapid way to determine these compounds without sample manipulation (i.e. minimizing potential analytical errors). However, if extraction recoveries are satisfactory, SPE is the preferred approach that allow reaching lower concertation levels. Six isotopically labelled internal standards were evaluated and used to correct for matrix effects. Due to the difficulties associated with this type of analysis, special emphasis has been placed on the analytical challenges encountered. The satisfactory validated methodology was applied to urban wastewater samples collected from different locations across Europe revealing the presence of 2,6-EA, 3,4-DCA, 3-PBA and 4-HSA i.e, metabolites of metolachlor-s, urea herbicides, pyrethroids and chlorpropham, respectively. Preliminary data reported in this paper illustrate the applicability of this analytical approach for assessing human exposure to pesticides through WBE.
... Such methods do not enable quantification of in vivo H 2 S levels at the time of sample collection, but instead measure subsequent synthetic enzyme capacity of the sample collected. Other methods for H 2 S quantification involve derivatization with monobromobimane, fluorescent probes, or alternative reagents, and detection with either fluorescence or mass spectrometry; however, these methods all rely on the use of external calibration curves [18,19,23], and lack the many potential benefits conferred by an internal standard and isotope dilution methodology [24,25]. Further, while one of the most commonly used derivatization reagents for H 2 S quantification (i.e. ...
Background
Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices.
Methods
After synthesizing [³⁴S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota.
Results
Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r² > 0.997), recovery (90.9%–110%) and stability (90.0%–100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0–66.7% reduction with antibiotics).
Conclusions
A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.
Schizophrenia is a serious mental disorder that significantly affects the social and professional life of patients, causing distortion of reality and loss of identity and cognitive abilities. Psychopharmacological treatment is an integral part of modern psychiatry, and the introduction of new “atypical” antipsychotic drugs has brought significant progress in the treatment of this disorder. One of these drugs is olanzapine, which has an effective effect on the productive symptoms of schizophrenia while having an almost minimal potential to cause extrapyramidal syndrome. However, its effectiveness is confronted with frequent side effects, referred to as “metabolic disorders”. Therefore, to ensure the effectiveness of treatment and to minimize the side effects caused by olanzapine, it is recommended to monitor the drug level during therapy. This article reviews the bioanalytical methodologies that enable efficient extraction and sensitive analysis of olanzapine. We considered the advantages and disadvantages of different sample pretreatment methods, including traditional and novel strategies. The analytical conditions required for the separation and detection of olanzapine and its metabolites were analyzed using chromatographic methods combined with various detectors.
The combination of three types of catalysts in one pot, including borate, palladium, and lipase, enabled a one-pot β-specific methylene C(sp ³ )–H deuteration reaction of aliphatic acids using D 2 O.
The different topics and synthetic approaches in an isotope chemistry laboratory of a pharma company are described. Besides the challenges in the synthesis of long-lived isotopes such as 3H or 14C, short-lived isotopes such as 68Ga and stable isotopes such as 15N, 13C or 2H approaches for the isotopic labeling are also demonstrated. Furthermore, method development with emphasis on collaborations with academic groups to tackle the future challenges are discussed.
1 Introduction
2 Isotopic Labeling with Hydrogen Isotopes Deuterium (2H, D) and Tritium (3H, T)
2.1 Deuterium Labeling for MS Standards
2.1.1 Labeled Nitrosamines – The Hunt to Quantify Hazardous Impurities
2.1.2 Deuterated Drugs, an Approach To Improve Existing Drugs or To Find Opportunities in Drug Discovery
2.2 Tritium-Labeling Methods – The Fast Approach to Radioactively Labeled Compounds
2.2.1 Hydrogen Isotope Exchange by Iridium Catalysis
2.2.2 Ruthenium-Catalyzed HIE
2.2.3 Nanoparticles as Catalysts in HIE
2.2.4 Photoredox-Catalyzed HIE
2.2.5 HIE via Classical Radical Mechanism
2.2.6 Beyond HIE – Halogen–Tritium Exchange
3 Challenges in 14C-Synthesis Projects
4 Short-Lived Isotopes – The Need for Speed
5 Beyond Isotope Science – Late-Stage Functionalization
5.1 Examples of Late-Stage Functionalization for Peptides
5.2 Examples of Catalyst-Controlled Late-Stage Functionalization
6 Conclusion
The field of plant hormonomics focuses on the qualitative and quantitative analysis of the hormone complement in plant samples, akin to other omics sciences. Plant hormones, alongside primary and secondary metabolites, govern vital processes throughout a plant's lifecycle. While active hormones have received significant attention, studying all related compounds provides valuable insights into internal processes. Conventional single-class plant hormone analysis employs thorough sample purification, short analysis and triple quadrupole tandem mass spectrometry. Conversely, comprehensive hormonomics analysis necessitates minimal purification, robust and efficient separation and better-performing mass spectrometry instruments. This review summarizes the current status of plant hormone analysis methods, focusing on sample preparation, advances in chromatographic separation and mass spectrometric detection, including a discussion on internal standard selection and the potential of derivatization. Moreover, current approaches for assessing the spatiotemporal distribution are evaluated. The review touches on the legitimacy of the term plant hormonomics by exploring the current status of methods and outlining possible future trends.
In diesem Überblick diskutieren wir die Einsatzmöglichkeiten von isotopenmarkierten Substanzen und betrachten ihre zukünftige Relevanz für die Biowissenschaften. Insbesondere wird ihre Verwendung in der Pharma‐ und Pflanzenschutz‐industrie erörtert. Ihre wichtigste Anwendung ist die Verwendung als Marker in Wirkstoffen. Ihre strukturelle Veränderung wird in der Umwelt, in vivo oder in komplexer Umgebung verfolgt. Dies dient dazu, Auswirkungen neuer Wirkstoffe zu ermitteln und die Sicherheit für die Menschheit und Umwelt zu steigern.
In this review the applications of isotopically labeled compounds are discussed and put into the context of their future impact in the life sciences. Especially discussing their use in the pharma and crop science industries to follow their fate in the environment, in vivo or in complex matrices to understand the potential harm of new chemical structures and to increase the safety of human society.
Objective:
The paper reports an attempt to develop and validate a HILIC UPLC/ QTof MS method for quantifying N-ε-carboxymethyl-L-lysine (CML) in vitro, testing N-ε-carboxy[D2]methyl-L-lysine (d2-CML), and N-ε-carboxy[4,4,5,5-D4]methyl-L-lysine (d4-CML) as internal standards.
Method:
During the method development, several challenging questions occurred that hindered the successful completion of the method. The study emphasizes the impact of issues, generally overlooked in the development of similar analytical protocols. For instance, the use of glassware and plasticware was critical for the accurate quantification of CML. Moreover, the origin of atypical variation in the response of the deuterated internal standards, though widely used in other experimental procedures, was investigated.
Result:
A narrative description of the systematic approach used to address the various drawbacks during the analytical method development and validation is presented.
Conclusion:
Reporting those findings can be considered beneficial while bringing an insightful notion about critical factors and potential interferences. Therefore, some conclusion and ideas can be drawn from these trouble-shooting questions, which might help other researchers to develop more reliable bioanalytical methods, or to raise their awareness of stumbling blocks along the way.
Natalizumab is a humanized recombinant monoclonal IgG4 antibody used in the treatment of multiple sclerosis. Commonly used methods for natalizumab and anti-natalizumab antibodies quantification are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Measurement of therapeutic monoclonal antibodies can be challenging due to the resemblance to human plasma immunoglobulins. Recent developments in mass spectrometry enables to analyze vast variety of large protein molecules. The aim of this study was to develop a LC-MS/MS method for determining natalizumab in human serum and cerebrospinal fluid (CSF) and apply it to clinical settings. For successful quantification, it was necessary to find specific sequences of peptides in natalizumab. This immunoglobulin was treated with dithiothreitol and iodoacetamide, cleaved with trypsin into short specific peptides and determined on a UPLC-MS/MS system. An Acquity UPLC BEH C18 column at 55 °C and gradient elution was used for analysis. Intra- and interassay accuracies and precisions were tested at four concentration levels. Precision was determined by coefficients of variation and was in the range of 0.8-10.2 %, with accuracy in the range of 89.8-106.4 %. The concentration of natalizumab in patient samples ranged from 1.8 to 193.3 μg/mL. The method was validated according to the European Medicines Agency (EMA) guideline, met all acceptance criteria for accuracy and precision, and is suitable for clinical applications. In comparison to immunoassay, which can be elevated by cross-reaction with endogenous immunoglobulins, the results of developed LC-MS/MS method are more accurate and specific.
Therapeutic monoclonal antibodies (mAbs) provide effective treatments for many diseases, including cancer, autoimmune disorders, and, lately, COVID-19. Monitoring the concentrations of mAbs is important during their production and subsequent processing. This work demonstrates a 5 min quantitation of most human immunoglobulin G (IgG) antibodies through capture of mAbs in membranes modified with ligands that bind to the fragment crystallizable (Fc) region. This enables binding and quantitation of most IgG mAbs. Layer-by-layer (LBL) adsorption of carboxylic acid-rich polyelectrolytes in glass-fiber membranes in 96-well plates allows functionalization of the membranes with Protein A or a peptide, oxidized Fc20 (oFc20), with high affinity for the Fc region of human IgG. mAb capture occurs in <1 min during the flow of solutions through modified membranes, and subsequent binding of a fluorophore-labeled secondary antibody enables quantitation of the captured mAbs using fluorescence. The intra- and inter-plate coefficients of variations (CV) are <10 and 15%, respectively, satisfying the acceptance criteria for many assays. The limit of detection (LOD) of 15 ng/mL is on the high end of commercial enzyme-linked immunosorbent assays (ELISAs) but certainly low enough for monitoring of manufacturing solutions. Importantly, the membrane-based method requires <5 minutes, whereas ELISAs typically take at least 90 min. Membranes functionalized with oFc20 show greater mAb binding and lower LODs than membranes with Protein A. Thus, the membrane-based 96-well-plate assay, which is effective in diluted fermentation broths and in mixtures with cell lysates, is suitable for near-real-time monitoring of the general class of human IgG mAbs during their production.
Determining the purity of deuterium labelled compounds is important due to the increasing use of these compounds in mass spectrometry (MS) based quantitative analyses for targeting metabolic flux, reducing toxicity,...
This work presents the evaluation of one- and two-dimensional liquid chromatography for the quantification of three stroke outcome predictors in plasma. Isotopically labelled analogues of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are used to quantify the three analytes by isotope dilution and tandem mass spectrometry. Chromatographic isotope effects were not observed between natural L-Arg and its 15N-labelled analogue but they were observed between natural ADMA and SDMA and their multiple deuterated analogues. Under these conditions, bidimensional chromatography through the use of an automated multiple heart cutting mode provided unsatisfactory results for ADMA and SDMA due to the different amounts of natural and labelled compounds transferred from the first to the second chromatographic dimension. In contrast, using one dimensional liquid chromatography after a derivatization step to esterify carboxylic groups, chromatographic isotope effects did not alter the initial mass balance as full coelution of natural and labelled analogues or baseline resolution between the analytes was not required. This method was successfully validated following the Clinical & Laboratory Standards Institute guidelines and applied to the analysis of plasma samples from patients who had suffered from an intraparenchymal haemorrhagic stroke.
The high interest of isotopically labelled compounds is induced by two important factors: (i) improvement of highly sensitive and precise analytical methods for isotope identification and (ii) development of new synthetic approaches for isotopically labelled compounds. However, there is still a lack of efficient and cheap methods for the design of deuterium labelled liquid crystalline materials. Herein, the continuous flow system was adapted for the synthesis of deuterated Liquid Crystals using the H-Cube Pro reactor, where deuterium gas was generated in situ from heavy water. We designed and developed the synthesis of homologous series of 4,4ʺ-dialkyl-2′,3′-difluoro-[1,1′:4′,1ʺ]terphenyls where deuterium atoms are placed at carbon α and carbon β positions or only at carbon α positions of alkyl terminal chains. The synthetic strategy involves mainly selective deuteration reactions of C≡C bonds as well as reduction of carbonyl groups C=O using batch or continuous flow conditions. Theoretical calculation and experimental study show that deuterium labelled Liquid Crystals exhibit increased photochemical stability compared to protonated ones. Moreover, the comparison of physicochemical properties between deuterium labelled and non-labelled 4,4ʺ-dialkyl-2′,3′-difluoro-[1,1′:4′,1ʺ]terphenyls is presented. This work provides efficient methods to obtain deuterated liquid crystalline materials with much better photochemical stability compared to their fully protonated isotopologues.
Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Initially identified in Wuhan (China) in December 2019, COVID-19 rapidly spread globally, resulting in the COVID-19 pandemic. Carriers of the SARS-CoV-2 can experience symptoms ranging from mild to severe (or no symptoms whatsoever). Although vaccination provides extra immunity toward SARS-CoV-2, there has been an urgent need to develop treatments for COVID-19 to alleviate symptoms for carriers of the disease. In seeking a potential treatment, deuterated compounds have played a critical role either as therapeutic agents or as internal MS standards for studying the pharmacological properties of new drugs by quantifying the parent compounds and metabolites. We have identified >70 examples of deuterium-labeled compounds associated with treatment of COVID-19. Of these, we found 9 repurposed drugs and >20 novel drugs studied for potential therapeutic roles along with a total of 38 compounds (drugs, biomarkers, and lipids) explored as internal mass spectrometry standards. This review details the synthetic pathways and modes of action of these compounds (if known), and a brief analysis of each study.
Hydrogen isotope exchange (HIE) represents one of the most attractive labeling methods to synthesize deuterium‐ and tritium‐labeled compounds. Catalytic HIE methods that enable site‐selective C–H bond activation and exchange labeling with gaseous isotopes D2 and T2 are of vital importance, in particular for high‐specific‐activity tritiation of pharmaceuticals. As part of our interest in exploring s‐block metals for catalytic transformations, we found CsN(SiMe3)2 to be an efficient catalyst for selective HIE of benzylic C–H bonds with D2 gas. The reaction proceeds through a kinetic deprotonative equilibrium that establishes an exchange pathway between C–H bonds and D2 gas. By virtue of multiple C–H bonds activation and high activity (isotope enrichment up to 99%), the simple cesium amide catalyst provided a very powerful and practically convenient labeling protocol for synthesis of highly deuterated compounds and high‐specific‐activity tritiation of pharmaceuticals.
Nirmatrelvir/ritonavir association has been authorized for conditional use in the treatment of COVID-19, especially in solid-organ transplant recipients who did not respond to vaccine and are still at high risk of severe disease. This combination remains at risk of drug interactions with immunosuppressants, so monitoring drug levels seems necessary. After a simple protein precipitation of plasma sample, analytes were analyzed using an ultrahigh performance liquid chromatography system coupled with tandem mass spectrometry in a positive ionization mode. Validation procedures were based on the guidelines on bioanalytical methods issued by the European Medicine Agency. The analysis time was 4 min per run. The calibration curves were linear over the range from 10 to 1000 ng/mL for ritonavir and 40 to 4000 ng/mL for nirmatrelvir, with coefficients of correlation above 0.99 for all analytes. Intra-/interday imprecisions were below 10%. The analytical method also meets criteria of matrix effect, carryover, dilution integrity, and stability. In the context of a SARS-CoV-2 infection in a renal transplant recipient, we present a case of tacrolimus overdose with serious adverse events despite discontinuation of nirmatrelvir and ritonavir. The patient had still effective concentrations of nirmatrelvir and tacrolimus 4 days after drug discontinuation. This method was successfully applied for therapeutic drug monitoring in clinical practice.
Bioanalysis describes quantitative estimation of chemicals or drug substances as well as their metabolic products in large variety of bio-samples. It is an integrated technique that has been employed in preclinical stages of drug-discovery process to further support the clinical phases of drug discovery. However, a bioanalytical method must be optimized, characterized, and validated following documented procedures according to United States Pharmacopeia (USP)/International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines to comply with the regulatory guidelines and acceptance criteria. Bioanalytical studies should therefore provide accurate and reproducible estimation of drugs or metabolites in biological samples at great level of sensitivity, selectivity, and specificity. In this chapter, various advanced techniques commonly employed in bioanalysis have been summarized. Different techniques of extraction and an array of advanced hyphenated techniques are employed for the evaluation of bioanalytes in biofluids, with improved analytical specificity and sensitivity. The applications of bioanalysis in biomedical, pharmaceutical, and other allied areas have been systematically reviewed in this chapter.
Validation of analytical methodologies is widely accepted as pivotal before they are put into routine use. Within the guidelines issued by Regulatory Authorities, there still exists scope for individual interpretation with respect to their conduct and acceptance criteria. The intention of this paper is to review the performances used and to provide practical approaches for determining selectivity, specificity, limit of detection, lower limit of quantitation, linearity, range, accuracy, precision, recovery, stability, ruggedness, and robustness of liquid chromatographic methods to support pharmacokinetic studies. A survey of recent literature on liquid chromatographic procedures used in the bioanalysis of anticancer drugs revealed that very variable standards were employed for validation.
N-(pyridin-4-yl)-[1-(4-chlorbenzyl)-indol-3-yl]-glyoxyl-amid (D-24851) is a novel synthetic compound that was identified in a cell-based screening assay to discover cytotoxic drugs. D-24851 destabilizes microtubules and blocks cell cycle transition specifically at G2-M phase. The binding site of D-24851 does not overlap with the tubulin binding sites of known microtubule-destabilizing agents like vincristine or colchicine. In vitro, D-24851 has potent cytotoxic activity toward a panel of established human tumor cell lines including SKOV3 ovarian cancer, U87 glioblastoma, and ASPC-1 pancreatic cancer cells. In vivo, oral D-24851 treatment induced complete tumor regressions (cures) in rats bearing Yoshida AH13 sarcomas. Of importance is that the administration of curative doses of D-24851 to the animals revealed no systemic toxicity in terms of body weight loss and neurotoxicity in contrast to the administration of paclitaxel or vincristine. Interestingly, multidrug-resistant cell lines generated by vincristine-driven selection or transfection with the Mr 170,000 P-glycoprotein encoding cDNA were rendered resistant toward paclitaxel, vincristine, or doxorubicin but not towards D-24851 when compared with the parental cells. Because of its synthetic nature, its oral applicability, its potent in vitro and in vivo antitumoral activity, its efficacy against multidrug-resistant tumors, and the lack of neurotoxicity, D-24851 may have significant potential for the treatment of various malignancies.
This book describes the full range of mass spectrometry techniques and applications. This versatile technique is in ubiquitous use in universities and industry laboratories because of its ability to identify and quantify materials quickly and, if necessary, in minute amounts, and solve analytical problems in a huge variety of fields. The authors adopt an instructional approach and make use of recent examples to illustrate important points. This second edition includes new methods and applications that have developed in the last ten years. Powerful methods combining mass spectrometry with newer separation techniques, the increased use of computers, and analysis of once difficult polar and large-mass compounds such as proteins using new ionisation methods are all discussed. Requiring no previous knowledge of mass spectrometry, this is an ideal teaching text at both undergraduate and postgraduate level, and will also be of considerable interest to research workers.
Over the years of chromatographic evolution, the purpose of using internal standards for quantitative determinations in bioanalysis has shifted from covering (correcting for) random and systematic errors of the entire method (including sample preparation, chromatography and detection) to currently covering (correcting for) random and systematic errors of the detection, by MS-MS especially. However, the need for internal standards in current LC-MS-MS is also dependent on the degree of chromatographic separation and the degree of sample clean-up. This emphasizes the need for integrated method development, including selection of an internal standard. Available options for internal standard selection are (1) no internal standard, (2) a structurally related compound, (3) a structurally similar compound or (4) a stable isotope labelled compound. An overview of the purposes of internal standards in bioanalytical LC-MS-MS is given and this overview is supported by various illustrations using different types of internal standards.
An overview of the purposes of internal standards in bioanalytical LC-MS-MS is given. It is supported by various illustrations using different types of internal standards.
The option of using deuterated internal standards has given us surprising results in some cases, where chromatographic and/or extraction behaviour of deuterated internal standards was different from the analyte.
A high-performance liquid chromatographic (HPLC) assay is described for the determination of the new, investigational antitumour drug SK&F 104864-A and its lactone ring opened form (SK&F 105992). The analytical methodology reported here involves a protein precipitation step with methanol as sample pretreatment procedure. The instability of the drug necessitates that the plasma fraction is obtained within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (-30 degrees C). The methanolic extract can be stored at -30 degrees C for several days without deterioration of the analyses. Stability data of the drug and its lactone ring opened metabolite in plasma and after methanolic extraction are discussed. The parent drug and the metabolite are separated by reversed-phase ion-pair liquid chromatography on a LiChrosorb RP-18 column, using methanol-water eluent (pH 6.0) with sodium dioctylsulphosuccinate (DOSS) as ion-pairing agent and fluorescence detection. The proposed method has been validated and, subsequently, implemented in a phase I clinical trial for pharmacokinetic evaluation of the new cytotoxic agent.
The invasion and metastasis of cancer cells is a complex multistep process involving attachment of tumor cells to the basement membrane, proteolysis of the local connective tissue stroma, and migration through the proteolyzed stroma. Recent evidence implicates metalloproteinases such as type IV collagenase and transin/stromelysin in the proteolytic aspects of this process. Type IV collagenase activity is modulated by tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). Immunohistochemical and biochemical studies of several human tumors show correlations between invasive potential and type IV collagenase activity.
Activated rhoA, a ras-related GTP-binding protein, stimulates the appearance of stress fibers, focal adhesions, and tyrosine phosphorylation in quiescent cells (Ridley, A.J., and A. Hall, 1992. Cell. 70:389-399). The pathway by which rho triggers these events has not been elucidated. Many of the agents that activate rho (e.g., vasopressin, endothelin, lysophosphatidic acid) stimulate the contractility of smooth muscle and other cells. We have investigated whether rho's induction of stress fibers, focal adhesions, and tyrosine phosphorylation is the result of its stimulation of contractility. We demonstrate that stimulation of fibroblasts with lysophosphatidic acid, which activates rho, induces myosin light chain phosphorylation. This precedes the formation of stress fibers and focal adhesions and is accompanied by increased contractility. Inhibition of contractility by several different mechanisms leads to inhibition of rho-induced stress fibers, focal adhesions, and tyrosine phosphorylation. In addition, when contractility is inhibited, integrins disperse from focal adhesions as stress fibers and focal adhesions disassemble. Conversely, upon stimulation of contractility, diffusely distributed integrins are aggregated into focal adhesions. These results suggest that activated rho stimulates contractility, driving the formation of stress fibers and focal adhesions and elevating tyrosine phosphorylation. A model is proposed to account for how contractility could promote these events.
A method was developed for the bio-analysis of Ecteinascidin 743 (ET-743) using miniaturized liquid chromatography (LC) coupled to an electrospray ionization sample inlet (TurbolonSpray) and two quadrupole mass analyzers (LC/ESI-MS/MS). Solid-phase extraction was used as a sample pretreatment procedure. Ecteinascidin 743 is a very potent anticancer compound and is administered in microgram m-2 dosages, which demands special requirements in terms of sensitivity for the analytical method supporting clinical pharmacokinetic studies. Using conventional LC/UV, a lower limit of quantitation (LLQ) of 1 ng ml-1 plasma was reached using a 500 microliters sample volume, but LC/ESI-MS/MS permitted an LLQ of 10 pg ml-1. The latter method was found to be accurate and precise, and provided a broad linear concentration range of 0.010-2.50 ng ml-1.
The HPLC/tandem mass spectrometric (LC/MS/MS) behavior of indinavir, an HIV protease inhibitor, in human urine is presented as an example of a case where endogenous matrix components were found to interfere with the ionization of the target analyte. The MS/MS system used for these experiments was equipped with a turbo ion spray LC interface. Results from two sample preparation procedures (direct dilution of urine vs urine extraction) and two chromatographic systems (low vs. high capacity factor (k')) for the analytes were compared. Additionally, the precision of the analysis that was achieved while using a stable isotope labeled internal standard is contrasted with the results obtained using an analog of indinavir as internal standard. The results obtained indicated that during development and validation of LC/MS/MS based assays the potential effect of co-eluting 'unseen' endogenous species should be evaluated to ensure that sample preparation and chromatography is adequate to overcome the matrix effect problems.
The basis of all pharmacokinetic evaluations are powerful assays to quantify drugs and/or metabolites in biological matrices using modern sensitive instrumental analytical techniques, such as capillary gas chromatography and high-performance liquid chromatography (HPLC). Being both specific and universal, mass spectrometry (MS) is an ideal chromatographic detector. Due to recent exciting achievements in the interfacing of liquid chromatography (LC) and MS, LC-MS, like the successfully preceding hyphenated technique gas chromatography-mass spectrometry (GC-MS), has now become a valuable technique in the analyst's toolbox. The key features of LC-MS are explained and four examples demonstrating its potential for highly specific and sensitive routine drug assays with the option of high sample throughput in pharmacokinetic investigations are presented.
Spisulosine is a novel antiproliferative (antitumoral) compound of marine origin. In this work the molecular target for this toxic agent has been analyzed. In the presence of spisulosine, cultured cells change their morphology, first acquiring a fusiform morphology, and later becoming rounded without focal adhesions. Analysis of the cytoskeleton of treated cells indicate the absence of actin stress fibers.
A novel metabolite-screening procedure for pibutidine. an H2-receptor antagonist, which uses high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS), demonstrated the presence of pibutidine and its four metabolites in plasma from volunteers who received a single dose of pibutidine hydrochloride. In order to quantitatively examine the metabolism of pibutidine, an assay based on LC-MS/MS was subsequently developed for the simultaneous determination of its metabolites in human plasma. Target analytes consisted of M-5, M-7 and M-8, which were prominently detected by the screening procedure, and M-9, which has pharmacological activity as an H2-receptor antagonist. Metabolites and their deuterated internal standards were extracted from human plasma using an Oasis HLB extraction cartridge, and chromatographed on a Monitor C18M column. No isotope effects on chromatographic retention time were observed for any deuterated compounds, which were ionized using an electrospray ionization (ESI)- interface and detected by MS/MS in the selected reaction-monitoring (SRM) mode simultaneously with the corresponding metabolites. The assay was validated over the concentration range of 0.1 to 25.6 ng ml(-1) and used to determine the plasma levels of metabolites in volunteers following oral administration of a 20-mg dose of pibutidine hydrochloride.
A method for the simultaneous determination of a cyclooxygenase-2 inhibitor, 4-(4-methanesulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib, I) and [13C7]rofecoxib, (II), in human plasma has been developed to support the clinical oral bioavailability (BA) study of I. The method is based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in the negative ionization mode using a heated nebulizer interface. Two different stable isotope labeled analogs of I were initially evaluated for their use as intravenous (i.v.) markers in the BA study. [13CD3]Rofecoxib was shown to be isotopically unstable in plasma and water containing solvent and an efficient deuterium exchange prevented its use in the study. On the other hand, the isotopic integrity of the subsequently synthesized [13C7]rofecoxib (II) was maintained, as expected, in plasma and other solvent systems. The results of these experiments clearly demonstrated the need for the careful evaluation of the isotopic integrity of the stable isotope labeled compound for the successful utilization of these compounds in BA studies and also as internal standards in the quantitative analysis of drugs in biological fluids. After liquid-liquid extraction of I, II, and internal standard (III) from plasma, the analytes were chromatographed on a narrow bore (100 mm x 3.0 mm) C18 analytical column, with mobile phase consisting of acetonitrile-water (1:1, v/v) at a flow-rate of 0.5 ml/min. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in the selected reaction monitoring mode. The precursor-->product ion combinations of m/z 313-->257, 320-->292, and 327-->271 were used to quantify I, II, and III, respectively. The assay was validated in the concentration range of 0.1 to 100 ng/ml of plasma for both I and II. The precision of the assay (expressed as relative standard deviation) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. The assay was utilized to support the clinical BA study in which oral doses of I were administered together with an i.v. dose of II to determine the oral BA of rofecoxib at 12.5- and 25-mg doses.
The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub‐ppb level is explored. Metabolites from two organophosphorous pesticides, 4‐nitrophenol (from parathion and parathion‐methyl) and 3‐methyl‐4‐nitrophenol (from fenitrothion), are taken as model analytes to conduct this study.
After direct injection of the urine sample (10 µL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope‐labeled internal standards. The advantages of applying coupled‐column liquid chromatography (LC/LC) as an efficient clean‐up without any type of sample manipulation are also discussed.
The combination of LC/LC with ESI‐MS/MS allows the direct analysis of free metabolites in urine, as the automated clean‐up performed by the coupled‐column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI‐MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4‐nitrophenol and 3‐methyl‐4‐nitrophenol in human urine without any sample treatment. Copyright © 2002 John Wiley & Sons, Ltd.
Kahalalide F (KF) is a novel cyclic depsipeptide anticancer drug, which has shown anticancer activity both in vitro and in vivo especially against human prostate cancer cell lines. To characterize the pharmacokinetics of KF during a phase I clinical trial in patients with androgen refractory prostate cancer, a method was developed and validated for the quantitative analysis of KF in human plasma using high-performance liquid chromatography (HPLC) coupled to positive electrospray ionization tandem mass spectrometry (ESI-MS/MS). Microbore reversed-phase liquid chromatography (LC) performed with mobile phases containing trifluoroacetic acid, an additive commonly used for separating peptides, resulted in substantial suppression of the signal for KF on ESI-MS/MS. An alternative approach employing a basic mobile phase provided an excellent response for KF when detected in the positive ion mode. Plasma samples were prepared for LC MS/MS by solid-phase extraction on C(18) cartridges. The LC separation was performed on a Zorbax Extend C(18) column (150 x 2.1 mm i.d., particle size 5 micro m) with acetonitrile -10 mM aqueous ammonia (85 : 15, v/v) as the mobile phase, at a flow-rate of 0.20 ml min(-1). A butyric acid analogue of KF was used as the internal standard. The lower limit of quantitation (LLQ) using a 500 micro l sample volume was 1 ng ml(-1) and the linear dynamic range extended to 1000 ng ml(-1). The inter-assay accuracy of the assay was -15.1% at the LLQ and between -2.68 and -9.05% for quality control solutions ranging in concentration from 2.24 to 715 ng ml(-1). The inter-assay precision was 9.91% or better at these concentrations. The analyte was stable in plasma under all relevant conditions evaluated and for a period of 16 h after reconstituting plasma extracts for LC analysis at ambient temperature.
A method was developed for the quantitative analysis of the novel anticancer agent ES-285 (spisulosine; free base) in human, mouse, rat, and dog plasma using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in order to support pre-clinical and clinical studies with the drug. Sample preparation was carried out by protein precipitation with acetonitrile, containing isotopically labeled (d(3)) ES-285 as internal standard. Aliquots of 10 micro l of the supernatant were injected directly on to an Inertsil ODS-3 column (50 x 2.0 mm i.d., 5 micro m). Elution was carried out using methanol-10 mM ammonium formate (pH 4) in water (80 : 20, v/v) pumped at a flow-rate of 0.2 ml min(-1) with a run time of 8 min. Multiple reaction monitoring chromatograms obtained on an API365 triple-quadrupole mass spectrometer were used for quantification. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in human, mouse, rat, and dog plasma and the linear dynamic range extended to 500 ng ml(-1). A full validation of the method was performed in human plasma, and partial validations were performed in mouse, rat and dog plasma. Accuracies and precisions were <20% at the LLOQ concentration and <15% for all other concentrations in all matrices. ES-285 was stable during all steps of the assay. Thus far this method has been used successfully to analyze over 500 samples in pre-clinical trials, and will be implemented in the planned clinical phase I studies.
Selective, accurate, and reproducible liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of mevalonic acid, an intermediate in the biosynthesis of cholesterol and therefore a useful biomarker in the development of cholesterol lowering drugs, in human plasma and urine. A hepta-deuterated analog of mevalonic acid was used as the internal standard. For both methods, calibration standards were prepared in water, instead of human plasma and urine, due to unacceptably high levels of endogenous mevalonic acid. The lower quality control (QC) samples were prepared in water while the higher QC samples were prepared in the biological matrices. For the isolation/purification of mevalonic acid from the plasma and urine matrices, the samples were first acidified to convert the acid analyte into its lactone form. For the plasma samples, the lactone analyte was retained on and then eluted off a polymeric solid-phase extraction (SPE) sorbent. For the urine method, the sample containing the lactone analyte was passed through a C-18 SPE column, which did not retain the analyte, with the subsequent analyte retention on and then elution off a polymeric SPE sorbent. Chromatographic separation was achieved isocratically on a polar-endcapped C-18 analytical column with a water/methanol mobile phase containing 0.5 mM formic acid. Detection was by negative-ion electrospray tandem mass spectrometry. The standard curve range was 0.500-20.0 ng/mL for the plasma method and 25.0-1,000 ng/mL for the urine method. Excellent accuracy and precision were obtained for both methods at all concentration levels tested. It was interesting to note that for certain batches of urine, when a larger sample volume was used for analysis, a high degree of matrix effect was observed which resulted not only in the attenuation of the absolute response, but also in a change of analyte/internal standard response ratio. This demonstrated that, under certain conditions, the use of a stable isotope analog internal standard does not, contrary to conventional thinking, guarantee the constancy of the analyte/internal response ratio, which is a prerequisite for a rugged bioanalytical method. On the other hand, under conditions where the sample matrix does not have such a deleterious effect, we have found that a stable isotope analog could serve as a surrogate (substitute) analyte. Thus, we have shown that using calibration standards prepared by spiking plasma with tri-deuterated or tetra-deuterated mevalonic acid, instead of mevalonic acid itself (the analyte), plasma QC samples that contain mevalonic acid can be successfully analyzed for the accurate and precise quantitation of mevalonic acid. The use of a surrogate analyte provides the opportunity to gauge the daily performance of the method for the low concentration levels prepared in the biological matrix, which otherwise is not achievable because of the endogenous concentrations of the analyte in the biological matrices.
A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the quantitative analysis of the novel anticancer drug ABT-518 and the screening of six potential metabolites in human plasma has been developed and validated to support a phase I study with the drug. ABT-518 is an inhibitor of matrix metalloproteinases, which are associated with tumor growth and development of metastasis. Plasma samples were prepared for analysis using a simple solid-phase extraction method on phenyl cartridges. LC separation was performed on a Zorbax extend C18 column (150 x 2.1 mm i.d., 5 microm particle size) using a mobile phase of methanol-aqueous 10 mM ammonium hydroxide (80:20, v/v) pumped at a flow-rate of 0.2 ml min(-1). An API2000 triple-quadrupole mass spectrometer was used for specific and sensitive detection. The best chromatographic speed (total run time 8 min) and peak shapes were obtained by employing an alkaline mobile phase (pH in aqueous phase approximately 10). Furthermore, an alkaline eluent was favored in order to obtain a better overall sensitivity for the protonated analytes. The dynamic range was from 10 to 1000 ng ml(-1) from 500 microl of plasma for ABT-518 and the metabolites were detected at levels of the same order of magnitude. Inter-assay accuracies for ABT-518 at five concentration levels were between -9.24 and 6.93% and inter-assay precisions were always <10.7%. Analyte stability was not critical during either storage or processing. This method was successfully applied in a phase I clinical study of ABT-518. The active drug, ABT-518, and all of the metabolites included in the assay could be identified in plasma from dosed patients. We believe that the method described in this paper using an alkaline mobile phase in combination with a basic stable analytical column may also be generally useful for the bioanalysis of other basic drugs.
The development of a liquid chromatography/tandem mass spectrometric assay for the quantitative analysis of the novel tubulin inhibitor D-24851 in human plasma and urine is described. D-24851 and the deuterated internal standard were extracted from 250 microL of plasma or urine using hexane/ether (1:1, v/v). Subsequently, 10-microL aliquots of reconstituted extracts were injected onto an Inertsil ODS analytical column (50 x 2.0 mm i.d., 5 microm particle size). An eluent consisting of methanol/5 mM ammonium acetate, 0.004% formic acid in water (80:20, v/v) was pumped at a flow rate of 0.2 mL/min. An API 365 triple quadrupole mass spectrometer was used in the multiple reaction monitoring mode for sensitive detection. For human plasma a dynamic range of 1-1000 ng/mL was validated, and for human urine a range of 0.25-50 ng/mL. Validation was performed according to the most recent FDA guidelines and all results were within requirements. The assay has been successfully applied to support a phase I clinical trial with orally administered D-24851.
The importance of a stable isotopically labeled (SIL) internal standard for the quantitative LC-MS/MS assay for Kahalalide F in human plasma is highlighted. Similar results can be expected for other LC-MS/MS assays. Therefore, we emphasize the need for an SIL internal standard for accurate and precise LC-MS/MS assays of drugs in biological matrices.