Stable Isotope-labeled Internal Standards Research Articles

Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.

Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-βOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-βOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for βOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish βOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-βOHB from L-βOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.

B-180 A Component Equation Approach to Resolving Calibration Curve Nonlinearity When Using Stable Isotope-Labeled Internal Standards for Quantifying Amino Acids With Mass Spectrometry

Abstract Background Stable isotope-labeled internal standards (SIL-IS) have been widely used with mass spectrometry to accurately quantify analytes. However, over an extended dynamic range, calibration curves calculated with the relative ratio of analytes to their corresponding SIL-IS are typically nonlinear. This makes accurate estimation of sample concentration difficult, especially at lower concentrations. Highly multiplexed clinical mass spectrometry assays routinely face this challenge. The component equation assumes interaction between an analyte and its SIL-IS causes the nonlinearity. Modeling this resulted in a linear calibration curve and high accuracy across the dynamic range. We compared the component equation with other calibration methods to quantify amino acids. Methods A total of 47 compounds were serially diluted and added with extraction solution containing a SIL-IS mixture to prepare 7 levels of calibrators. Calibrators were chromatographically separated using a Sciex ExionLC system with an Imtakt Intrada Amino Acid column and injected into a Sciex Citrine triple-quadrupole mass spectrometer. Seven different fitting methods were compared for calibration of each amino acid: non-weighted linear, quadratic and cubic models, linear models with 1/X and 1/X2 weighting, Padé[1,1] approximation, and component equation. Results Results for an example amino acid, tyrosine, are shown in the table below. Most models yield high accuracy at high concentrations, however, all models recorded significantly higher bias for the lowest calibrator than the component equation (≥55.7% vs 11.8%). The component equation outperformed all other models for mean bias (≥16.6% vs 5.9%). Similar results were obtained for the complete set of analytes. Conclusions When compared to other popular calibration methods to quantify a panel of amino acids, the component equation generated a linear curve and the greatest accuracy across all concentrations. The application of this simple equation could overcome the limitations of existing weighting and non-linear fitting approaches when using SIL-IS for quantifying analytes with mass spectrometry.

B-174 Quantification of vitamin C in serum as a biomarker using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract Background Ascorbic acid (AA) or vitamin C, is a crucial substance that is involved in numerous physiological processes in living beings, including collagen production and antioxidant activity. When ascorbic acid is exposed to moisture, air, heat, light and oxygen, it oxidizes and turns into dehydroascorbic acid (DHAA). Our objective is to describe the sample preparation procedure and total quantification of AA and DHAA in human serum sample based on LDTD-MS/MS, and demonstrate its efficiency in quantification, combined with analysis selectivity and rapidity. Methods The Luxon is a sample introduction and ionization source that used a fiber-coupled laser diode to thermally desorb neutral molecules and charge them using a corona needle so they can be analyzed by mass spectrometry. Due to the instability of ascorbic acid, plasma samples were enriched and stabilized with an acidic solution. Sample preparation utilized a phosphate-containing buffer to minimize interferents and help volatize the analyte, followed by a precipitation step using acetonitrile to further clean the extract. Stable isotope-labeled internal standard (13C6 ascorbic acid) and homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) negative mode. Calibration curves ranging from 1 to 100 μg/mL and QCs were prepared in ascorbic acid-depleted serum (kept at room temperature/exposed to light for 4 days). The peak area against the internal standard (IS) ratio was used to normalize the signal. The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. The following parameters were assessed for quantitative analysis: linearity, precision, accuracy, carryover and parallelism. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the correlation coefficient of calibration curves prepared in water to six curves prepared with biological matrixes. Intra and inter assay precision and accuracy were determined by measuring QC samples ranging from low to high concentration in five replicates for 3 consecutive days. Results Calibration curves (n=3) showed excellent linearity. The calibration curve resulted in a linear response throughout the concentration range, the correlation coefficient was 0.99. For each quality control concentrations CVs were <10% complying with desirable specifications for imprecision and accuracy. Intra-assay CVs were 3.0-8.89% and inter-assay CVs were 7.64-9.79% for precision which reflects the agreement between repeated measurements. The results for accuracy showed similar results, intra-assay CVs were 3.83-9.20% and inter-assay CVs were 5.0-9.7%. Conclusions The new LDTD-MS/MS method described here has been validated as an effective technique to reliably and rapidly quantify ascorbic acid. Simple sample preparation, rapid acid stabilization combined with high yield allow analysis without degradation responses.

Simultaneous Determination of Tobacco Smoke Exposure and Stress Biomarkers in Saliva Using In-Tube SPME and LC-MS/MS for the Analysis of the Association between Passive Smoking and Stress.

Passive smoking from environmental tobacco smoke not only increases the risk of lung cancer and cardiovascular disease but may also be a stressor triggering neuropsychiatric and other disorders. To prevent these diseases, understanding the relationship between passive smoking and stress is vital. In this study, we developed a simple and sensitive method to simultaneously measure nicotine (Nic) and cotinine (Cot) as tobacco smoke exposure biomarkers, and cortisol (CRT), serotonin (5-HT), melatonin (MEL), dopamine (DA), and oxytocin (OXT) as stress-related biomarkers. These were extracted and concentrated from saliva by in-tube solid-phase microextraction (IT-SPME) using a Supel-Q PLOT capillary as the extraction device, then separated and detected within 6 min by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Kinetex Biphenyl column (Phenomenex Inc., Torrance, CA, USA). Limits of detection (S/N = 3) for Nic, Cot, CRT, 5-HT, MEL, DA, and OXT were 0.22, 0.12, 0.78, 0.39, 0.45, 1.4, and 3.7 pg mL-1, respectively, with linearity of calibration curves in the range of 0.01-25 ng mL-1 using stable isotope-labeled internal standards. Intra- and inter-day reproducibilities were under 7.9% and 14.6% (n = 5) relative standard deviations, and compound recoveries in spiked saliva samples ranged from 82.1 to 106.6%. In thirty nonsmokers, Nic contents positively correlated with CRT contents (R2 = 0.5264, n = 30), while no significant correlation was found with other biomarkers. The standard deviation of intervals between normal beats as the standard measure of heart rate variability analysis negatively correlated with CRT contents (R2 = 0.5041, n = 30). After passive smoke exposure, Nic levels transiently increased, Cot and CRT levels rose over time, and 5-HT, DA, and OXT levels decreased. These results indicate tobacco smoke exposure acts as a stressor in nonsmokers.

Determination of Taurine in Infant Formula and Adult/Pediatric Nutritionals by Ultra High-Performance Liquid Chromatography with Multiple Reaction Monitoring-Mass Spectrometry: Single-Laboratory Validation, First Action 2022.08.

Taurine (2-aminoethanesulfonic acid) is an essential β-amino acid, which is one of the most abundant intracellular amino acid component in humans. The level of free taurine in human milk is higher than in bovine milk. Since taurine is considered important for the development of a newborn, fortification of infant nutrition with taurine is common. This publication describes a rapid method for the determination of taurine in infant formula and adult/pediatric nutritionals. This publication demonstrates the suitability and applicability of the method to determine taurine content in infant formula and adult/pediatric nutritionals. Test portions are deproteinized by acid in an aqueous environment prior to analysis. The level of taurine is analyzed by hydrophilic interaction chromatography (HILIC) coupled with multiple reaction monitoring MS (MRM-MS). Quantitation is accomplished using a stable isotope-labeled internal standard (SILS) for taurine. The accuracy of the method was validated using standard reference materials and spike recovery studies. The average recoveries ranged between 92% and 105%. The relative standard deviation repeatability (RSDr) and relative standard deviation intermediate precision (RSDiR) were in the range of 0.7-4.6% (RSDr) and 1.3-6.7% (RSDiR). The limit of quantitation (LOQ) was below the requirement of 0.5 mg/100 g. The single-laboratory validation (SLV) of the method for determination of taurine content in infant formula and adult/pediatric nutritionals demonstrates that it meets the requirements per AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR) 2014.013. A fast method to selectively determine the content of taurine in infant formula and adult/pediatric nutritionals was evaluated and found to be fit for purpose for routine product compliance testing. Based on the results of this SLV, the method was approved by the AOAC Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel for SPIFAN Nutrient Methods for First Action Official MethodsSM status.

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.

Direct Multi-Deuterium Labelling of Pirtobrutinib.

Herein, we demonstrate an efficient method for multi-deuterium labelling of pirtobrutinib-a Bruton's tyrosine kinase inhibitor recently approved by the FDA-using a straightforward hydrogen isotope exchange (HIE) reaction. A remarkably high level of deuterium incorporation was achieved using an excess of a Kerr-type iridium catalyst. The key factor in the significant deuterium labelling was the decision to employ a deuterium uniformly labelled solvent, chlorobenzene-d5, at an elevated temperature. Virtually, no d0-d3 species were detected, with only traces of d4-d5 isotopomers (< 5%) observable in the mass spectrum of pirtobrutinib-d8, fulfilling requirements for stable isotope-labelled internal standard. The labelled compound-mainly consisting of isotopomers d6-d9 at 82.4% of the total abundance-was isolated in a high yield (73%) and purity (99%). Noteworthy, fluorine group acting as a directing group was observed for the first time. Significant incorporation of deuterium in ortho-positions, exceeding 87%, was observed. Interestingly, chlorinated solvent used in the HIE reactions was non-specifically deuterated yielding up to 0.42 deuterium per chlorobenzene molecule even at an exceptionally low iridium catalyst loading of 4.17 × 10-2 mol%.

Quantification of gut microbiome metabolites using chemical isotope derivatization strategy combined with LC-MS/MS: Application in neonatal hypoxic-ischemic encephalopathy rat model

The gut microbiome plays pivotal roles in various physiological and pathological processes, with key metabolites including short chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) derivatives gaining significant attention for their diverse physiological roles. However, quantifying these metabolites presents challenges due to structural similarity, low abundance, and inherent technical limitations in traditional detection methods. In this study, we developed a precise and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing a chemical isotope derivatization technique employing 4-(aminomethyl)-N,N-dimethylaniline-d0/d6 (4-AND-d0/d6) reagents to quantify 37 typical gut microbiome-derived metabolites. This method achieved an impressive 1500-fold enhancement in sensitivity for detecting metabolites, compared to methods using non-derivatized, intact molecules. Moreover, the quantitative accuracy of our chemical isotope derivatization strategy proved comparable to the stable isotope labeled internal standards (SIL-IS) method. Subsequently, we successfully applied this newly developed method to quantify target metabolites in plasma, brain, and fecal samples obtained from a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. The aim was to identify crucial metabolites associated with the progression of HIE. Overall, our sensitive and reliable quantification method holds promise in elucidating the role of gut microbiome metabolites in the pathogenesis of various diseases.

Investigation of variability in the matrix effect on stable isotope-labeled internal standards in liquid chromatography-tandem mass spectrometry analysis of 25 pesticides in vegetables.

The matrix effects (ME) in simultaneous analysis of pesticide residue using liquid chromatography-tandem mass spectrometry (LC-MS/MS) were evaluated by comparing the slopes of matrix-matched and reagent-only calibrations of four types of vegetable samples. Both the sampling and measurement variances of the ME were also determined using one-way analysis of variance. Substantial ion suppression (ME<-20%) was observed in komatsuna, spinach, and tomato when a modified Japanese official method was implemented. The ME magnitude varied significantly due to sample variability for some pesticides, but it varied by no more than 4% as a result of analytical procedure variance. This study also showed that the addition of stable isotope-labeled internal standards at low concentrations improved the recovery of pesticides from samples at various residue levels. The findings of this study highlight the importance and practical application of internal standards and the matrix-matched calibration method in residue analysis using LC-MS/MS.

Development of Noninvasive Method for the Automated Analysis of Nine Steroid Hormones in Human Saliva by Online Coupling of In-Tube Solid-Phase Microextraction with Liquid Chromatography–Tandem Mass Spectrometry

Accurate measurement of steroid hormones is crucial to elucidate new mechanisms of action and diagnose steroid metabolism-related diseases. This study presents a simple, sensitive, and automated analytical method for nine representative steroid hormones. The method involves on-line coupling of in-tube solid-phase microextraction (IT-SPME) with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The steroid hormones were extracted and enriched on a Supel-Q PLOT capillary column using IT-SPME. Subsequently, they were separated and detected within 6 min using a Discovery HS F5-3 column and positive ion mode multiple reaction monitoring system via LC–MS/MS. Calibration curves of these compounds using each stable isotope-labeled internal standard (IS) showed linearity with correlation coefficients greater than 0.9990 in the range of 0.01–40 ng/mL, with limits of detection (S/N = 3) of 0.7–21 pg/mL. Moreover, intra- and inter-day variations were lower than 8.1 and 15% (n = 6), respectively. The recoveries of these compounds from saliva samples were in the range of 82–114%. The developed IT-SPME/LC–MS/MS method of steroid hormones is a highly sensitive, specific, and non-invasive analytical method that allows extraction and enrichment with no organic solvents, and enables direct automated online analysis by simply ultrafiltrating a small sample of saliva.

Miniature mass spectrometer-based point-of-care assay for quantification of metformin and sitagliptin in human blood and urine.

Metformin (MET) and sitagliptin (STG) are widely used as the first-line and long-term oral hypoglycemic agents for managing type 2 diabetes mellitus (T2DM). However, the current lack of convenient and rapid measurement methods poses a challenge for individualized management. This study developed a point-of-care (POC) assay method utilizing a miniature mass spectrometer, enabling rapid and accurate quantification of MET and STG concentrations in human blood and urine. By combining the miniature mass spectrometer with paper spray ionization, this method simplifies the process into three to four steps, requires minimal amounts of bodily fluids (50 μL of blood and 2 μL of urine), and is able to obtain quantification results within approximately 2min. Stable isotope-labeled internal standards were employed to enhance the accuracy and stability of measurement. The MS/MS responses exhibited good linear relationship with concentration, with relative standard deviations (RSDs) below 25%. It has the potential to provide immediate treatment feedback and decision support for patients and healthcare professionals in clinical practice.

Therapeutic Drug Monitoring of 6 New-Generation Antiseizure Medications Using a Mass Spectrometry Method: Analysis of 2-Year Experience in a Large Cohort of Korean Epilepsy Patients.

New-generation antiseizure medications (ASMs) are increasingly prescribed, and therapeutic drug monitoring (TDM) has been proposed to improve clinical outcome. However, clinical TDM data on new-generation ASMs are scarce. To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for therapeutic drug monitoring (TDM) of 6 new-generation ASMs in serum and analyze the clinical TDM data from a large cohort of Korean patients with epilepsy. Stable isotope-labeled internal standards were added to protein precipitations of serum. One microliter of sample was separated on Agilent Poroshell EC-C18 column, and lacosamide, perampanel, gabapentin, pregabalin, vigabatrin, and rufinamide were simultaneously quantified by Agilent 6460 triple-quad mass spectrometer in multiple-reaction monitoring mode. Linearity, sensitivity, precision, accuracy, specificity, carryover, extraction recovery, and matrix effect were evaluated. TDM data of 458 samples from 363 Korean epilepsy patients were analyzed. The method was linear with limit of detection less than 0.05 μg/mL in all analytes. Intraassay and interassay imprecisions were less than 5% coefficient of variation. Accuracy was within ±15% bias. Extraction recovery ranged from 85.9% to 98.8%. A total of 88% (403 of 458) were on polypharmacy, with 29% (118 of 403) using concomitant enzyme inducers. Only 38% (175 of 458) of the concentrations were therapeutic, with 53% (244 of 458) being subtherapeutic. Drug concentration and concentration-to-dose ratio were highly variable among individuals in all 6 ASMs. A simple and rapid LC-MS/MS method for TDM of 6 ASMs was developed and successfully applied to clinical practice. This large-scale TDM data could help establish an effective monitoring strategy for these drugs.

LC-MS/MS method for quick detection of vonoprazan fumarate in human plasma: Development, validation and its application to a bioequivalence study.

A liquid chromatography-tandem mass spectrometry method with vonoprazan fumarate-d4 as a stable isotope-labeled internal standard was developed and validated aiming at quantification of vonoprazan fumarate in human plasma for a bioequivalence study. Chromatographic separation was achieved by acetonitrile one-step protein precipitation using a gradient elution of 0.1% formic acid aqueous solution and acetonitrile with a run time of 3.65 min. Detection was carried out on a tandem mass spectrometer in multiple reaction monitoring mode via a positive electrospray ionization interface. The multiple reaction monitoring mode of precursor-product ion transitions for vonoprazan fumarate and vonoprazan fumarate-d4 were m/z 346.0 → 315.1 and 350.0 → 316.0, respectively. The linear range was 0.150-60.000 ng/ml. This method was fully validated with acceptable results in terms of selectivity, carryover, lower limit of quantification, calibration curve, accuracy, precision, dilution effect, matrix effect, stability, recovery and incurred sample reanalysis. A successful application of this method was realized in the bioequivalence study of vonoprazan fumarate tablet (20 mg) among healthy Chinese volunteers.

Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

Quantifying payloads of antibody‒drug conjugates using a postcolumn infused-internal standard strategy with LC‒MS

BackgroundAntibody‒drug conjugates (ADCs) are innovative biopharmaceutics consisting of a monoclonal antibody, linkers, and cytotoxic payloads. Monitoring circulating payload concentrations has the potential to identify ADC toxicity; however, accurate quantification faces challenges, including low plasma concentrations, severe matrix effects, and the absence of stable isotope-labeled internal standards (SIL-IS) for payloads and their derivatives. Previous studies used structural analogs as internal standards, but different retention times between structural analogs and target analytes may hinder effective matrix correction. Therefore, a more flexible approach is required for precise payload quantification. ResultsWe developed an LC‒MS/MS method incorporating a postcolumn-infused internal standard (PCI-IS) strategy for quantifying payloads and their derivatives of trastuzumab emtansine, trastuzumab deruxtecan, and sacituzumab govitecan, including DM1, MCC-DM1, DXd, SN-38, and SN-38G. Structural analogs (maytansine, Lys-MCC-DM1, and exatecan) were selected as PCI-IS candidates, and their accuracy performance was evaluated based on the percentage of samples within 80%–120% quantification accuracy. Compared to the approach without PCI-IS correction, exatecan enhanced the accuracy performance from 30-40%–100% for SN-38 and DXd, while maytansine and Lys-MCC-DM1 showed comparable accuracy for DM1 and MCC-DM1. This validated PCI-IS analytical method showed superior normalization of matrix effect in all analytes compared to the conventional internal standard approach. The clinical application of this approach showed pronounced differences in DXd and SN-38 concentrations before and after PCI-IS correction. Moreover, only DXd concentrations after PCI-IS correction were significantly higher in patients with thrombocytopenia (p = 0.037). SignificanceThis approach effectively addressed the issue of unavailability of SIL-IS for novel ADC payloads and provided more accurate quantification, potentially yielding more robust statistical outcomes for understanding the exposure-toxicity relationship in ADCs. It is anticipated that this PCI-IS strategy may be extrapolated to quantify payloads and derivatives in diverse ADCs, thereby providing invaluable insights into drug toxicity and fortifying patient safety in ADC usage.

A Simplified Proteomics LC-MRM-MS Assay for Determination of apoE Genotypes in Plasma Samples.

Apolipoprotein E (apoE), a polymorphic plasma protein, plays a pivotal role in lipid transportation. The human apoE gene possesses three major alleles (ε2, ε3, and ε4), which differ by single amino acid (cysteine to arginine) substitutions. The ε4 allele represents the primary genetic risk factor for Alzheimer's disease (AD), whereas the ε2 allele protects against the disease. Knowledge of a patient's apoE genotype has high diagnostic value. A recent study has introduced an LC-MRM-MS-based proteomic approach for apoE isoform genotyping using stable isotope-labeled peptide internal standards (SIS). Here, our goal was to develop a simplified LC-MRM-MS assay for identifying apoE genotypes in plasma samples, eliminating the need for the use of SIS peptides. To determine the apoE genotypes, we monitored the chromatographic peak area ratios of isoform-specific peptides relative to a peptide that is common to all apoE isoforms. The assay results correlated well with the standard TaqMan allelic discrimination assay, and we observed a concordance between the two methods for all but three out of 172 samples. DNA sequencing of these three samples has confirmed that the results of the LC-MRM-MS method were correct. Thus, our simplified UPLC-MRM-MS assay is a feasible and reliable method for identifying apoE genotypes without using SIS internal standard peptides. The approach can be seamlessly incorporated into existing quantitative proteomics assays and kits, providing additional valuable apoE genotype information. The principle of using signal ratios of the protein isoform-specific peptides to the peptide common for all of the protein isoforms has the potential to be used for protein isoform determination in general.

Mitigating matrix effects for LC-MS/MS quantification of nitrosamine impurities in rifampin and rifapentine

Since the discovery of potentially mutagenic nitrosamine impurities in pharmaceuticals in 2018, liquid chromatography-mass spectrometry (LC-MS) methods have been widely applied for the trace level quantification of nitrosamines. As more nitrosamines and drug samples are analyzed, method accuracy is increasingly recognized as a major analytical challenge. Matrix effects can have a significant adverse impact on method accuracy but have not attracted much attention in nitrosamine quantification literature. In this study, we observed severe matrix effects when adapting LC-MS method conditions from a published and validated method for the analysis of nitrosamines in tuberculosis drugs. Several approaches were explored to mitigate these matrix effects, including using stable isotope-labeled internal standards, modifying LC conditions and sample concentration, and quantifying with standard addition instead of an external calibration method. The results underscore the importance of monitoring matrix effects and validating nitrosamine quantification procedures for specific sample matrices. Finally, two LC-MS/MS methods are presented with validation data for the quantification of 1-methyl-4-nitrosopiperazine (MNP) in rifampin and 1-cyclopentyl-4-nitrosopiperazine (CPNP) in rifapentine and revealed high nitrosamine levels in the tested samples that far exceeded recommended acceptable intake limits.

Targeted Mass Spectrometry for Quantification of Receptor Tyrosine Kinase Signaling.

Targeted proteomics enables sensitive and specific quantification of proteins and post-translational modifications. By coupling peptide immunoaffinity enrichment with targeted mass spectrometry, we have developed the methodology for multiplexed quantification of proteins and phosphosites involved in the RAS/MAPK signaling network. The method uses anti-peptide antibodies to enrich analytes and heavy stable isotope-labeled internal standards, spiked in at known concentrations. The enriched peptides are directly measured by multiple-reaction monitoring (MRM), a well-characterized quantitative mass spectrometry-based method. The analyte (light) peptide response is measured relative to the heavy standard. The method described provides quantitative measurements of phospho-signaling and is generally applicable to other phosphopeptides and sample types.

Probing Liquid Chromatography-Tandem Mass Spectrometry Response Dynamics and Nonlinear Effects for Response Level Defined Calibration Strategies with Simple Methods To Expand Linear Dynamic Ranges.

The response characteristics of liquid chromatography-tandem mass spectrometry (LC-MS/MS) serve as the basis for selecting calibration methods in quantitative analysis. LC-MS/MS inherently exhibits nonlinear detection behavior, primarily attributed to the disproportionate growth observed between peak area and peak height at elevated response levels, potentially leading to signal saturation. This disproportionate peak growth results in reduced unit response (UR), which quantifies the instrument's detection sensitivity. LC-MS/MS typically operates within a narrow near-linear response range (NLRR) due to approximately proportional peak growth, yet the NLRR width varies across different analytes or platforms. Although the inclusion of stable isotope-labeled (SIL) internal standards (IS) in LC-MS/MS analysis can mitigate certain instrument response variations, it does not eliminate the fundamental cause of nonlinearity. Moreover, the concentration range accommodated by the NLRR can significantly fluctuate at different sensitivity levels. LC-MS/MS also encounters various other nonlinear effects, including ion suppression during ionization, signal cross-contribution between the analyte/IS, and matrix effects (ME). Consequently, quadratic regression emerges as a more adaptable approach to LC-MS/MS nonlinear response dynamics, offering a broader calibration range. The application of linear regression, on the other hand, requires strict conditions. Although the signal saturation zone typically remains inaccessible to calibration methods, reducing responses by employing less-optimal selected reaction monitoring (SRM) transitions and/or lower detection gain can facilitate fitting a wide concentration range into the NLRR, thereby enabling accurate linear regression calibration. This report delves into examining the LC-MS/MS response profile, its dynamics, and major nonlinear effects through instrument response mapping to elucidate their influence on the selection of calibration methods.

Multiplexed Antibody Glycosylation Profiling Using Dual Enzyme Digestion and Liquid Chromatography-Triple Quadrupole Mass Spectrometry Method

Antibody glycosylation plays a crucial role in the humoral immune response by regulating effector functions and influencing the binding affinity to immune cell receptors. Previous studies have focused mainly on the immunoglobulin G (IgG) isotype owing to the analytical challenges associated with other isotypes. Thus, the development of a sensitive and accurate analytical platform is necessary to characterize antibody glycosylation across multiple isotypes. In this study, we have developed an analytical workflow using antibody-light-chain affinity beads to purify IgG, IgA, and IgM from 16 μL of human plasma. Dual enzymes, trypsin and Glu-C, were used during on-bead digestion to obtain enzymatic glycopeptides and protein-specific surrogate peptides. Ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used in order to determine the sensitivity and specificity. Our platform targets 95 glycopeptides across the IgG, IgA, and IgM isotypes, as well as eight surrogate peptides representing total IgG, four IgG classes, two IgA classes, and IgM. Four stable isotope-labeled internal standards were added after antibody purification to calibrate the preparation and instrumental bias during analysis. Calibration curves constructed using serially diluted plasma samples showed good curve fitting (R2 > 0.959). The intrabatch and interbatch precision for all the targets had relative standard deviation of less than 29.6%. This method was applied to 19 human plasma samples, and the glycosylation percentages were calculated, which were comparable to those reported in the literature. The developed method is sensitive and accurate for Ig glycosylation profiling. It can be used in clinical investigations, particularly for detailed humoral immune profiling.