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Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 μM up to 2.5 μM. The detection limit was 0.08 μM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 μM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples., (© 2024 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

The carryover of trace allergens in complex food matrices poses challenges for detection techniques. Here, we demonstrate an accurate UPLC-MS/MS quantification assay for the shrimp allergen tropomyosin with a full-length isotope-labelled recombinant tropomyosin (TM-I) internal standard in complex food matrices. The TM-I, expressed based on the SILAC technique, exhibited a high isotope labelling ratio (>99%), purity, and alignment with the natural sequence. This method determined the tropomyosin ranging from 0.2 to 100 ng/mL. Mean recoveries ranged from 89 to 116%, with intra- and inter-day RSDs below 12%, for three signature peptides across three types of commercially processed food matrices. The limits of quantitation were 1 μg/g in pop food and sauce, and 10 μg/g in surimi product, respectively. This study supports the use of recombinant full-length isotope-labelled proteins rather than stable-isotope labelling peptides as internal standards to achieve more accurate quantitation of food allergens as the digestion error is corrected., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC)-MS/MS. Then, natural angiotensin I and 13 C 1 -labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13 C 1 -labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13 C 1 -labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as "in-house" standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC-MS and LC-MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments., (© 2024. The Author(s).)

1,3-dichlorobenzene (1,3-DCB) is an aromatic solvent that might be formed during thermal decomposition of bis(2,4-dichlorobenzoyl)peroxide used as initiator in silicone rubber production with many workers exposed worldwide. During metabolism of 1,3-DCB, two isomeric mercapturic acids can be formed from ring oxidation of 1,3-DCB in the liver, namely 2,4-dichlorophenylmercapturic acid (24CPhMA) and 3,5-dichlorophenylmercapturic acid (35CPhMA). These urinary mercapturic acids might serve as biomarkers of the toxicologically relevant absorbed dose of 1,3-DCB and have not been determined so far. Thus, we were aimed to develop an analytical method for quantification of these biomarkers. Authentic standards of both mercapturic acids as well as deuterium-labelled analogues were self-synthesized. A method for the quantification of both CPhMAs in human urine using online-SPE LC/MS/MS was developed and validated with an LOQ of 0.1 ng mL -1 for both CPhMAs. The analytes were extracted from urine by online-SPE on a restricted access material phase, transferred to the analytical column and quantified by tandem mass spectrometry. Interday (n = 6) and Intraday (n = 10) precision for both CPhMAs ranged from 1.7 to 4.3 % with accuracies between 99.4 and 109.9 % at concentrations of 0.6 and 3 ng mL -1 . We applied the method on post-shift urine samples of 16 workers of the silicone rubber industry with occupational exposure to 1,3-DCB. Both CPhMAs were above LOQ in 15 of 16 urine samples with median levels (range) for 24CPhMA and 35CPhMA of 1.64 ng mL -1 (<0.1 - 8.2 ng mL -1 ) and 3.98 ng mL -1 (0.36 - 24.1 ng mL -1 ), respectively. This is the first report on specific urinary mercapturic acids of 1,3-DCB in humans. Our results show that ring oxidation of 1,3-DCB is considered to be a toxicologically relevant metabolic pathway in humans. This might improve risk assessment of 1,3-DCB-emissions in silicone rubber industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

We describe the simultaneous quantification of six antiviral drugs in serum based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The target drugs-hydroxychloroquine, chloroquine, favipiravir, umifenovir, ritonavir, and lopinavir-were extracted and purified from serum with 75 % v/v methanol as the precipitant reagent. The six analytes were clearly separated within 15 min using gradient elution and mixed-mode stationary phase. The measurement accuracy and precision were assured by adopting isotopes as internal standards. The optimized measurement procedure was strictly validated in linearity, sensitivity, accuracy, and precision. To confirm the robustness of the method in matrix, the method was additionally applied to various types of serum, namely hyperlipidemic and hyperglycemic serum. The method was then applied to assess the stability of the drugs in serum in order to set sample handling and storage guides for laboratory testing. Lastly, the method was implemented in different LC-MS systems to confirm its applicability across similar equipment commonly used in clinical testing laboratories. The overall results show that the optimized protocol is suitable for the accurate, simultaneous quantification of the six antiviral drugs in serum, and it is anticipated to satisfactorily serve as a reference protocol for the analysis of a wide range of other antiviral drugs for drug monitoring with various purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Although different quantification methods are extensively used in environmental chemistry, the impact of the choice of method on the quality and range of analytical results is understudied. This two-part study consists of (a) in-lab evaluation and (b) a traditional meta-analysis (n = 66) of commonly used quantification methods): (i) external calibration; (ii) isotope dilution method with authentic target analogs; (iii) isotope dilution with non-target standards; and (iv) standard addition prior to LC-MS/MS in liquid chromatography tandem mass spectrometry (LC-MS/MS) by example of antibiotics in sewage sludge from across the U.S. Using method (i) as the benchmark quantification method for the antibiotic erythromycin in biosolids, other quantification methods resulted in an overestimation (110-450 %) or an underestimation (10-60 %). Using the method (iv) as the benchmark for other compounds resulted in an overestimation (101-14,700 %) or an underestimation (6-98 %). Matrix effects were also observed and were dependent on the matrix and analyte type. For example, in the case of erythromycin, all sample matrices showed signal suppression. This study showed that in the absence of isotopically labeled analogs, the most accurate alternate quantification method may need to be experimentally determined depending on the analyte. Analysis of published literature on pharmaceuticals in sewage sludge indicated that isotope dilution with authentic target analog is most commonly used, followed by non-target isotope standards, standard addition, and finally external calibration., Competing Interests: Declaration of competing interest R.U.H. is a cofounder of AquaVitas, an Arizona State University startup company providing commercial services in wastewater-based epidemiology. R.U.H. also is the founder of OneWaterOneHealth, a nonprofit project of the Arizona State University Foundation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 β-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 β-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 ± 39.0 pmol/L, and 2017012 was 761.9 ± 35.5 pmol/L., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Previous methods for vitamin B12 (B12) analysis have extensively used cyanidation conversion with the intention of converting all cobalamins to cyanocobalamin (CNCbl) for total B12 determination. This approach has been favored for its advantages in reducing the number of analytes, increasing analyte concentration, and improving analyte stability. However, the present study revealed underlying limitations associated with this approach. First, a stable isotope dilution assay (SIDA) determining total B12 as CNCbl after cyanidation conversion (conversion SIDA method) was developed. Method validation demonstrated good sensitivity, recovery, accuracy, and reproducibility for the target analyte CNCbl. However, subsequent application of the conversion method to real meat samples showed incomplete conversions of cobalamins. These inconsistencies revealed day-to-day variability and reliability challenges associated with the cyanidation process. It was not possible to identify this issue during method validation as CNCbl was spiked as the sole analyte and it requires no further cyanidation conversion. The application of LC-MS/MS enabled the detection of trace amounts of unconverted cobalamins. Nevertheless, this approach remains restricted by instrument sensitivity and stability as well as the performance of immunoaffinity purification for different vitamers. Further development of a reliable monitoring method is a prerequisite for further optimization of the cyanidation process. However, significant improvements of analytical instrumentation in terms of sensitivity and stability are required to overcome the current limitations., (© 2023. The Author(s).)

Carbonyl compounds are among the most important flavor substances that affect the taste of Baijiu. However, high coverage analysis of carbonyl compounds is obstructed due to the poor ionization efficiency of these compounds. Here we report a chlorine isotope labeling-assisted ultrahigh-performance liquid chromatography-high resolution mass spectrometry-based method (CIL-UHPLCHRMS) for profiling and annotation of carbonyl compounds in sauce flavored-Baijiu Daqu. 4-Chloro-2-hydrazinylpyridine was demonstrated to be a good labeling reagent that could achieve highly sensitive profiling and high-coverage screening of carbonyl compounds in the absence of heavy isotope labeling reagents. In the analysis of eight carbonyl standards representing different carbonyl categories, l-(-)-fucose, 2-carboxybenzaldehyde, 2-hydroxyacetophenone and heptan-2-one could be ionized only after labeling and MS signals were significantly increased for other 4 standards with an enhancement factor ranging from 181-fold for 3-methoxysalicylaldehyde to 3141-fold for tridecan-2-one. The annotation was achieved based on multidimensional information including MS1, predicted t R , in silico MS/MS and manually annotated fragments. In total, 487 carbonyl compounds were detected in Baijiu Daqu, among which, 314 (64.5%) of them were positively or putatively identified. The outcome of the linearity (with a linear range of 2, 3 orders of magnitude), precision (less than 10%), and limit of detection (varied from 0.07 to 0.10 nM) indicated that the method was adequate for profiling carbonyl compounds in complex biological samples. The established method was successfully applied to study carbonyl compounds in Baijiu Daqu with different colors and different seasons. Taken collectively, the present work provides an effective, simple and economic strategy for comprehensive analysis of carbonyl compounds in complex matrix samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Aldehyde-containing metabolites are reactive electrophiles that have attracted extensive attention due to their widespread occurrence in organisms and natural foods. Herein we described a newly-designed Girard's reagent, 1-(4-hydrazinyl-4-oxobutyl)pyridin-1-ium bromide (HBP), as charged tandem mass (MS/MS) tags to facilitate selective capture, sensitive detection and semi-targeted discovery of aldehyde metabolites via hydrazone formation. After HBP labeling, the detection signals of the test aldehydes were increased by 21-2856 times, with the limits of detection were 2.5-7 nM. Upon isotope-coded derivatization with a pair of labeling reagents, HBP-d 0 and its deuterium-labeled counterpart HBP-d 5 , the aldehyde analytes were converted to hydrazone derivatives, which generated characteristic neutral fragments of 79 Da and 84 Da, respectively. The isobaric HBP-d 0 /HBP-d 5 labeling based LC-MS/MS method was validated by relative quantification of human urinary aldehydes (slope=0.999, R 2 > 0.99, RSDs ≤ 8.5%) and discrimination analysis between diabetic and control samples. The unique isotopic doubles (Δm/z = 5 Da) by dual neutral loss scanning (dNLS) provided a generic reactivity-based screening strategy that allowed non-targeted profiling and identification of endogenous aldehydes even amidst noisy data. The LC-dNLS-MS/MS screening of cinnamon extracts led to finding 61 possible natural aldehydes and guided discovery of 10 previously undetected congeners in this medicinal plant., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Objectives: Topiramate is an antiepileptic drug (AED) used for the monotherapy or adjunctive treatment of epilepsy and for the prophylaxis of migraine. It has several pharmacodynamic properties that contribute to both its clinically useful properties and observed adverse effects. Accurate measurement of its concentration is therefore essential for dose adjustment/optimisation of AED therapy. Our aim was to develop and validate a novel reference measurement procedure (RMP) for the quantification of topiramate in human serum and plasma., Methods: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method in combination with a protein-precipitation-based sample preparation allows for quantification of topiramate in human serum and plasma. To assure traceability to SI units, quantitative nuclear magnetic resonance (qNMR) was applied to characterize the reference material used as primary calibrator for this RMP. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Accuracy and precision was evaluated performing an extensive five day precision experiment and measurement uncertainty was evaluated according Guide to the Expression of Uncertainty in Measurement (GUM)., Results: The method enabled topiramate quantification within the range of 1.20-36.0 μg/mL without interference from structurally related compounds and no evidence of a matrix effect. Intermediate precision was ≤3.2 % and repeatability was 1.4-2.5 % across all concentration levels. The relative mean bias was -0.3 to 3.5 %. Expanded measurement uncertainties for target value assignment (n=6) were found to be ≤2.9 % (k=2) independent of the concentration level and the nature of the sample., Conclusions: In human serum and plasma, the RMP demonstrated high analytical performance for topiramate quantification and fulfilled the requirements on measurement uncertainty. Traceability to SI units was established by qNMR content determination of the topiramate, which was used for direct calibration of the RMP. This RMP is, therefore, fit for purpose for routine assay standardization and clinical sample evaluation., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Purpose: Detection of Clostridium perfringens enterotoxin (CPE) in human stool is critical evidence of food poisoning. However, processing patient-derived samples is difficult and very few methods exist to confirm the presence of CPE. In this study, a technique was developed using proteomic analysis to identify and quantify CPE in artificial gut fluid as an alternative., Methods: The standard CPE was spiked into artificial gut fluids, and effective methods were developed by employing both a stable isotope-labelled internal standard peptide and liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Proteotypic peptide EILDLAAATER formed by tryptic digestion was selected for quantitation of CPE. The peptide was identified using product ion spectra. Although the nontoxic peptides originating from CPE showed very low detectability in extraction and tryptic digestion, they could be detected with sufficient sensitivity using the method we developed. Based on a spiked recovery test at two concentrations (50 and 200 µg/kg), the recovery values were 85 and 78%, respectively. The relative standard deviations of repeatability and within-laboratory reproducibility were less than 8 and 11%, respectively. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification (LOQ) was estimated to be 50 µg/kg. The combination of the product ion spectra and relative ion ratio supported CPE identification at the LOQ level., Conclusions: To the best of our knowledge, this is the first report of proteomic analysis of CPE using LC-MS/MS. The method would greatly help in assessing CPE reliably., (© 2023. The Author(s), under exclusive licence to Japanese Association of Forensic Toxicology.)

Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability., (© 2023 Wiley-VCH GmbH.)

Objectives: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for levetiracetam quantification in human serum and plasma., Methods: Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to characterize the RMP material to ensure traceability to SI units. To quantify levetiracetam, an LC-MS/MS method was optimized using a C8 column for chromatographic separation following protein-precipitation-based sample preparation. Spiked matrix samples of serum and plasma were used to test selectivity and specificity. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Precision and accuracy were evaluated over 5 days. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM)., Results: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of levetiracetam within the range of 1.53-90.0 μg/mL. Intermediate precision was <2.2% and repeatability was 1.1-1.7% across all concentrations. The relative mean bias ranged from -2.5% to -0.3% across all levels and matrices within the measuring range. Diluted samples were found with a mean bias ranging from -0.1 to 2.9%. The predefined acceptance criterion for measurement uncertainty was met and determined for individual measurements independently of the concentration level and sample type to be ≤4.0% (k=2)., Conclusions: We present a novel LC-MS/MS)-based candidate RMP for levetiracetam in human serum and plasma. Its expanded measurement uncertainty of ≤4.0% meets the clinical needs in levetiracetam monitoring. Utilizing qNMR to characterize levetiracetam reference materials allowed metrological traceability to SI units., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, we describe our FT4 candidate cRMP and its validation in clinical samples., Methods: This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera., Results: It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers., Conclusions: Our candidate cRMP measures FT4 accurately and performs well in serum matrix., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented., Methods: The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5 days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days., Results: The RMP allowed aldosterone quantification within the range of 20-1,200 pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between -2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be ≤2.3%; expanded uncertainties were ≤4.6% (k=2) for all levels., Conclusions: The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objective: To establish a method for the determination of three furfural compounds in coffee and its products by gas chromatography-tandem mass spectrometry., Methods: The samples were extracted with ethanol water(1∶2, V∶V) solution, ultrasonic with 10% Na&#95;2CO&#95;3 solution for 5 min, purified with 100 mg C&#95;(18), 50 mg Srong Cation exchang(SCX), 150 mg anhydrous MgSO&#95;4, separated by HP-INNOWAX capillary column(30 m×0.25 mm, 0.25 μm), detected by gas chromatography-tandem mass spectrometry, and quantified by isotope internal standard method., Results: The correlation coefficients(r) of the three furfural compounds were all greater than 0.999, the limits of detection were 0.004-0.011 mg/kg, and the limits of quantitation were 0.013-0.031 mg/kg. The average recoveries were 86.0%-112% and the relative standard deviations were 4.1%-10.6%(n=6), at 3 supplemental levels in 3 different coffee substrates. Nine samples of coffee beans, instant coffee and coffee drinks were tested, and all three components to be tested were detected., Conclusion: The method is simple, rapid, sensitive, with good accuracy and precision. It is suitable for the determination of three furfural compounds in coffee and its products.

This study aimed to establish an isotope dilution LC-MS/MS method for the determination of folic acid, 5-formyltetrahydrofolate and 5-methyltetrahydrofolate in human serum. This method was then used to quantify these three folate forms in the healthy adult population and supplement users. A stable 96-well solid-phase extraction system was used to prepare serum samples. The highly sensitive method was established using a Shimadzu LCMS-8060NX. The linearity was good in the range of 0.1-10 nmol/l for folic acid and 5-formyltetrahydrofolate and 1.0-100 nmol/l for 5-methyltetrahydrofolate. The accuracy and precision were good. The method was sensitive, robust and high-throughput and could be used for the routine clinical monitoring of these three folate forms in the Chinese population.

Objectives: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma., Methods: Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines., Results: The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200-5,700 ng/mL (0.01584-12.54 μmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was ≤4.3% and repeatability 1.5-2.1% for all analyte concentrations. Bias was -3.0 to 2.1% for samples within the measuring range and 0.8-4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was ≤3.4%., Conclusions: This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: We developed an isotope dilution (ID)-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for lamotrigine in human serum and plasma, using quantitative nuclear magnetic resonance-characterized reference standards to ensure traceability to the International System of Units., Methods: A sample preparation protocol based on protein precipitation combined with LC-MS/MS analysis using a C18 column for chromatographic separation was established for the quantification of lamotrigine in human serum and plasma. Assay validation was performed according to current guidelines. Spiked serum and plasma samples were used to assess selectivity and specificity; a post-column infusion experiment and comparison of standard line slopes were performed to ascertain possible matrix effects. Precision and accuracy were determined in a 5 days validation experiment. Measurement uncertainty was determined per the Guide to the Expression of Uncertainty in Measurement., Results: The method allowed the quantification of lamotrigine in serum and plasma in a range of 0.600-24.0 μg/mL without any observable matrix effects. The relative mean bias (n=6) ranged from 1.7 to 3.7%; intermediate precision, including variances in between-day, -calibration, and -injection, was ≤2.4%, independent of the level and matrix. Total measurement uncertainty for a single measurement was ≤2.6%; expanded uncertainty was ≤5.2% (coverage factor k=2)., Conclusions: This candidate RMP based on ID-LC-MS/MS provides a traceable and reliable platform for the standardization of routine assays and the evaluation of clinical samples., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Retention time (RT) alignment has been important for robust protein identification and quantification in proteomics. In data-dependent acquisition mode, whereby the precursor ions are semistochastically chosen for fragmentation in MS/MS, the alignment is used in an approach termed matched between runs (MBR). MBR transfers peptides, which were fragmented and identified in one experiment, to a replicate experiment where they were not identified. Before the MBR transfer, the RTs of experiments are aligned to reduce the chance of erroneous transfers. Despite its widespread use in other areas of quantitative proteomics, RT alignment has not been applied in data analyses for protein turnover using an atom-based stable isotope-labeling agent such as metabolic labeling with deuterium oxide, D 2 O. Deuterium incorporation changes isotope profiles of intact peptides in full scans and their fragment ions in tandem mass spectra. It reduces the peptide identification rates in current database search engines. Therefore, the MBR becomes more important. Here, we report on an approach to incorporate RT alignment with peptide quantification in studies of proteome turnover using heavy water metabolic labeling and LC-MS. The RT alignment uses correlation-optimized time warping. The alignment, followed by the MBR, improves labeling time point coverage, especially for long labeling durations.

Ceramides are important intermediates in the metabolism of sphingolipids. High-throughput liquid chromatography-mass spectrometry has been used extensively for monitoring the levels of serological ceramides, but is still limited by inadequate coverage or lack of sensitivity. Herein, a rapid, sensitive, and high-throughput isotope dilution liquid chromatography-negative ion electrospray tandem mass spectrometry (IDLC-nESI-MS/MS) method was developed and verified for accurate quantification of 41 ceramides, involving ceramides with C16-20 sphingosine, dihydro-ceramide, and dehydro-ceramide. This method was validated with excellent linearity (R 2  > 0.99) and good recovery in the range of 90-110%. Intra- and inter-day imprecision were below 5.57% and 7.83% respectively. The improved high-throughput quantitative method developed in this study may aid in the accurate characterization of ceramides for understanding ceramide biology and application in disease diagnosis., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

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 15 N-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.

Plasma renin activity (PRA) is recommended as the first screening indicator for primary aldosteronism. Immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for quantifying PRA, but the interchangeability across assays and laboratories was suboptimal, which predominantly related to the differences in the plasma incubation strategy. This study aims to establish and validate a designed comparison method based on LC-MS/MS. The sensitivity, matrix effect, precision, accuracy, and storage stability were validated according to the Clinical Laboratory Standard Institution (CLSI) C-62A guidelines. The plasma incubation procedure was optimized to achieve maximum PRA results. The short-term stability of PRA plasma was assessed at 4 °C and room temperature (RT) for specific time points. Differences from the baseline were calculated using a one-way analysis of variance. The designed comparison method for PRA measurement exhibits excellent performance characteristics. The results from the 2022 national external quality assessment scheme for PRA showed good consistency of the developed method with other LC-MS/MS methods (relative biases: -6.8% to 4.6%), which demonstrated the reliability of the established method. Two sets of generation buffers were optimized to maximize the renin activity. The acetate buffer was recommended to be used in laboratory practice due to better metrological sensitivity. PRA plasma is stable for one day at 4 °C and RT. In summary, a reliable, traceable, and reproducible LC-MS/MS method for determining PRA was well-established and validated. The recommended incubation protocol is hoped to reduce the discrepancy in Ang1 generation. The evaluated short-term stability for PRA plasma could provide flexibility in clinical practice.

Glycated albumin (GA) in human serum is tested clinically as a short-term indicator for glucose monitoring. Here, we evaluated a candidate serum reference material (RM) at three different GA concentrations to help standardize serum GA measurements. Both albumin and GA were quantitatively determined using isotope-dilution liquid chromatography/tandem mass spectrometry with lysine-4,4,5,5-D 4 ·2HCl (D 4 -lysine) and N ε - l3 C 6 -(l-deoxy-d-fructose-1-yl)-l-lysine ( 13 C 6 -DOF-lysine) as internal standards and lysine and synthetic DOF-lysine as calibration standards. The method was evaluated with the RM, JCCRM611-1, from the Reference Material Institute for Clinical Chemistry Standards. The homogeneity and stability of the candidate RMs were examined using a commercial biochemical analyzer. Fifteen units were randomly selected, and statistical analysis showed no inhomogeneity. The candidate RMs were stable for at least 6 months at -80 °C. The coefficients of variation (CVs) for the JCCRM611-1 RM ranged from 3.2% to 2.3%, and the biases ranged from 4.12% to -1.84%. GA was tested at low, medium, and high concentrations, which were quantified as 249.53 ± 13.29, 408.02 ± 11.70, and 637.22 ± 17.03 mmol/mol, respectively. The overall CVs ranged from 0.99% to 2.51%. The candidate RMs can potentially be used to develop a traceability chain to improve the accuracy of GA measurements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objectives: To describe and validate a reference measurement procedure (RMP) for gabapentin, employing quantitative nuclear magnetic resonance (qNMR) spectroscopy to determine the absolute content of the standard materials in combination with isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) to accurately measure serum and plasma concentrations., Methods: A sample preparation protocol based on protein precipitation in combination with LC-MS/MS analysis using a C8 column for chromatographic separation was established for the quantification of gabapentin. Assay validation and determination of measurement uncertainty were performed according to guidance from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. ID-LC-MS/MS parameters evaluated included selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement., Results: The use of qNMR provided traceability to International System (SI) units. The chromatographic assay was highly selective, allowing baseline separation of gabapentin and the gabapentin-lactam impurity, without observable matrix effects. Variability between injections, preparations, calibrations, and days (intermediate precision) was <2.3%, independent of the matrix, while the coefficient of variation for repeatability was 0.9-2.0% across all concentration levels. The relative mean bias ranged from -0.8-1.0% for serum and plasma samples. Passing-Bablok regression analysis indicated very good inter-laboratory agreement; the slope was 1.00 (95% confidence interval [CI] 0.98 to 1.03) and the intercept was -0.05 (95% CI -0.14 to 0.03). Pearson's correlation coefficient was ≥0.996. Expanded measurement uncertainties for single measurements were found to be ≤5.0% (k=2)., Conclusions: This analytical protocol for gabapentin, utilizing traceable and selective qNMR and ID-LC-MS/MS techniques, allows for the standardization of routine tests and the reliable evaluation of clinical samples., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been widely used in the quantitative analysis of drugs. The ubiquitous concomitant drug scenario in the clinic has spawned a large number of co-analyses based on this technique. However, signal suppression caused by concomitant drugs during electrospray ionization may affect the quantification accuracy of analytes, which has not received enough attention. In this study, metformin (MET) and glyburide (GLY) were co-eluted by the conventional optimization of chromatographic conditions to illustrate the effect of signal suppression caused by the combined drugs on the quantitative analysis. The response of MET was not affected by GLY over the investigated concentration range. However, the GLY signal could be suppressed by about 30% in the presence of MET, affecting its pharmacokinetic analysis in simulated samples. As an attempt to solve the suppression of GLY by co-eluting MET, dilution can alleviate the suppression. However, this method still has limitations due to the sacrifice of sensitivity. The stable isotope-labeled internal standard could play a role in correction and improve the quantitative accuracy of GLY, which was further confirmed in the pharmacokinetic study of simulated samples. This study provided an example model to illustrate the possible effect of clinical drug combination on LC-MS/MS drug quantitative analysis and investigated the effective methods to solve this problem., Competing Interests: The authors declare no conflict of interest.

Accurate identification and quantification of drugs and their metabolites (analytes) in biological matrices is an analytical foundation of clinical and forensic toxicology. For decades, liquid chromatography interfaced by electrospray ionization with tandem mass spectrometry (LC-ESI-MS/MS) has been a widely used technology for analysis in the field of toxicology, as well as in many other fields of bioscience. It is also known that ion response in LC-ESI-MS/MS analysis is influenced by coeluting biological compounds and that preanalytical sample clean-up is often insufficient in removing these interferences. As a result, a normalization technique is commonly used for assessment and compensation of matrix effects encountered in routine analysis. Internal standardization with a stable isotope analog of the analyte is the predominant normalization technique used in LC-ESI-MS/MS analysis. The technique, however, requires commercial availability or costly custom synthesis of an isotopic analog specific for each analyte. Here we describe an alternative technique for matrix normalization for use in high-volume, multianalyte testing without the need for isotope analogs. The technique involves analysis of the original sample (neat analysis) followed by analysis of a second sample aliquot (spike analysis) that has been fortified with a controlled amount of reference analyte. A calibration procedure similar to internal standardization is employed, using an ion response ratio of neat to fortified analyte. As a demonstration of the technique in multianalyte testing, we provide a detailed protocol for simultaneous detection and quantification of 102 drugs and drug metabolites in human urine. We also provide a support protocol for addition of new analytes to the multianalyte panel, allowing convenient collection of the validation data during routine testing. The matrix normalization technique and testing principles may be applicable to a wide range of analytes and biological matrices, not only those encountered in toxicology but also in other fields of bioscience. © 2023 Wiley Periodicals LLC. Basic Protocol: Detection and quantification of 102 toxicology analytes in urine by LC-ESI-MS/MS analysis using the threshold accurate calibration technique Support Protocol: Method for addition and validation of new analytes to expand the Basic Protocol., (© 2023 Wiley Periodicals LLC.)

Rationale: The in-sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked-in stable isotope labeled internal standard (SIL-IS) to build a standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high-resolution mass spectrometers, quantitation on high-resolution mass spectrometer with product ions has not been tested. We tested the feasibility of this approach using product ions on an high-resolution mass spectrometer equipped with an Orbitrap detector., Methods: Using a proteomics workflow for sample preparation, two surrogate peptides were quantified from a complex matrix of protein digest from human peripheral blood mononuclear cells (hPBMCs). SIL-IS was spiked in at different levels to construct calibration curves in a traditional manner. ISCCs were prepared using extracted ion chromatograms from isotopically resolved mass spectra and compared with traditional calibration curves., Results: A linear response was observed with ISCC approach for at least two to three orders of magnitude in MS1 as well as targeted MS2 (tMS2). From protein digests, isobaric interferences were observed for endogenous peptides on the MS1 level; this was circumvented with product-ion-based quantitation where for one peptide, %CV for endogenous levels was more than 20% with ISCC but higher with the traditional calibration curve approach. For the second peptide, endogenous levels could not be determined in the traditional approach as calibrant levels did not bracket the lower end, and with the ISCC approach, isotopologues at abundances lower than the endogenous level allowed for quantitative assessments., Conclusions: ISCC demonstrated improved precision across surrogate peptides from endogenous protein digests. In samples where endogenous analyte concentrations were low in abundance, ISCC rescued what would have been a non-reportable result in a traditional bioanalytical assay as calibrant levels were not prepared at adequately low levels to bracket unknowns. ISCC using high-resolution mass spectrometer is feasible and ideal compared to unit resolution mass spectrometers. High-resolution mass spectrometer allows for isotopic resolution for analytes with > + 2 charge state and provides flexibility in quantification using multiple product ions. ISCC using high-resolution mass spectrometer allows for simultaneous assaying of low abundance isotopologues, the signal acquisition of which is not constrained by limits in data acquisition or calibrant preparation as with other approaches but rather limited by platform sensitivity. In contrast to unit resolution mass spectrometers, these features offered by high-resolution mass spectrometer could be especially useful for the drug discovery assay support where there is less lead time for assay development than for the assays to support the drug development studies., (© 2022 John Wiley & Sons Ltd.)

Purpose: The present study aimed to establish and validate an isotope-dilution-UHPLC-MS/MS method for the determination of perampanel (PER) concentration and investigate the effect of eslicarbazepine acetate (ESL) on the pharmacokinetics of PER in rats., Methods: The rats were randomly divided into the control (0.5% CMC-Na) and experimental groups (ESL, 72 mg/kg), with six rats in each group. A single dose of PER (1 mg/kg) was administered after a week of repetitive 0.5% CMC-Na or ESL dosing (72 mg/kg); then, plasma samples were collected. Perampanel-d 5 (PER-d 5 ) was used as the internal standard (IS), liquid-liquid extraction of plasma samples was carried out using ethyl acetate, and chromatographic separation was carried out on a Titank C18 column (2.1 mm × 50 mm, 3.0 μm) using a gradient mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.3 mL/min., Results: PER had good linearity (0.3-600 ng/mL, r >0.999), and the accuracy, precision, recovery rate, and matrix effects (ME) met the Food and Drug Administration (FDA) guidelines. Compared to the control group, the area under the curve AUC 0→t, AUC 0→∞ , and Cmax of PER in the experimental group decreased by 30.28%, 30.34%, and 46.94%, respectively, and CL increased by 32.08%., Conclusion: ESL could induce the metabolism of PER in rats and decreases plasma exposure to PER. Thus, the concomitant treatment with ESL may require a high dose of PER to achieve the same efficacy., Competing Interests: The authors report no conflicts of interest in this study., (© 2022 Liu et al.)

Beta 2-microglobulin (B2M) is a commonly used detection index in clinical laboratories. Currently, it is used as a sensitive indicator for the early detection of kidney disease. Immunoassay is the most commonly used method for B2M detection in clinical practice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has changed the face of laboratory testing by providing high-throughput analysis with better specificity than detection methods using only antibodies. An isotope-dilution LC-MS/MS (ID-LC-MS/MS) method was developed for serum B2M quantification. After B2M denaturation, reduction and alkylation of cysteine residues, trypsin was added for digestion of B2M. Then, it was purified by a solid-phase extraction column, and the sample was injected into a high-performance LC-MS/MS for measurement. A signature peptide (VNHVTLSQPK) was selected as a surrogate for B2M. A stable isotope-labeled peptide (VNHVT[ 13 C 6 15 N]LSQP) was used as the internal standard to quantify B2M based on the calibration curve method. The linear range of serum B2M calibration curve was from 0.25 to 40 mg/L. The limit of quantification was 0.25 mg/L, and limit of detection was 0.06 mg/L. At different concentrations of serum B2M, the precision (coefficients of variation, CV%) ranged from 1.47% to 3.97%, and accuracy (relative error, RE%) was within -3.15% and 6.80% of nominal values. The applicability and reliability of the method were verified by measuring B2M in the ERM-DA470k/IFCC and serum samples. The validated LC-MS/MS method was successfully applied to a clinical study involving quantification of serum B2M in patients with acute renal insufficiency and healthy individuals. Deming analysis showed that the ID-LC-MS/MS and immunoassay were in good agreement. The LC-MS/MS method we developed is sensitive and reliable for the absolute quantification of serum B2M., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In inductively coupled plasma mass spectrometry, the most abundant Ca isotope ( 40 Ca) suffers from isobaric interference with argon, hindering the potential for low detection limits of Ca. A powerful approach is to remove the interference by using a reaction gas in a reaction cell. Ammonia (NH 3 ) has proven to be an effective reaction gas by process of a charge transfer reaction. However, NH 3 is highly corrosive and toxic and cannot remove isobaric 40  K. Therefore, this work proposes the use of nitrous oxide (N 2 O) to mass shift the target analyte 40 Ca to 40 Ca 16 O + as a non-corrosive and non-toxic alternative. Instrument performance testing demonstrated that N 2 O was capable of reaching equivalent detection limits (0.015 ng g -1 ) and background equivalence concentrations (0.041 ng g -1 ) to that of NH 3 and limited by the blank only. Further investigation of matrix interferences with synthetic standards highlighted that the N 2 O approach supports the separation of potassium (K) and magnesium (Mg)-based interferences at tested concentrations of more than 600 times and almost 800 times higher than Ca respectively, whereas NH 3 was found to only support the removal of Mg. This work highlights a clear advantage of N 2 O for low-level Ca determinations with high matrix loads, as well as compatibility with other instrumentation sensitive to corrosion that supports reaction cell technology., (© 2022. The Author(s).)

Background: Vitamin D deficiency/insufficiency and toxicity are worldwide issues; thus, accurate diagnostic assays are required to measure vitamin D. We evaluated the performance of the new Elecsys ® Vitamin D total III assay (Roche Diagnostics International Ltd)., Methods: Repeatability and intermediate precision of the Elecsys Vitamin D total III assay (cobas e 601 analyzer) were evaluated at three sites using five human serum pools (HSPs) and two PreciControls (five-day model, one reagent lot [CLSI-EP05-A3]) and compared against prespecified acceptance criteria. A serum verification panel, with reference isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) values, was used for comparator assay/concordance studies at two sites, assessed using unweighted Deming regression. Testing of serum vs. plasma on the Elecsys assay was conducted at one site using samples from healthy adults; assessed using Passing-Bablok regression., Results: Repeatability (HSP1 [16.8-18.4 ng/ml], SD 0.87-1.07; HSP5 [94.5-98.0 ng/ml], CV 1.58%-2.76%) and intermediate precision (HSP1, SD 1.14-1.77; HSP5, CV 2.00%-4.13%) met acceptance criteria across sites. Agreement was observed between the Elecsys assay and (i) the ID-LC-MS/MS verification panel (slope, 0.936-1.01; Pearson's r, 0.960-0.986) and (ii) comparator assays (slope, 0.921-1.15; Pearson's r, 0.958-0.982). The Elecsys assay correctly assigned the highest combined percentage of samples to deficient (100%) and insufficient (89.5%) vitamin D categories vs. comparator assays and demonstrated comparable performance in serum and plasma (y = 0.103 + 0.984x)., Conclusions: The Elecsys Vitamin D total III assay demonstrated good analytical performance and compared favorably with other assays, supporting its use in clinical practice., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

This study developed a sensitive and easy method to directly analyze N ε -(carboxymethyl)lysine (CML) by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Acetonitrile and water were used as the binary mobile phase without ion pair reagents, while BEH Amide column was applied to detect CML. The target substance without derivatization could be well retained and separated after the pretreatment conditions were optimized. Oasis MCX solid phase extraction (SPE) cartridge showed the best recovery rate of CML 96.7 % and purification ability among the tested SPE cartridges. There was no obvious difference in pretreatment abilities with or without protein precipitation on the CML quantitative analysis. The recovery rates 97-98 % for CML were achieved by UPLC-MS/MS, as well as the detection limit and quantification limit were 0.05 mg/kg and 0.15 mg/kg sample, respectively. This method was suitable for quantifying CML content with high recovery rate and low detection and quantification limit in sterilized milk., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Various genotoxic carcinogens ubiquitously present in the human environment or respective reactive metabolites form adducts in DNA and proteins, which can be used as biomarkers of internal exposure. For example, the mass spectrometric determination of Val adducts at the N-termini of hemoglobin (Hb) peptide chains after cleavage by an Edman degradation has a long tradition in occupational medicine. We developed a novel isotope-dilution UHPLC-MS/MS method for the simultaneous quantification of Val adducts of eight genotoxic substances in Hb after cleavage with fluorescein-5-isothiocyanate (FIRE procedure™). The following adducts were included [sources in square brackets]: N-(2,3-dihydroxypropyl)-Val [glycidol], N-(2-carbamoylethyl)-Val [acrylamide], N-(2-carbamoyl-2-hydroxyethyl)-Val [glycidamide], N-((furan-2-yl)methyl)-Val [furfuryl alcohol], N-(trans-isoestragole-3'-yl)-Val [estragole/anethole], N-(3-ketopentyl)-Val [1-penten-3-one], N-(3-ketooctanyl)-Val [1-octene-3-one], and N-benzyl-Val [benzyl chloride], each of which was quantified with a specific isotope-labeled standard. The limits of quantification were between 0.014 and 3.6 pmol/g Hb (using 35 mg Hb per analysis); other validation parameters were satisfactory according to guidelines of the U.S. Food and Drug Administration. The quantification in erythrocyte samples of human adults (proof of principle) showed that the median levels of Hb adducts of acrylamide, glycidamide, and glycidol were found to be significantly lower in six non-smokers (25.9, 12.2, and 4.7 pmol/g Hb, respectively) compared to those of six smokers (69.0, 44.2, and 8.6 pmol/g Hb, respectively). In summary, the method surpasses former techniques of Hb adduct quantification due to its simplicity, sensitivity, and accuracy. It can be extended continuously with other Hb adducts and will be used in epidemiological studies on internal exposure to carcinogens., (© 2022. The Author(s).)

Purpose: An analytical method for quantitation of sibutramine in human hair using gas chromatography (GC)-isotope dilution tandem mass spectrometry (MS/MS) was newly established. In this article, a case is presented, in which a 3.5-year-old male child accidentally ingested chocolate-like product containing sibutramine, showing various symptoms; he could survived the crisis. About 1 month after the incident, his scalp hair sample was subjected to analysis for the causative sibutramine., Method: After cryo-grinding for the hair sample, target compound was extracted with methanol, and the solvent layer was evaporated to dryness. The residue was reconstituted in methanol and analyzed by GC-MS/MS, using the selected reaction monitoring (SRM) mode with a deuterated isotope internal standard., Results: The substance was identified as sibutramine; its concentration in the hair sample of the child was 58.6 pg/mg. The calibration curve of sibutramine in hair samples had a good linear relationship in the concentration range of 20-200 pg/mg (r > 0.99); the extraction recovery rate 85.2-91.8%; the interday and intraday precision and accuracy (bias) examined not greater than 9.6%. Sibutramine in human hair had good stability under 3 different storage conditions at room (20 °C), refrigerated (4 °C) and frozen ( - 20 °C) temperatures for at least 7 days., Conclusions: It should be expected that the method established in this study would contribute to rapid determinations of sibutramine. To our knowledge, this is the first report describing quantitation of sibutramine in an authentic human hair sample by GC-MS/MS., (© 2021. Japanese Association of Forensic Toxicology.)

Objectives: Accurate measurement of serum folate is essential for the diagnosis and management of various disorders. This study aims to investigate the between-method differences of four immunoassays and a rapid isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method., Methods: Roche Cobas (USA), Abbott Alinity i2000 (USA), Beckman Coulter Access (USA), Mindray CL-6000i (China), and the ID-LC-MS/MS method were compared using 46 human serum samples. The results were analysed by Passing-Bablok regressions and Bland-Altman plots. A bias of 13.31% based on biological variation was used as the bias criterion., Results: All the within-run and total coefficients of variation (CVs) met the specification. The folate concentrations determined by all the assays were significantly different (p=0.0028). All assays had correlation coefficients over 0.97 with each other. The 95% confidence intervals (CIs) for the slope seldom contained 1 and few 95% CIs for the intercept contained 0 in the regression equations. Compared to ID-LC-MS/MS, the biases of all assays ranged from -20.91 to 13.56 nmol/L, and the mean relative biases ranged from -9.85 to 40.33%. The predicted mean relative biases at the medical decision levels rarely met the criterion., Conclusions: Assays for serum folate had good correlations with each other but lacked good agreement. The accuracy and consistency of assays for serum folate should be measured and assessed routinely. Standardization work to improve the accuracy of serum folate assays, such as the extension of traceability to reference methods or materials, calibration standardization efforts, and assay-adjusted cut-offs should be promoted., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

We present the results of a multi-disciplinary investigation on a deciduous human tooth (Pradis 1), recently recovered from the Epigravettian layers of the Grotte di Pradis archaeological site (Northeastern Italian Prealps). Pradis 1 is an exfoliated deciduous molar (Rdm 2 ), lost during life by an 11-12-year-old child. A direct radiocarbon date provided an age of 13,088-12,897 cal BP (95% probability, IntCal20). Amelogenin peptides extracted from tooth enamel and analysed through LC-MS/MS indicate that Pradis 1 likely belonged to a male. Time-resolved 87 Sr/ 86 Sr analyses by laser ablation mass spectrometry (LA-MC-ICPMS), combined with dental histology, were able to resolve his movements during the first year of life (i.e. the enamel mineralization interval). Specifically, the Sr isotope ratio of the tooth enamel differs from the local baseline value, suggesting that the child likely spent his first year of life far from Grotte di Pradis. Sr isotopes are also suggestive of a cyclical/seasonal mobility pattern exploited by the Epigravettian human group. The exploitation of Grotte di Pradis on a seasonal, i.e. summer, basis is also indicated by the faunal spectra. Indeed, the nearly 100% occurrence of marmot remains in the entire archaeozoological collection indicates the use of Pradis as a specialized marmot hunting or butchering site. This work represents the first direct assessment of sub-annual movements observed in an Epigravettian hunter-gatherer group from Northern Italy., (© 2022. The Author(s).)

Serum retinol binding protein 4 levels play critical roles in the early diagnosis and therapeutic monitoring of diabetic kidney disease. In this paper, an liquid chromatography-tandem mass spectrometry approach for the absolute quantification of serum RBP4 with high sensitivity and specificity was presented. Following series of procedures including denaturation, reduction, alkylation and trypsin digestion, the signature peptides of RBP4 were separated on the HPLC system by gradient elution. Quantitative analysis was achieved by a triple quadrupole mass spectrometer under multiple reaction monitoring in the positive ion (ESI+) mode. The calibration range was from 6.25 to 125 mg/L (R 2  > 0.993), the lower limit of quantification was 2.50 mg/L, and the lower limit of detection reached 0.0150 mg/L. In the study, the accuracy ranged from 94.6% to 107%, and the relative standard deviation of intra-assay and inter-assay imprecision was less than 5%. The method was demonstrated to realize sensitive and reliable absolute quantification of serum RBP4 conforming to guidelines for bioanalytical method validation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Matrix effects are a great challenge for the quantitative analysis of complex food samples by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS). Stable isotope labeling (SIL) has been widely used as an effective strategy to eliminate matrix effects. Herein, a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click-reaction-based SIL method was proposed for a highly sensitive and selective determination of six synthetic steroid hormones in three different food samples (milk, yogurt, and eggs) by high-performance liquid chromatography (HPLC)-MS/MS. A pair of novel SIL agents, N -(2-azidyl ethyl) aniline ( d 0 -AEA) and d 5 - N -(2-azidyl ethyl) aniline ( d 5 -AEA) were synthesized to label steroid hormones in the samples and standard solution, respectively. The reaction accomplishes in 30 min at 60 °C. The heavy labeled standards were used as internal standards (ISs), which experience the identical ionization processes with light labeled samples to minimize matrix effects. After derivatization, the ionization efficiencies of steroid hormones were greatly improved by 2-54-folds, and the matrix effects ranged from 88.6 to 99.8%. The established method achieved satisfactory detection limits (0.1-2.5 μg L -1 ) and high recoveries (85-102%). These results demonstrated that the proposed method holds unique advantages for trace steroid hormones analysis in foodstuffs.

Objective: To establish a rapid, accurate and sensitive method by liquid chromatography-tandem mass spectrometry with isotope internal standard dilution technique for the determination of chlorpromazine and promethazine and their metabolites in swine tissues., Methods: The swine tissues sample was extracted with acetonitrile and purified on MCX cartridge. The liquid chromatography separation was performed on an ACQUITY UFLC® HSS T3(100 mm×2.1 mm, 1.8 μm) with a linear gradient elution program of 0.1%(V/V) fomic acid-acetonitrile and 0.1%(V/V) formic acid-water solution as the mobile phase. The analytes were analyzed using ESI operating in the positive multiple reaction monitoring(MRM) mode., Results: The limits of quantitation(LOQs) and limits of detection(LODs) for the target objects were 0.12-0.51 μg/kg and 0.04-0.17 μg/kg, respectively. The calibration curves were linear in range of 0.1-20.0 μg/L for chlorpromazine and promethazine, and 0.5-100.0 μg/L for their metabolites(chlorpromazine sulfoxide and isopropyl sulfoxide). The recoveries were between 90.8%-106.0%, and the relative standard deviations(RSDs) were between 1.9%-6.2%(n=6)., Conclusion: The method is highly sensitive and accurate, and is suitable for the analysis of chlorpromazine and promethazine and their metabolites(chlorpromazine sulfoxide and isopropyl sulfoxide) in swine tissues.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used quantitative method in small-molecule pharmacokinetic sample analysis. The linear dynamic range of mass analyzers, typically spanning 3 orders of magnitude, is usually insufficient for this purpose. Utilization of multiple isotopologues has been proposed as a compelling approach to expand the linear dynamic range of LC-MS/MS assays, particularly when the detector is saturated. Isotopologues are a statistical mixture of molecules of the same compound but of different exact masses due to the presence of natural chemical isotopes. While the concept of isotopologues is widely recognized in large-molecule bioanalysis and small-molecule metabolite profiling, it has not been commonly implemented in small-molecule targeted quantification. To increase the awareness of the value of isotopologues in small-molecule LC-MS/MS analysis, this minireview provides the basis of isotopologue distribution in MS/MS and summarizes published studies as well as our own experience in utilizing multiple isotopologues to expand the linear dynamic range of small-molecule LC-MS/MS assays. Considering that utilizing natural isotopologue transitions in the LC-MS/MS assays represents an easy, straightforward, and robust way to expand the linear dynamic range, we believe this method deserves wide application in small-molecule pharmacokinetic sample analysis and can particularly benefit people working in pharmacokinetic labs as well as the GLP bioanalytical labs in pharmaceutical industry., Competing Interests: Conflict of Interest No conflict of interest., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

The determination of amino acids in biological samples is central to the diagnosis of inherited metabolic disorders and also gives significant information about the metabolisms in the cells and living body. The development of analytical method for reliable quantification of amino acids in biological samples is still challenging because of the polar nature of amino acids and complex nature of biological samples causing a high degree of interferences during analysis. In the present study, a pre-column derivatization method using 2-naphtoyl chloride combined with liquid chromatography-tandem mass spectrometry method was developed for the determination of 17 amino acids in human serum and urine matrices. Low detection limits were obtained in the range of 0.015 - 0.266 µmol kg -1 and acceptable recovery results were obtained in human serum and urine samples. Isotopically labelled ( 15 N labelled) amino acids were spiked to standards and samples before derivatization to compensate for the analytical errors in the whole procedure. The combination of quadrupole isotope dilution strategy with the derivatization based reversed phase chromatography allowed to improve method accuracy and precision., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Accumulating evidence suggests that DNA lesion-induced DNA-protein cross-links (DPCs) interrupt normal DNA metabolic processes, such as transcription, replication, and repair, resulting in profound biological consequences, including the development of many human diseases, such as cancers. Although apurinic/apyrimidinic (AP) sites are among the most predominant DNA lesions and are in close proximity to the histone proteins that they wrap around in the nucleosome, knowledge of the chemical structure or biological consequences of their associated DPCs is limited in part due to a lack of sensitive and selective analytical methods. We developed liquid chromatography-tandem mass spectrometry coupled with a stable isotope dilution method for rigorous quantitation of DPCs formed by reacting a DNA AP site with a lysine residue. In combination with chemical derivatization with fluorenylmethoxycarbonyl chloride to form a hydrophobic conjugate, the developed LC-MS/MS method allows sensitive detection of AP site-Lys cross-links down to sub-1 adduct per 10 6 nt. After validation using a synthetic AP site-lysine-cross-linked peptide and an oligodeoxyribonucleotide, the method was used to determine the concentration of AP site-lysine cross-links in hot acid-treated DNA and in human cells exposed to methyl methanesulfonate.

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) lactate as a clinical diagnostic test. Lactate is produced from cellular metabolism, primarily in muscle cells, and provides a source of energy especially during instances of low oxygen levels. Measurement of lactate in CSF provides diagnostic information regarding the body's oxidative metabolism including diagnosis of lactate acidosis, aiding in the diagnosis of blood-brain barrier glucose transporter defect and differentiation between bacterial and viral meningitis. Determination of lactate in CSF (20 μL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Lactate in CSF is determined by a 1:10 dilution with internal standard (sodium lacate-d3) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a six-point standard curve (0.625-20 mM) and has an analytical measurement range of 0.3-20 mM., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The amyloid β peptide, as one of the main components in senile plaque, represents a defining pathological feature for Alzheimer's disease, and is therefore commonly used as a biomarker for this disease in clinical analysis. However, the selection of suitable standards is limited here, since only a few are commercially available, and these suffer from varying purity. Hence, the accurate characterization of these standards is of great importance. In this study, we developed a method for the traceable quantification of the peptide content using species-specific isotope dilution and ICP-MS/MS detection. It is based on the separation of the sulfur-containing amino acids methionine and cysteine after oxidation and hydrolysis of the peptide. Using a strong anion exchange column, both amino acids could be separated from each other, as well as from their oxidized forms and sulfate. The sulfur content was determined via ICP-MS/MS using oxygen as reaction gas. Species-specific isotope dilution was enabled by using a 34 S-labeled yeast hydrolysate, containing methionine sulfone and cysteic acid with different isotopic composition. The peptide contents of Aβ standards (Aβ40,42), as well as myoglobin and lysozyme with different degrees of purity, were determined. For validation purposes, the standard reference material NIST 2389a, which contains the amino acids in a similar concentration, was subjected to the developed sample preparation and analysis method. In addition to accounting for errors during sample preparation, high levels of accuracy and precision could be obtained using this method, making it fit-for-purpose for the characterization of peptide standards., (© 2021. The Author(s).)

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folate and is involved in the regulation of homocysteine and numerous methylation reactions, including synthesis of neurotransmitters, lipids, DNA, and RNA. Measurement of 5-MTHF in CSF provides diagnostic information regarding disorders affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism and cerebral folate deficiency. Determination of 5-MTHF in CSF (50 μL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF- 13 C 5 ) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Acrylamide (AA) is a toxicant in high-temperature processed foods and an animal carcinogen. Upon absorption, AA is metabolized to glycidamide (GA) or conjugates with glutathione (AA-GSH). Important advantages of microdialysis coupled with liquid chromatography-tandem mass spectrometry (MD-LC-MS/MS) include its minimization of potential losses during sample collection, storage and preparation, as well as an improvement in temporal resolution for toxicokinetics (TKs). We aimed to simultaneously study the TKs of AA and products of its primary metabolism using an isotope-dilution (ID) MD-LC-MS/MS method. MD probes implanted into the jugular vein/right atrium of anesthetized Sprague Dawley rats were connected to the ID-LC-MS/MS for continuous monitoring of AA, GA and AA-GSH in the blood every 15 min over 8 h following intraperitoneal AA administration (0.1 mg/kg or 5 mg/kg). AA, GA, and AA-GSH TKs followed linear kinetics: GA AUC/AA AUC = 0.11 and AA-GSH AUC/AA AUC = 0.011 at 5 mg/kg. Elimination half-life (T e1/2 ) values were 2.44 ± 0.70, 4.93 ± 2.37 and 3.47 ± 1.47 h for AA, GA and AA-GSH, respectively. GA TKs reached a plateau at 3-6 h, suggesting that metabolic saturation of AA and T e1/2 values of the analytes were prolonged with AA at 5 mg/kg. Our results demonstrate that oxidation of AA to GA overwhelmed the conjugation of AA with GSH. Our innovative MD-ID-LC-MS/MS method facilitates the simultaneous characterization of multiple TKs associated with toxicants and their active metabolites with excellent temporal resolution to capture metabolic saturation of AA to GA., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of CSF GABA has clinical utility in diagnosing inborn errors of GABA metabolism, specifically for deficiencies of GABA-transaminase and succinic semialdehyde dehydrogenase. Quantitation of CSF GABA is performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D 2 ) and injected directly onto the HPLC-ESI-MS/MS system. Quantitation of total GABA in CSF requires additional sample preparation in order to hydrolyze all the conjugated GABA in the sample to free GABA. Complete hydrolysis is performed incubating sample at >100 °C in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90% acetonitrile/0.1% formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 to 1000 nM and 0.63 to 80 μM for free and total GABA, respectively., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)