Corrigendum to "Development and validation of a multiplexed LC-MS/MS ketone body assay for clinical diagnostics" [J. Mass Spectrom. Adv. Clin. Lab 31 (2024) 49-58].

[This corrects the article DOI: 10.1016/j.jmsacl.2024.01.004.].

Development and validation of a multiplexed LC-MS/MS ketone body assay for clinical diagnostics.

Objectives: Ketone bodies (KBs) serve as important energy sources that spare glucose, providing the primary energy for cardiac muscle, skeletal muscle during aerobic exercise, and the brain during periods of catabolism. The levels and relationships between the KBs are critical indicators of metabolic health and disease. However, challenges in separating isomeric KBs and concerns about sample stability have previously limited their clinical measurement. Methods: A novel 6.5-minute liquid chromatography-mass spectrometry-based assay was developed, enabling the precise measurement of alpha-, beta- and gamma-hydroxybutyrate, beta-hydroxyisobutyrate, and acetoacetate. This method was fully validated for human serum and plasma samples by investigating extraction efficiency, matrix effects, accuracy, recovery, intra- and inter-precision, linearity, lower limit of quantitation (LLOQ), carryover, specificity, stability, and more. From 107 normal samples, reference ranges were established for all analytes and the beta-hydroxybutyrate/acetoacetate ratio. Results: All five analytes were adequately separated chromatographically. An extraction efficiency between 80 and 120 % was observed for all KBs. Accuracy was evaluated through spike and recovery using 10 random patient samples, with an average recovery of 85-115 % for all KBs and a coefficient of variation of ≤ 3 %. Coefficients of variation for intra- and inter-day imprecision were < 5 %, and the total imprecision was < 10 %. No significant interferences were observed. Specimens remained stable for up to 6 h on ice or 2 h at room temperature. Conclusions: The developed method is highly sensitive and robust. It has been validated for use with human serum and plasma, overcoming stability concerns and providing a reliable and efficient quantitative estimation of ketone bodies.

Characterization of Bile Acid Isomers and the Implementation of High-Resolution Demultiplexing with Ion Mobility-Mass Spectrometry.

Bile acids (BAs) are a complex suite of clinically relevant metabolites that include many isomers. Liquid chromatography coupled to mass spectrometry (LC-MS) is an increasingly popular technique due to its high specificity and sensitivity; nonetheless, acquisition times are generally 10-20 min, and isomers are not always resolved. In this study, the application of ion mobility (IM) spectrometry coupled to MS was investigated to separate, characterize, and measure BAs. A subset of 16 BAs was studied, including three groups of isomers belonging to unconjugated, glycine-conjugated, and taurine-conjugated BA classes. A variety of strategies were explored to increase BA isomer separation such as changing the drift gas, measuring different ionic species (i.e., multimers and cationized species), and enhancing the instrumental resolving power. In general, Ar, N2, and CO2 provided the best peak shape, resolving power (Rp), and separation, especially CO2; He and SF6 were less preferable. Furthermore, measuring dimers versus monomers improved isomer separation due to enhanced gas-phase structural differences. A variety of cation adducts other than sodium were characterized. Mobility arrival times and isomer separation were affected by the choice of adduct, which was shown to be used to target certain BAs. Finally, a novel workflow that involves high-resolution demultiplexing in combination with dipivaloylmethane ion-neutral clusters was implemented to improve Rp dramatically. A maximum Rp increase was observed with lower IM field strengths to obtain longer drift times, increasing Rp from 52 to 187. A combination of these separation enhancement strategies demonstrates great potential for rapid BA analysis.

A rapid and robust method for amino acid quantification using a simple N-hydroxysuccinimide ester derivatization and liquid chromatography-ion mobility-mass spectrometry.

The vast majority of mass spectrometry (MS)-based metabolomics studies employ reversed-phase liquid chromatography (RPLC) to separate analytes prior to MS detection. Highly polar metabolites, such as amino acids (AAs), are poorly retained by RPLC, making quantitation of these key species challenging across the broad concentration ranges typically observed in biological specimens, such as cell extracts. To improve the detection and quantitation of AAs in microglial cell extracts, the implementation of a 4-dimethylaminobenzoylamido acetic acid N-hydroxysuccinimide ester (DBAA-NHS) derivatization agent was explored for its ability to improve both analyte retention and detection limits in RPLC-MS. In addition to the introduction of the DBAA-NHS labeling reagent, a uniformly (U) 13C-labeled yeast extract was also introduced during the sample preparation workflow as an internal standard (IS) to eliminate artifacts and to enable targeted quantitation of AAs, as well as untargeted amine submetabolome profiling. To improve method sensitivity and selectivity, multiplexed drift-tube ion mobility (IM) was integrated into the LC-MS workflow, facilitating the separation of isomeric metabolites, and improving the structural identification of unknown metabolites. Implementation of the U-13C-labeled yeast extract during the multiplexed LC-IM-MS analysis enabled the quantitation of 19 of the 20 common AAs, supporting a linear dynamic range spanning up to three orders of magnitude in concentration for microglial cell extracts, in addition to reducing the required cell count for reliable quantitation from 10 to 5 million cells per sample.

Separation of Structurally Similar Anabolic Steroids as Cation Adducts in FAIMS-MS.

Novel synthetic anabolic androgenic steroids have been developed not only to dodge current antidoping tests at the professional sports level, but also for consumption by noncompetitive bodybuilders. These novel anabolic steroids are commonly referred to as "designer steroids" and pose a significant risk to users because of the lack of testing for toxicity and safety in animals or humans. Manufacturers of designer steroids dodge regulation by distributing them as nutritional or dietary supplements. Improving the throughput and accuracy of screening tests would help regulators to stay on top of illicit anabolic steroids. High-field asymmetric-waveform ion mobility spectrometry (FAIMS) utilizes an alternating asymmetric electric field to separate ions by their different mobilities at high- and low-fields as they travel through the separation space. When coupled to mass spectrometry (MS), FAIMS enhances the separation of analytes from other interfering compounds with little to no increase in analysis time. Here we investigate the effects of adding various cation species to sample solutions for the separation of structurally similar or isomeric anabolic androgenic steroids. FAIMS-MS spectra for these cation-modified samples show an increased number of compensation field (CF) peaks, some of which are confirmed to be unique for one steroid isomer over another. The CF peaks observed upon addition of cation species correspond to both monomer steroid-cation adduct ions and larger multimer ion complexes. Notably, the number of CF peaks and their CF shifts do not appear to have a straightforward relationship with cation size or electronegativity. Future directions aim at investigating the structures for these analyte-cation adduct ions for building a predictive model for their FAIMS separations.

Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/Drift Tube Ion Mobility-Mass Spectrometry.

Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D (25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD), only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal m/z. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a 2 min LC/IM-MS method. 25-Hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.

Effects of Solvent Vapor Modifiers for the Separation of Opioid Isomers in Micromachined FAIMS-MS.

Opioid addiction is an escalating problem that is compounded by the introduction of synthetic opiate analogues such as fentanyl. Screening methods for these compound classes are challenged by the availability of synthetically manufactured analogues, including isomers of existing substances. High-field asymmetric-waveform ion mobility spectrometry (FAIMS) utilizes an alternating asymmetric electric field to separate ions by their different mobilities at high and low fields as they travel through the separation space. When coupled to mass spectrometry (MS), FAIMS enhances the separation of analytes from other interfering compounds with little to no increase in analysis time. Addition of solvent vapor into the FAIMS carrier gas has been demonstrated to enable and improve the separation of isomers. Here we investigate the effects of several solvents for the separation of four opioids. FAIMS-MS spectra with added solvent vapors show dramatic compensation field (CF) shifts for opioid [M+H]+ ions when compared to spectra acquired using dry nitrogen. Addition of vapor from aprotic solvents, such as acetonitrile and acetone, produces significantly improved resolution between the tested opioids, with baseline resolution achieved between certain opioid isomers. For protic solvents, notable CF shift differences were observed in FAIMS separations between addition of water vapor and vapors from small alcohols. Graphical Abstract.

Measuring the Integrity of Gas-Phase Conformers of Sodiated 25-Hydroxyvitamin D3 by Drift Tube, Traveling Wave, Trapped, and High-Field Asymmetric Ion Mobility.

Quantitation of the serum concentration of 25-hydroxyvitamin D is a high-demand assay that suffers from long chromatography time to separate 25-hydroxyvitamin D from its inactive epimer; however, ion mobility spectrometry can distinguish the epimer pair in under 30 ms due to the presence of a unique extended or "open" gas-phase sodiated conformer, not shared with the epimer, reducing the need for chromatographic separation. Five ion mobility mass spectrometers utilizing commercially available IMS technologies, including drift tube, traveling wave, trapped, and high-field asymmetric ion mobility spectrometry, are evaluated for their ability to resolve the unique open conformer. Additionally, settings for each instrument are evaluated to understand their influence on ion heating, which can drive the open conformer into a compact or "closed" conformer shared with the epimer. The four low-field instruments successfully resolved the open conformer from the closed conformer at baseline or near-baseline resolution at typical operating parameters. High-field asymmetric ion mobility was unable to resolve a unique peak but detected two peaks for the epimer, in contrast to the low-field methods that detected one conformer. This study seeks to expand the instrument space by highlighting the potential of each platform for the separation of 25-hydroxyvitamin D epimers.

Cation-Dependent Conformations in 25-Hydroxyvitamin D3-Cation Adducts Measured by Ion Mobility-Mass Spectrometry and Theoretical Modeling.

Ion mobility-mass spectrometry is a useful tool in separation of biological isomers, including clinically relevant analytes such as 25-hydroxyvitamin D3 (25OHD3) and its epimer, 3-epi-25-hydroxyvitamin D3 (epi25OHD3). Previous research indicates that these epimers adopt different gas-phase sodiated monomer structures, either the "open" or "closed" conformer, which allow 25OHD3 to be readily resolved in mixtures. In the current work, alternative metal cation adducts are investigated for their relative effects on the ratio of "open" and "closed conformers. Alkali and alkaline earth metal adducts caused changes in the 25OHD3 conformer ratio, where the proportion of the "open" conformer generally increases with the size of the metal cation in a given group. As such, the ratio of the "open" conformer, which is unique to 25OHD3 and absent for its epimer, can be increased from approximately 1:1 for the sodiated monomer to greater than 8:1 for the barium adduct. Molecular modeling and energy calculations agree with the experimental results, indicating that the Gibbs free energy of conversion from the "closed" to the "open" conformation decreased with increasing cation size, correlating with the variation in ratio between the conformers. This work demonstrates the effect of cation adducts on gas-phase conformations of small, flexible molecules and offers an additional strategy for resolution of clinically relevant epimers.

Portable FAIMS: Applications and Future Perspectives.

Miniaturized mass spectrometry (MMS) is optimal for a wide variety of applications that benefit from field-portable instrumentation. Like MMS, field asymmetric ion mobility spectrometry (FAIMS) has proven capable of providing in situ analysis, allowing researchers to bring the lab to the sample. FAIMS compliments MMS very well, but has the added benefit of operating at atmospheric pressure, unlike MS. This distinct advantage makes FAIMS uniquely suited for portability. Since its inception, FAIMS has been envisioned as a field-portable device, as it affords less expense and greater simplicity than many similar methods. Ideally, these are simple, robust devices that may be operated by non-professional personnel, yet still provide adequate data when in the field. While reducing the size and complexity tends to bring with it a loss of performance and accuracy, this is made up for by the incredibly high throughput and overall convenience of the instrument. Moreover, the FAIMS device used in the field can be brought back to the lab, and coupled to a conventional mass spectrometer to provide any necessary method development and compound validation. This work discusses the various considerations, uses, and applications for portable FAIMS instrumentation, and how the future of each applicable field may benefit from the development and acceptance of such a device.

Ion Mobility in Clinical Analysis: Current Progress and Future Perspectives.

BACKGROUND: Ion mobility spectrometry (IMS) is a rapid separation tool that can be coupled with several sampling/ionization methods, other separation techniques (e.g., chromatography), and various detectors (e.g., mass spectrometry). This technique has become increasingly used in the last 2 decades for applications ranging from illicit drug and chemical warfare agent detection to structural characterization of biological macromolecules such as proteins. Because of its rapid speed of analysis, IMS has recently been investigated for its potential use in clinical laboratories. CONTENT: This review article first provides a brief introduction to ion mobility operating principles and instrumentation. Several current applications will then be detailed, including investigation of rapid ambient sampling from exhaled breath and other volatile compounds and mass spectrometric imaging for localization of target compounds. Additionally, current ion mobility research in relevant fields (i.e., metabolomics) will be discussed as it pertains to potential future application in clinical settings. SUMMARY: This review article provides the authors' perspective on the future of ion mobility implementation in the clinical setting, with a focus on ambient sampling methods that allow IMS to be used as a "bedside" standalone technique for rapid disease screening and methods for improving the analysis of complex biological samples such as blood plasma and urine.