Novel methods for the rapid and selective analysis of biological samples using hyphenated ion mobility-mass spectrometry with ambient ionization

The increased use of mass spectrometry in the clinical setting has led to a demand for high sample throughput. Developments such as ultra high performance liquid chromatography and the ambient ionization techniques enable high sample throughput by reducing chromatographic run times or by removing the requirement for sample preparation and fractionation prior to analysis. This thesis assesses the reproducibility and robustness of these high throughput techniques for the analysis of clinical and pharmaceutical samples by ion mobility-mass spectrometry. The rapid quantitative analysis of the urinary biomarkers of chronic obstructive pulmonary disease, desmosine and isodesmosine has been performed by ultra high performance liquid chromatography combined with ion mobility-mass spectrometry. The determination of health status based on the free unbound fraction rather than the total bound and unbound desmosine and isodesmosine, significantly reduces the time taken in sample preparation. The potential for direct analysis of the urinary metabolites from undeveloped TLC plates using a solvent extraction surface sample probe is demonstrated. The use of a solvent gradient for the extraction separates urinary metabolites from salts and other matrix components and allows fractionation of the sample as a result of differential retention on the undeveloped RP-TLC plate. This separation, combined with ion mobility-mass spectrometry provides a rapid ambient ionization method for urinary profiling. The combination of a thermal desorption probe with extractive electrospray ionization has been applied to the direct detection of a known genotoxic impurity from a surrogate active pharmaceutical ingredient. The volatility of the impurity compared to the matrix, allowed selective thermal desorption of the analyte, which was ionized by extractive electrospray and detected by mass spectrometry. The use of a rapid on-probe derivatisation reaction, combined with thermal desorption is demonstrated for the direct determination of urinary creatinine. The aqueous acylation of creatinine significantly increases the volatility of the analyte enabling separation from the urine matrix and analysis by thermal desorption extractive electrospray combined with ion mobility-mass spectrometry

Direct determination of urinary creatinine by reactive-thermal desorption-extractive electrospray-ion mobility-tandem mass spectrometry.

A direct, ambient ionization method has been developed for the determination of creatinine in urine that combines derivatization and thermal desorption with extractive electrospray ionization and ion mobility-mass spectrometry. The volatility of creatinine was enhanced by a rapid on-probe aqueous acylation reaction, using a custom-made thermal desorption probe, allowing thermal desorption and ionization of the monoacylated derivative. The monoacyl creatinine [M + H] ion (m/z 156) was subjected to mass-to-charge selection and collision induced dissociation to remove the acyl group, generating the protonated creatinine [M + H] product ion at m/z 114 before an ion mobility separation was applied to reduce chemical noise. Stable isotope dilution using creatinine-d as internal standard was used for quantitative measurements. The direct on-probe derivatization allows high sample throughput with a typical cycle time of 1 min per sample. The method shows good linearity (R = 0.986) and repeatability (%RSD 8-10%) in the range of 0.25-2.0 mg/mL. The creatinine concentrations in diluted urine samples from a healthy individual were determined to contain a mean concentration of 1.44 mg/mL creatinine with a precision (%RSD) of 9.9%. The reactive ambient ionization approach demonstrated here has potential for the determination of involatile analytes in urine and other biofluids. © 2013 American Chemical Society

Determination of free desmosine and isodesmosine as urinary biomarkers of lung disorder by ultra performance liquid chromatography-ion mobility-mass spectrometry

The elastin degradation products, desmosine (DES) and isodesmosine (IDES) are highly stable, cross-linking amino-acids that are unique to mature elastin. The excretion of DES/IDES in urine, in the free form and with associated peptide fragments, provides an indicator of lung damage in chronic obstructive pulmonary disease (COPD). A quantitative ion mobility-mass spectrometry (IM-MS) method has been developed for the analysis of free DES/IDES in urine with deuterated IDES as an internal standard. Resolution of DES/IDES isomers was achieved in less than five minutes using ultra performance liquid chromatography (UPLC) combined with ion pairing. The optimized UPLC-IM-MS method provided a linear dynamic range of 10-300 ng/mL and a limit of quantitation of 0.028 ng/mL for IDES and 0.03 ng/mL for DES (0.55 ng and 0.61 ng on column respectively). The method reproducibility (%RSD) was < 4% for DES and IDES. The UPLC-IM-MS method was applied to the analysis of urine samples obtained from healthy volunteers and COPD patients. The DES/IDES concentrations in healthy and COPD urine showed an increase in DES (79%) and IDES (74%) in the COPD samples, relative to healthy controls. The incorporation of an IM separation prior to m/z measurement by MS was shown to reduce non-target ion responses from the bio-fluid matrix

Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography-ion mobility-mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time

Oral abstracts 1: SpondyloarthropathiesO1. Detecting axial spondyloarthritis amongst primary care back pain referrals

Background: Inflammatory back pain (IBP) is an early feature of ankylosing spondylitis (AS) and its detection offers the prospect of early diagnosis of AS. However, since back pain is very common but only a very small minority of back pain sufferers have ASpA or AS, screening of back pain sufferers for AS is problematic. In early disease radiographs are often normal so that fulfilment of diagnostic criteria for AS is impossible though a diagnosis of axial SpA can be made if MRI evidence of sacroiliitis is present. This pilot study was designed to indicate whether a cost-effective pick up rate for ASpA/early AS could be achieved by identifying adults with IBP stratified on the basis of age. Methods: Patients aged between 18 and 45 years who were referred to a hospital physiotherapy service with back pain of more than 3 months duration were assessed for IBP. All were asked to complete a questionnaire based on the Berlin IBP criteria. Those who fulfilled IBP criteria were also asked to complete a second short questionnaire enquiring about SpA comorbidities, to have a blood test for HLA-B27 and CRP level and to undergo an MRI scan of the sacroiliac joints. This was a limited scan, using STIR, diffusion-weighted, T1 and T2 sequences of the sacroiliac joints to minimize time in the scanner and cost. The study was funded by a research grant from Abbott Laboratories Ltd. Results: 50 sequential patients agreed to participate in the study and completed the IBP questionnaire. Of these 27 (54%) fulfilled criteria for IBP. Of these, 2 patients reported a history of an SpA comorbidity - 1 psoriasis; 1 ulcerative colitis - and 3 reported a family history of an SpA comorbidity - 2 psoriasis; 1 Crohn's disease. 4 were HLA-B27 positive, though results were not available for 7. Two patients had marginally raised CRP levels (6, 10 -NR ≤ 5). 19 agreed to undergo MRI scanning of the sacroiliac joints and lumbar spine; 4 scans were abnormal, showing evidence of bilateral sacroiliitis on STIR sequences. In all cases the changes met ASAS criteria but were limited. Of these 4 patients 3 were HLA-B27 positive but none gave a personal or family history of an SpA-associated comorbidity and all had normal CRP levels. Conclusions: This was a pilot study yielding only limited conclusions. However, it is clear that: Screening of patients referred for physiotherapy for IBP is straightforward, inexpensive and quick. It appears that IBP is more prevalent in young adults than overall population data suggest so that targeting this population may be efficient. IBP questionnaires could be administered routinely during a physiotherapy assessment. HLA-B27 testing in this group of patients with IBP is a suitable screening tool. The sacroiliac joint changes identified were mild and their prognostic significance is not yet clear so that the value of early screening needs further evaluation. Disclosure statement: C.H. received research funding for this study from Abbott. A.K. received research funding for this study, and speaker and consultancy fees, from Abbott. All other authors have declared no conflicts of interes

Efficacy and safety of baricitinib or ravulizumab in adult patients with severe COVID-19 (TACTIC-R): a randomised, parallel-arm, open-label, phase 4 trial

Background From early in the COVID-19 pandemic, evidence suggested a role for cytokine dysregulation and complement activation in severe disease. In the TACTIC-R trial, we evaluated the efficacy and safety of baricitinib, an inhibitor of Janus kinase 1 (JAK1) and JAK2, and ravulizumab, a monoclonal inhibitor of complement C5 activation, as an adjunct to standard of care for the treatment of adult patients hospitalised with COVID-19. Methods TACTIC-R was a phase 4, randomised, parallel-arm, open-label platform trial that was undertaken in the UK with urgent public health designation to assess the potential of repurposing immunosuppressants for the treatment of severe COVID-19, stratified by a risk score. Adult participants (aged ≥18 years) were enrolled from 22 hospitals across the UK. Patients with a risk score indicating a 40% risk of admission to an intensive care unit or death were randomly assigned 1:1:1 to standard of care alone, standard of care with baricitinib, or standard of care with ravulizumab. The composite primary outcome was the time from randomisation to incidence (up to and including day 14) of the first event of death, invasive mechanical ventilation, extracorporeal membrane oxygenation, cardiovascular organ support, or renal failure. The primary interim analysis was triggered when 125 patient datasets were available up to day 14 in each study group and we included in the analysis all participants who were randomly assigned. The trial was registered on ClinicalTrials.gov (NCT04390464). Findings Between May 8, 2020, and May 7, 2021, 417 participants were recruited and randomly assigned to standard of care alone (145 patients), baricitinib (137 patients), or ravulizumab (135 patients). Only 54 (39%) of 137 patients in the baricitinib group received the maximum 14-day course, whereas 132 (98%) of 135 patients in the ravulizumab group received the intended dose. The trial was stopped after the primary interim analysis on grounds of futility. The estimated hazard ratio (HR) for reaching the composite primary endpoint was 1·11 (95% CI 0·62–1·99) for patients on baricitinib compared with standard of care alone, and 1·53 (0·88–2·67) for ravulizumab compared with standard of care alone. 45 serious adverse events (21 deaths) were reported in the standard-of-care group, 57 (24 deaths) in the baricitinib group, and 60 (18 deaths) in the ravulizumab group. Interpretation Neither baricitinib nor ravulizumab, as administered in this study, was effective in reducing disease severity in patients selected for severe COVID-19. Safety was similar between treatments and standard of care. The short period of dosing with baricitinib might explain the discrepancy between our findings and those of other trials. The therapeutic potential of targeting complement C5 activation product C5a, rather than the cleavage of C5, warrants further evaluation

Direct Detection of a Sulfonate Ester Genotoxic Impurity by Atmospheric-Pressure Thermal Desorption–Extractive Electrospray–Mass Spectrometry

This article is closed access.A direct, ambient ionization method has been developed using atmospheric pressure thermal desorption−extractive electrospray−mass spectrometry (AP/TD-EESI-MS) for the detection of the genotoxic impurity (GTI) methyl p-toluenesulfonate (MTS) in a surrogate pharmaceutical matrix. A custom-made thermal desorption probe was used to the desorb and vaporize MTS from the solid state, by rapid heating to 200 °C then cooling to ambient temperature, with a cycle time of 6 min. The detection of MTS using EESI with a sodium acetate doped solvent to generate the [MTS+Na]+ adduct ion provided a significant sensitivity enhancement relative to the [M+H]+ ion generated using a 0.1% formic acid solvent modifier. The MTS detection limit is over an order of magnitude below the longterm daily threshold of toxicological concern (TTC) of 1.5 μg/g and the potential for quantitative analysis has been determined using starch as a surrogate active pharmaceutical ingredient (API)

Direct extraction of urinary analytes from undeveloped reversed-phase thin layer chromatography plates using a solvent gradient combined with on-line electrospray ionisation ion mobility-mass spectrometry

The direct extraction of urinary analytes deposited on reversed-phase thin-layer chromatography (RP-TLC) plates is demonstrated using a solvent gradient extraction procedure without prior chromatographic development. The surface sample probe TLC-MS interface used for the gradient extraction is compared to direct loop injection into the electrospray ion source for biofluid profiling. The gradient elution is shown to enhance ion intensities, as urinary salts are eluted in aqueous formic acid in the early part of the gradient reducing ion suppression. The retention of urinary components on the C18 RP-TLC plate was confirmed by monitoring analyte responses with, and without, an aqueous wash phase prior to the solvent gradient extraction. The use of gradient elution allows fractionation of the complex biological matrix as a result of differential retention of urine components on the undeveloped RP-TLC plate. The direct gradient analysis of TLC plates has also been combined with ion mobility-mass spectrometry to further resolve the complex urinary profile and identify co-eluting compounds

Direct detection of a sulfonate ester genotoxic impurity by atmospheric-pressure thermal desorption−extractive electrospray−mass spectrometry

A direct, ambient ionization method has been developed using atmospheric pressure thermal desorption−extractive electrospray−mass spectrometry (AP/TD-EESI-MS) for the detection of the genotoxic impurity (GTI) methyl p-toluenesulfonate (MTS) in a surrogate pharmaceutical matrix. A custom-made thermal desorption probe was used to the desorb and vaporize MTS from the solid state, by rapid heating to 200 °C then cooling to ambient temperature, with a cycle time of 6 min. The detection of MTS using EESI with a sodium acetate doped solvent to generate the [MTS+Na]+ adduct ion provided a significant sensitivity enhancement relative to the [M+H]+ ion generated using a 0.1% formic acid solvent modifier. The MTS detection limit is over an order of magnitude below the longterm daily threshold of toxicological concern (TTC) of 1.5 μg/g and the potential for quantitative analysis has been determined using starch as a surrogate active pharmaceutical ingredient (API)

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection