Journal of Mass Spectrometry and Advances in the Clinical Lab最新文献

Background

Malaria is a parasitic disease that affects many of the poorest economies, resulting in approximately 241 million clinical episodes and 627,000 deaths annually. Piperaquine, when administered with dihydroartemisinin, is an effective drug against the disease. Drug concentration measurements taken on day 7 after treatment initiation have been shown to be a good predictor of therapeutic success with piperaquine. A simple capillary blood collection technique, where blood is dried onto filter paper, is especially suitable for drug studies in remote areas or resource-limited settings or when taking samples from children, toddlers, and infants.

Methods

Three 3.2 mm discs were punched out from a dried blood spot (DBS) and then extracted in a 96-well plate using solid phase extraction on a fully automated liquid handling system. The analysis was performed using LC-MS/MS with a calibration range of 3 – 1000 ng/mL.

Results

The recovery rate was approximately 54–72 %, and the relative standard deviation was below 9 % for low, middle and high quality control levels. The LC-MS/MS quantification limit of 3 ng/mL is sensitive enough to detect piperaquine for up to 4–8 weeks after drug administration, which is crucial when evaluating recrudescence and drug resistance development. While different hematocrit levels can affect DBS drug measurements, the effect was minimal for piperaquine.

Conclusion

A sensitive LC-MS/MS method, in combination with fully automated extraction in a 96-well plate format, was developed and validated for the quantification of piperaquine in DBS. The assay was implemented in a bioanalytical laboratory for processing large-scale clinical trial samples.

Introduction

The accurate quantification of amyloid beta (Aβ) peptides in cerebrospinal fluid (CSF) is crucial for Alzheimer's disease (AD) research, particularly in terms of preclinical and biomarker studies. Traditional methods, such as the enzyme-linked immunosorbent assay (ELISA), have limitations. These include high costs, labor intensity, lengthy processes, and the possibility of cross-reactivity.

Objectives

The primary objectives of this research were twofold: to comprehensively characterize Aβ peptides and to develop a reliable and accurate method for the simultaneous quantification of Aβ 1–40 and Aβ 1–42 peptides in surrogate CSF that is traceable to the International System of Units (SI).

Methods

We developed a novel method that combined solid phase extraction (SPE) with isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC/MSMS). SPE was employed to efficiently eliminate matrix interferences, while [15N] Aβ1-40 and [15N] Aβ1-42 served as internal standards to improve accuracy. In addition, we introduced Peptide Impurity Corrected Amino Acid Analysis (PICAA) to ensure traceability to the SI and reliable quantification of Aβ peptides.

Results

The developed platform demonstrated a linear calibration range of 300–20000 pg/ml for both Aβ1-42 and Aβ1-40 peptides, accompanied by strong correlation coefficients greater than 0.995. Quality Control (QC) samples demonstrated an accuracy of at least 90.0 %.

Conclusion

The enhanced specificity and flexibility of the developed platform potentially have implications for Alzheimer's disease diagnosis and future investigations of novel Aβ peptide biomarkers.

Therapeutic drug monitoring (TDM) is a critical clinical tool used to optimize the safety and effectiveness of drugs by measuring their concentration in biological fluids. These fluids are primarily plasma or blood. TDM, together with real-time dosage adjustment, contributes highly to the successful management of glycopeptide antimicrobial therapies. Understanding pharmacokinetic/pharmacodynamic (PK/PD) properties is vital for optimizing antimicrobial therapies, as the efficacy of these therapies depends on both the exposure of the patient to the drug (PK) and pharmacodynamic (PD) parameters such as the in vitro estimated minimum drug concentration that inhibits bacterial growth (MIC). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is widely recognized as the gold standard for measuring small molecules, such as antibiotics. This review provides a comprehensive overview of LC-MS/MS methods available for TDM of glycopeptide antibiotics, including vancomycin, teicoplanin, dalbavancin, oritavancin, and telavancin.

Introduction

Human saliva contains a wealth of proteins that can be monitored for disease diagnosis and progression. Saliva, which is easy to collect, has been extensively studied for the diagnosis of numerous systemic and infectious diseases. However, the presence of amylase, the most abundant protein in saliva, can obscure the detection of low-abundance proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF MS), thus reducing its diagnostic utility.

Objectives

In this study, we used a device to deplete salivary amylase from water-gargle samples by affinity adsorption. Following depletion, saliva proteome profiling was performed using MALDI-ToF MS on gargle samples from individuals confirmed to have COVID-19 based on nasopharyngeal (NP) swab reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Results

The depletion of amylase led to increased signal intensities of various peaks and the detection of previously unobserved peaks in the MALDI-ToF MS spectra. The overall specificity and sensitivity after amylase depletion were 100% and 85.17%, respectively, for detecting COVID-19.

Conclusion

This simple, rapid, and inexpensive technique for depleting salivary amylase can reveal spectral diversity in saliva using MALDI-ToF MS, expose low-abundance proteins, and assist in establishing novel biomarkers for diseases.

Objective

To develop and validate an assay for the analysis of bedaquiline and its M2 metabolite in human breast milk.

Methods

The analytes were extracted using solid phase extraction following protein precipitation. Quantification was performed with liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was achieved using gradient chromatography on a Poroshell 120 SB-C18 analytical column at 40 °C, with a flow rate of 350 µL/minute and a total run time of eight minutes. An AB Sciex 3000 mass spectrometer with electrospray ionization in the positive mode was used for detection, employing multiple reaction monitoring scan mode. Bedaquiline-d6 and M2-d3-¹³C were used as internal standards.

Results

Calibrations curves for bedaquiline and M2 exhibited quadratic (weighted 1/x concentration) regressions over the respective concentration ranges of 0.0780 to 5.00 µg/mL and 0.0312 to 2.00 µg/mL. Inter- and intra-day validation accuracies ranged between 96.7 % and 103.5 % for bedaquiline, and 104.2 % to 106.5 % for M2, with a coefficient of variation below 9.2 % for both compounds.

Conclusion

The developed assay demonstrated selectivity and robustness, enabling differentiation between bedaquiline and M2 within the context of endogenous compounds from six separate lots of breast milk samples. Successful application was observed in the analysis of breast milk samples sourced from patients treated for multidrug-resistant tuberculosis within a clinical study setting.

Background

Measurement of trough levels for calcineurin inhibitors by venipuncture sampling is a mainstay of patient management in solid organ transplant recipients but challenging in pediatric patients. Volumetric Absorptive Microsampling (VAMS) is a patient-friendly, minimally invasive sampling technique to accurately collect blood. An assay for measurement of tacrolimus in blood using VAMS, coupled with parallel reaction monitoring (PRM) mass spectrometry, was validated in pediatric heart transplant patients.

Methods

Tacrolimus was measured by a newly developed high-resolution PRM assay and compared with low-resolution tandem mass spectrometry (MRM). Dried blood samples were collected from pediatric heart transplant patients (n = 35) using VAMS devices and a satisfaction survey was completed by patients/guardians. Tacrolimus concentrations were compared across whole liquid blood, dried blood spots, and capillary blood, and shipping stability determined.

Results

The PRM assay was linear over a range 1–50 ng/mL, similar to MRM but had greater specificity due to reduced background noise. No significant differences in tacrolimus concentrations were observed between VAMS and venous blood. Tacrolimus dried on VAM tips was stable for 14 days and concentrations were unaffected by postal shipping. The variability in two simultaneously collected at-home patient samples was minimal – average concentration difference was 0.12 ± 0.94 ng/mL (p = 0.6) between paired samples.

Conclusion

A high resolution PRM mass spectrometry assay was developed for home-based dried blood collections for therapeutic monitoring of tacrolimus. The advantage of PRM was enhanced specificity and the VAMS devices provided a simple and convenient approach to blood sampling at home in pediatric heart transplant patients.

Introduction

LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin digestion, which has primarily focused on advancements in research-based biomarker discovery applications that rely on protein/peptide identifications rather than clinical biomarker quantification. However, there is a need for simple, fast, and reproducibly efficient surrogate peptide recovery in clinical biomarker quantification.

Methods

Multiple methodologies were evaluated to enhance tryptic digestion for the analysis of thyroglobulin, a prototypical serum protein biomarker. The main criteria for assessment were the yield and speed of formation of surrogate peptides. Various factors such as different additives, types of trypsin, microwave- and pressure-assisted systems, and enzyme concentration were considered as key variables, in addition to digestion time.

Results

It was observed that digestion additives/denaturants had a significant impact on the speed and yield of digestion for each surrogate peptide. Increasing the concentration of trypsin alone was found to accelerate digestions appreciably for most surrogate peptides, without affecting the yield. However, the use of sequencing-grade trypsins and microwave/pressure-assisted systems did not offer significant advantages over the use of 'standard-grade' TPCK-treated trypsin in combination with a conventional incubator, once digestion time and additive had been optimized.

Conclusion

We have dispelled the notion that trypsin digestion is inherently slow and expensive for targeted quantification of serum proteins. Additionally, we have established a groundwork for experimentation that can pave the way for the creation of efficient trypsin digestion protocols, aiming to optimize yield, speed, and cost. It is our hope that these advancements will promote the wider incorporation of such assays in clinical laboratories.

Introduction

The VeriStrat® test (VS) is a blood-based assay that predicts a patient's response to therapy by analyzing eight features in a spectrum obtained from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of human serum and plasma. In a recent analysis of the INSIGHT clinical trial (NCT03289780), it was found that the VS labels, VS Good and VS Poor, can effectively predict the responsiveness of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitor (ICI) therapy. However, while VS measures the intensities of spectral features using MALDI-TOF analysis, the specific proteoforms underlying these features have not been comprehensively identified.

Objectives

The objective of this study was to identify the proteoforms that are measured by VS.

Methods

To resolve the features obtained from the low-resolution MALDI-TOF procedure used to acquire mass spectra for VS DeepMALDI® analysis of serum was employed. This technique allowed for the identification of finer peaks within these features. Additionally, a combination of reversed-phase fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then used to identify the proteoforms associated with these peaks.

Results

The analysis revealed that the primary constituents of the spectrum measured by VS are serum amyloid A1, serum amyloid A2, serum amyloid A4, C-reactive protein, and beta-2 microglobulin.

Conclusion

Proteoforms involved in host immunity were identified as significant components of these features. This newly acquired information improves our understanding of how VS can accurately predict patient response to therapy. It opens up additional studies that can expand our understanding even further.