Clinical Mass Spectrometry最新文献

Presented are the validation results of a second-generation assay for determining the relative abundances of two protein biomarkers found in maternal serum that predict an individual’s risk of spontaneous preterm birth. The sample preparation workflow is complex, consisting of immuno-depletion of high-abundance serum proteins, tryptic digestion of the immuno-depleted fraction to generate surrogate peptide analytes, and detection by tandem mass spectrometry. The method was determined to be robust on observation of the following characteristics: classifier peptide detection precision was excellent; results were accurate when compared to a reference method; results were linear over a clinically relevant range; the limits of quantitation encompassed the range of expected results; and the method demonstrated analytical specificity and resilience to differences in patient serum and common endogenous interferents.

Mass spectrometry provides considerable benefits over other analytical techniques due to advantages imparted by the use of isotopically labeled internal standards. In some analytical approaches, the internal standard provides reference points for quantitative data reduction. However, there is an important phenomenon that occurs in the development of an internal standard, namely the distribution of naturally occurring isotopes is altered by artificial labeling. As a result, the number of molecules measured in each isotopic state (e.g., +0, +1, +2) varies between the manufactured internal standard and the naturally occurring unmodified analyte. This can create inaccuracies that are a function of the abundance, type and location of the isotopic labeling when internal standards act as the calibration material. In this study, theoretical examples calculated with naturally occurring isotopic incorporation, manufactured isotopic enrichment, and experimental data from comparative analysis of naturally and artificially labeled peptides were explored to demonstrate the variation in measurement between analytes and their stable, isotopically labeled internal standards.

Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with high reproducibility (CV ⩽ 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. This SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure.

Polyunsaturated fatty acids (PUFAs), including essential omega-3 and omega-6 fatty acids, play important roles in diverse physiological and pathological processes. Diligent monitoring of PUFAs is recommended for individuals with increased risk of developing essential fatty acid deficiency (EFAD), including premature and very low birth weight infants, patients on prolonged parenteral nutrition, and those with dietary restrictions, for example due to inborn errors of metabolism. Here, we present a gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI‐MS) method for the quantitation of total levels of twenty-two fatty acids (C12‐C22) in serum/plasma, including omega-3 and omage-6 PUFAs. Hydrolysis was used to release esterified fatty acids, which were analyzed by GC-NCI-MS as pentafluorobenzyl esters in selected-ion monitoring (SIM) mode. The calibration curves for all analytes had consistent slopes with R² of ⩾0.990. Intra- and inter-assay precision CVs were ⩽9.0% and ⩽13.2%, respectively. Samples were found to be stable for 24 h at room temperature, at least 7 days at 4 °C, at least 75 days at −20 °C, and for three freeze/thaw cycles. No matrix effects or interferences were observed. This method offers improvements over published studies including smaller sample volume, inclusion of additional internal standards, analysis in a single injection, and use of methane reagent gas. This method could be used in a clinical laboratory setting for the diagnosis of EFAD, evaluation of nutritional status, and diet monitoring.

More than seventy-five isotypes of α-1-antitrypsin (AAT) have been described. To assess risks associated with AAT deficiency, isotype identification is necessary. Isoelectric focusing (IEF) is traditionally used for isotype differentiation, however, IEF has limited scope since it is a manual procedure that is not suitable for automation, and antitrypsin variants must differ in net charge in order to be resolved. In comparison, mass spectrometric assays are easily automated and offer a more complete solution for characterization of proteins. To capitalize on these advantages, we have developed a qualitative top-down liquid chromatography–mass spectrometry (LC–MS) method for selective phenotyping of AAT. This technique requires no sample pretreatment, and has the potential for use in routine clinical diagnostics. We have validated our LC–MS results against both DNA sequencing and IEF. Thus far, this method has identified the AAT variants P_Lowell, S and Z, as well as unique fragments shared by different M alleles. Its high selectivity is indirectly illustrated by the detection of a variant carrying the amino acid substitution p.Ala308Ser, which cannot be visualized by IEF.

Drug monitoring of biofluids is often time consuming and prohibitively expensive. Analysis of dried blood spots offers advantages, such as reduced sample volume, but depends on extensive sample preparation and the presence of a trained lab technician. Paper spray mass spectrometry allows rapid analysis of small molecules from blood spots with minimal sample preparation, however, plasma is often the preferred matrix for bioanalysis. Plasma spots can be analyzed by paper spray MS, but a centrifugation step to isolate the plasma is required. We demonstrate here the development of a paper spray cartridge containing a plasma fractionation membrane to perform automatic on-cartridge plasma fractionation from whole blood samples. Three commercially available blood fractionation membranes were evaluated based on: 1) accuracy of drug concentration determination in plasma, and 2) extent of cell lysis and/or penetration. The accuracy of drug concentration determination was quantitatively determined using high performance liquid chromatography–mass spectrometry (HPLC–MS). While the fractionation membranes were capable of yielding plasma samples with low levels of cell lysis, the membranes did exhibit drug binding to varying degrees, as indicated by a decrease in the drug concentration relative to plasma obtained by centrifugation. Using the membrane exhibiting the lowest binding, we developed a composite paper spray cartridge incorporating the selected fractionation membrane. Quantitative analysis of the plasma samples by paper spray MS yielded results similar to those found with HPLC–MS, but without the need for offline extraction or chromatography.

Instrument calibration, required for any accurate quantitative calculation, is a trivial process when performed correctly, but is also full of easily overlooked stumbling blocks. To minimize the risk of error associated with improper calibrations, national and international guidance dictates a minimum number of calibrators and the threshold at which a measurement becomes an outlier. Evidence from industry practice, which conflicts with regulatory guidance, suggests that most groups are focused on remapping their detector with each run. We present a post facto explanation for the calibrator minimum and provide recommendations for curve building, which include improved outlier detection for high-volume mass spectrometry laboratories.

Introduction

Burn injury inevitably leads to changes in the endogenous production of cytokines, as well as adrenal and gonadal steroids. Previous studies have reported gender-related differences in outcome following burn injury, which suggests that gonadal steroids may play a role. The aim of this study was to assess alterations in concentration of endogenous steroids in patients with burn injury.

Methods

For this single-center, prospective descriptive study, high-sensitivity liquid chromatography tandem mass spectrometry (LC-MS/MS)-based steroid quantification was used to determine longitudinal profiles of the concentrations of endogenous steroids in plasma from sixteen adult male patients with burn injury (14.5–72% of total body surface area). Steroids were extracted from plasma samples and analyzed using multiple reaction monitoring acquisition, with electrospray ionization on a triple quadruple mass spectrometer. Total protein concentration was measured in the samples using spectrophotometry.

Results

Steroid and total protein concentration distributions were compared to reference intervals characteristic of healthy adult men. Concentrations of the following steroids in plasma of burn injured patients were found to correlate positively to the area of the burn injury: cortisol (r = 0.84), corticosterone (r = 0.73), 11-deoxycortisol (r = 0.72), androstenedione (r = 0.72), 17OH-progesterone (r = 0.68), 17OH-pregnenolone (r = 0.64) and pregnenolone (r = 0.77). Concentrations of testosterone decreased during the acute phase and were up to ten-times lower than reference values for healthy adult men, while concentrations of estrone were elevated. By day 21 after injury, testosterone concentrations were increased in younger, but not older, patients. The highest concentrations of estrone were observed on day 3 after the injury and then declined by day 21 to concentrations comparable to those observed on the day of the injury.

Conclusion

Burn injury alters endogenous steroid biosynthesis, with decreased testosterone concentrations and elevated estrone concentrations, during the first 21 days after the injury. Concentrations of glucocorticoids, progestagens and androgen precursors correlated positively with the area of burn injury. The finding of increased estrone following burn injury needs to be confirmed in a larger hypothesis-driven study.

The simple and rapid detection and identification of designer drugs is of substantial importance to forensic scientists and law enforcement. Although synthetic cathinones, cannabinoids, and other common novel psychoactive substances (NPS) are produced for purposes that do not include human consumption, they are regularly abused. The analysis of these compounds is often achieved using mass spectrometry, but can be complicated by the lack of spectral libraries and the scarcity of simple and reliable sample introduction techniques. PaperSpray® ionization is a new, automated technique for rapid analysis of samples, without chromatography or prior purification. Matrix, such as powder or plant material, is dissolved or extracted with common solvents and deposited directly on disposable PaperSpray® cartridges for high-throughput, automated analysis. The combination of PaperSpray® sample introduction and High Resolution Accurate Mass Spectrometry (HRAM) provides a powerful and simple tool for identification of new substances, without requiring reference standards.

Background

Cystatin C (CysC), a marker for chronic kidney disease, exists as three sequence proteoforms, in addition to the wild-type sequence: one contains hydroxyproline at position 3 (3Pro-OH), the two others have truncated sequences (des-S and des-SSP). Here, we examine correlations between each of these CysC proteoforms and estimated glomerular filtration rate (eGFR), a diagnostic criterion for chronic kidney disease (CKD).

Methods

CysC proteoform concentrations were determined from the plasma of 297 diabetes patients at a baseline time point and nine-months later, using a mass spectrometric immunoassay, and were correlated with eGFR calculations.

Results

In all samples, 3Pro-OH was the most abundant CysC proteoform, followed by the wild-type proteoform. Least abundant were the truncated CysC proteoforms, des-S and des-SSP, although they demonstrated stronger negative correlation with eGFR than the 3Pro-OH and wild-type proteoforms. The des-SSP truncated proteoform exhibited negative predictive value for eGFR.

Conclusions

The truncated CysC proteoforms show potential for clinical and prognostic utility in CKD staging. This could be useful in populations where current methods do not provide satisfactory solutions.