Quantification and identification analysis of Ziziphus jujuba Mill. peel pigmentation at different developmental stages.

The fruit peel of a color mutant jujube cultivar, 'Sanbianhong' (SBF), was investigated using an ultra-high performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) at five ripening stages (S1, Young fruit stage; S2, swelling stage; S3, white-mature stage; S4, pre-mature stage and S5, mature stage). Lutein, ß-carotene, chlorophyll a, chlorophyll b, and 13 anthocyanins were identified. Chlorophyll a and cyanidin 3-O-galactoside were considered key color metabolites in S1 with the content of 1.083 mg/g of fresh weight (FW) and 4.585 mg/g of FW, respectively. Delphinidin (0.488 mg/g FW) and cyanidin (6.259 mg/g FW) were identified as the key pigments in S3. Delphinidin 3-O-glucoside (0.256 mg/g FW) was identified as the key anthocyanin in maturity S5. Herein, the identification and quantitation of pigment-related metabolites of SBF were studied for the first time, and the results provide a theoretical basis for understanding the pigment changes of jujube fruit during ripening.

Development of a high-throughput differential mobility separation-tandem mass spectrometry (DMS-MS/MS) method for clinical urine drug testing.

INTRODUCTION: Differential mobility separation (DMS) is an analytical technique used for rapid separation of ions and isomers based on gas phase mobility prior to entering a mass spectrometer for analysis. The entire DMS process is accomplished in fewer than 20 ms and can be used as a rapid alternative to chromatographic separation. OBJECTIVE: The primary objective was to evaluate the utility of DMS-tandem mass spectrometry (DMS-MS/MS) as a replacement for immunoassay-based clinical toxicology testing. METHODS: A sensitive DMS-MS/MS method was developed and validated for simultaneous identification of 33 drugs and metabolites in human urine samples. After DMS optimization, the method was validated and used to screen 56 clinical urine samples. These results were compared to results obtained by immunoassay. RESULTS: The DMS-MS/MS method achieved limits of detection ranging from 5 to 100 ng/mL. Moreover, the total analysis time was 2 min per sample. For the method performance evaluation, DMS-MS/MS results were compared with previously obtained urine toxicology immunoassay results. DMS-MS/MS showed higher sensitivity and identified 20% more drugs in urine, which were confirmed by LC-MS/MS. CONCLUSION: The DMS-MS/MS as applied in our lab demonstrated the capability for rapid drug screening and provided better analytical performance than immunoassay.

A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease.

Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used. Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma. Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays. Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean. Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

Rapid identification of plasmalogen molecular species using targeted multiplexed selected reaction monitoring mass spectrometry.

Plasmalogens (Pls) levels are reported to be altered in several neurological and metabolic diseases. Identification of sn-1 fatty alcohols and sn-2 fatty acids of different Pls species is necessary to determine the roles and mechanisms of action of Pls in different diseases. Previously, full-scan tandem mass spectrometry (MS/MS) was used for this purpose but is not effective for low-abundance Pls species. Recently, multiplexed selected reaction monitoring MS (SRM/MS) was found to be more selective and sensitive than conventional full-scan MS/MS for the identification of low-abundance compounds. In the present study, we developed a liquid chromatography (LC)-targeted multiplexed SRM/MS system for the identification and quantification of different Pls choline (Pls-PC) and Pls ethanolamine (Pls-PE) species. We determined five precursor-product ion transitions to identify sn-1 and sn-2 fragments of each Pls species. Consequently, sn-1 and sn-2 fatty acyl chains of 22 Pls-PC and 55 Pls-PE species were identified in mouse brain samples. Among them, some species had C20:0 and C20:1 fatty alcohols at the sn-1 position. For quantification of Pls species in mouse brain samples, a single SRM transition was employed. Thus, our results suggest that the LC-targeted multiplexed SRM/MS system is very sensitive for the identification and quantification of low-abundance lipids such as Pls, and is thus expected to make a significant contribution to basic and clinical research in this field in the future.

LC-MS lipidomics of renal biopsies for the diagnosis of Fabry disease.

INTRODUCTION: Lipidomics analysis or lipid profiling is a system-based analysis of all lipids in a sample to provide a comprehensive understanding of lipids within a biological system. In the last few years, lipidomics has made it possible to better understand the metabolic processes associated with several rare disorders and proved to be a powerful tool for their clinical investigation. Fabry disease is a rare X-linked lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A (α-GAL A). This deficiency results in the progressive accumulation of glycosphingolipids, mostly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), as well as galabiosylceramide (Ga2) and their isoforms/analogs in the vascular endothelium, nerves, cardiomyocytes, renal glomerular podocytes, and biological fluids. OBJECTIVES: The primary objective of this study was to evaluate lipidomic signatures in renal biopsies to help understand variations in Fabry disease markers that could be used in future diagnostic tests. METHODS: Lipidomic analysis was performed by ultra-high pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) on kidney biopsies that were left over after clinical pathology analysis to diagnose Fabry disease. RESULTS: We employed UHPLC-HRMS lipidomics analysis on the renal biopsy of a patient suspicious for Fabry disease. Our result confirmed α-GAL A enzyme activity declined in this patient since a Ga2-related lipid biomarker was substantially higher in the patient's renal tissue biopsy compared with two controls. This suggests this patient has a type of LSD that could be non-classical Fabry disease. CONCLUSION: This study shows that lipidomics analysis is a valuable tool for rare disorder diagnosis, which can be conducted on leftover tissue samples without disrupting normal patient care.

Poisoning by organophosphorus nerve agents and pesticides: An overview of the principle strategies and current progress of mass spectrometry-based procedures for verification.

Intoxication by organophosphorus (OP) poisons, like nerve agents and pesticides, is characterized by the life-threatening inhibition of acetylcholinesterase (AChE) caused by covalent reaction with the serine residue of the active site of the enzyme (phosphylation). Similar reactions occur with butyrylcholinesterase (BChE) and serum albumin present in blood as dissolved proteins. For forensic purposes, products (adducts) with the latter proteins are highly valuable long-lived biomarkers of exposure to OP agents that are accessible by diverse mass spectrometric procedures. In addition, the evidence of poison incorporation might also succeed by the detection of remaining traces of the agent itself, but more likely its hydrolysis and/or enzymatic degradation products. These relatively short-lived molecules are distributed in blood and tissue, and excreted via urine. This review presents the mass spectrometry-based methods targeting the different groups of biomarkers in biological samples, which are already internationally accepted by the Organisation for the Prohibition of Chemical Weapons (OPCW), introduces novel approaches in the field of biomedical verification, and outlines the strict quality criteria that must be fulfilled for unambiguous forensic analysis.

Comparison of four clinically validated testosterone LC-MS/MS assays: Harmonization is an attainable goal.

INTRODUCTION: Immunoassays and liquid chromatography-tandem mass spectrometry assays are commonly employed in clinical laboratories for measurement of total testosterone in serum. Results obtained from either of these methodologies compare poorly due to differences in calibration and/or inadvertent detection of interfering substances by the immunoassays. Standardization efforts are underway, but recent studies indicate that accuracy remains an issue. METHODS: This study compares the results from four independently developed and validated LC-MS/MS assays for total testosterone. The calibration for each assay was verified using National Institute of Standards and Technology Standard Reference Material 971. RESULTS: Initially, one of the four assays had a mean percent difference of +11.44%, compared to the All Method Mean, but following re-verification of all five non-zero calibrator concentrations with the NIST SRM 971, the mean percent difference decreased to -4.88%. Subsequently, the agreement between all four assays showed a mean bias of <5% across the range of all testosterone concentrations (0.13-38.10 nmol/L; 3.7-1098 ng/dL), including at low concentrations of <1 nmol/L (<29 ng/dL). CONCLUSIONS: Excellent agreement between four independently developed LC-MS/MS assays demonstrates that harmonization using standard reference material is attainable. However, as we found in this study, to ensure accurate calibration it is critical to validate the concentrations of new lots of calibrators.

Validation data for the quantification of the Annonaceous acetogenin annonacin in Rat brain by UPLC-MS/MS.

Annonaceous acetogenins (AAGs) are environmental neurotoxins from the fruit pulp of several Annonaceae species, whose consumption was linked to the occurrence of sporadic atypical Parkinsonism with dementia. The quantification of the prototypical AAG annonacin in Rat brain homogenates was performed by UPLC-MS/MS in selected reaction monitoring (SRM) mode, using a triple quadrupole mass analyzer. A natural analog of annonacin was used as an internal standard. Analyzed data set of the analytical validation of this method is presented, including stability of the samples, extraction recovery and matrix effect, supporting the results described in the article "Quantification of the environmental neurotoxin annonacin in Rat brain by UPLC-MS/MS" N. Bonneau, I. Schmitz-Afonso, D. Touboul, A. Brunelle, P. Champy (2016) [1].

Discordance between protein and transcript levels detected by selected reaction monitoring.

Expression levels between transcript and protein are not always correlated. In the present study, the abundance of protein PDR9/ABCG37 in 3 Arabidopsis pdr9/abcg37 mutant alleles was evaluated using selected reaction monitoring analysis. The results showed that protein and mRNA expression levels were similar in 2 mutant alleles. The mRNA expression levels in another mutant, determined by both semi-quantitative and quantitative RT-PCR, were similar to the wild-type, although the abundance of protein was about half the abundance of the wild-type. These results suggested that using only mRNA expression levels to infer protein abundance, compare mutants or responses to various stimuli may lead to incorrect interpretation and conclusions.