High-throughput, low-cost quantification of 11 therapeutic antibodies using caprylic acid precipitation and LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring of treatment with therapeutic antibodies is hampered by the application of a wide range of different methods in the quantification of serum levels. LC-MS based methods could significantly improve comparability of results from different laboratories, but such methods are often considered complicated and costly. We developed a method for LC-MS/MS based quantification of 11 therapeutic antibodies concomitantly measured in a single run, with emphasis on simplicity in sample preparation and low cost. RESULTS: After a single-step sample purification using caprylic acid precipitation to remove interfering proteins, the sample underwent proteolysis followed by LC-MS/MS analysis. Infliximab is used as internal standard for sample preparation while isotope-labeled signature peptides identified for each analyte are internal standards for the LC-MS/MS normalization. The method was validated according to recognized guidelines, and pipetting steps can be performed by automated liquid handling systems. The total precision of the method ranged between 2.7 and 7.3 % (5.1 % average) across the quantification range of 4-256 µg/ml for all 11 drugs, with an average accuracy of 96.3 %. Matrix effects were xamined in 55 individual patient samples instead of the recommended 6, and 147 individual patient samples were screened for interfering compounds. SIGNIFICANCE AND NOVELTY: Our method for simultaneous quantification of 11 t-mAb in human serum allows an unprecedented integration of robustness, speed and reduced complexity, which could pave the way for uniform use in research projects and clinical settings alike. In addition, the first LC-MS protocol for signature peptide-based quantification of durvalumab is described. This high throughput method can be readily adapted to a drug panel of choice.

A Rapid and Simple UHPLC-MS/MS Method for Quantification of Plasma Globotriaosylsphingosine (lyso-Gb3).

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A gene (GLA) mutations, resulting in loss of activity of the lysosomal hydrolase, α-galactosidase A (α-Gal A). As a result, the main glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), accumulate in plasma, urine, and tissues. Here, we propose a simple, fast, and sensitive method for plasma quantification of lyso-Gb3, the most promising secondary screening target for FD. Assisted protein precipitation with methanol using Phree cartridges was performed as sample pre-treatment and plasma concentrations were measured using UHPLC-MS/MS operating in MRM positive electrospray ionization. Method validation provided excellent results for the whole calibration range (0.25-100 ng/mL). Intra-assay and inter-assay accuracy and precision (CV%) were calculated as <10%. The method was successfully applied to 55 plasma samples obtained from 34 patients with FD, 5 individuals carrying non-relevant polymorphisms of the GLA gene, and 16 healthy controls. Plasma lyso-Gb3 concentrations were larger in both male and female FD groups compared to healthy subjects (p < 0.001). Normal levels of plasma lyso-Gb3 were observed for patients carrying non-relevant mutations of the GLA gene compared to the control group (p = 0.141). Dropping the lower limit of quantification (LLOQ) to 0.25 ng/mL allowed us to set the optimal plasma lyso-Gb3 cut-off value between FD patients and healthy controls at 0.6 ng/mL, with a sensitivity of 97.1%, specificity of 100%, and accuracy of 0.998 expressed by the area under the ROC curve (C.I. 0.992 to 1.000, p-value < 0.001). Based on the results obtained, this method can be a reliable tool for early phenotypic assignment, assessing diagnoses in patients with borderline GalA activity, and confirming non-relevant mutations of the GLA gene.

A rapid and reliable liquid chromatography/mass spectrometry method for SARS-CoV-2 analysis from gargle solutions and saliva.

We describe a rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the direct detection and quantitation of SARS-CoV-2 nucleoprotein in gargle solutions and saliva. The method is based on a multiple-reaction monitoring (MRM) mass spectrometry approach with a total cycle time of 5 min per analysis and allows the detection and accurate quantitation of SARS-CoV-2 nucleoprotein as low as 500 amol/µL. We improved the sample preparation protocol of our recent piloting SARS-CoV-2 LC-MS study regarding sensitivity, reproducibility, and compatibility with a complementary reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of the same sample. The aim of this work is to promote diagnostic tools that allow identifying and monitoring SARS-CoV-2 infections by LC-MS/MS methods in a routine clinical environment.

Development and validation of a simple, selective, and sensitive LC-MS/MS assay for the quantification of remdesivir in human plasma.

Remdesivir, formerly GS-5734, has recently become the first antiviral drug approved by the U.S. Food and Drug Administration (FDA) to treat COVID-19, the disease caused by SARS-CoV-2. Therapeutic dosing and pharmacokinetic studies require a simple, sensitive, and selective validated assay to quantify drug concentrations in clinical samples. Therefore, we developed a rapid and sensitive LC-MS/MS assay for the quantification of remdesivir in human plasma with its deuterium-labeled analog, remdesivir-2H5, as the internal standard. Chromatographic separation was achieved on a Phenomenex® Synergi™ HPLC Fusion-RP (100 × 2 mm, 4 µm) column by gradient elution. Excellent accuracy and precision (<5.2% within-run variations and. <9.8% between-run variations) were obtained over the range of 0.5-5000 ng/mL. The assay met the FDA Bioanalytical Guidelines for selectivity and specificity, and low inter-matrix lot variability (<2.7%) was observed for extraction efficiency (77%) and matrix effect (123%) studies. Further, stability tests showed that the analyte does not degrade under working conditions, nor during freezing and thawing processes.

Rapid Shotgun Phosphoproteomics Analysis.

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.

Development of a multi-residue high-throughput UHPLC-MS/MS method for routine monitoring of SARM compounds in equine and bovine blood.

Selective androgen receptor modulators (SARMs) are a group of anabolic enhancer drugs posing threats to the integrity of animal sports and the safety of animal-derived foods. The current research describes for the first time the development of a semi-quantitative assay for the monitoring of SARM family compounds in blood and establishes the relative stability of these analytes under various storage conditions prior to analysis. The presented screening method validation was performed in line with current EU legislation for the inspection of livestock and produce of animal origin, with detection capability (CCß) values determined at 0.5 ng/mL (Ly2452473), 1 ng/mL (AC-262536 and PF-06260414), 2 ng/mL (bicalutamide, GLPG0492, LGD-2226, ostarine, S-1, S-6, and S-23), and 5 ng/mL (andarine, BMS-564929, LGD-4033, RAD140, and S-9), respectively. The applicability of the developed assay was demonstrated through the analysis of blood samples from racehorses and cattle. The developed method presents a high-throughput cost-effective tool for the routine screening for a range of SARM compounds in sport and livestock animals.

Determination of 42 mycotoxins in oats using a mechanically assisted QuEChERS sample preparation and UHPLC-MS/MS detection.

A method was developed and validated for the simultaneous determination of 42 mycotoxins in oats. The method includes all the mycotoxins listed under Commission Regulation 1881/2006 and Commission Recommendation 165/2013, the emerging mycotoxins (beauvericin, alternariol, alternariol-methyl-ether and enniatins), and two masked metabolites, namely deoxynivalenol-3-glucoside and T-2-glucoside. The method also focuses on a wide range of analytes of toxicological interest. The sample preparation involved extraction with an aqueous acetic acid solution and acetonitrile, followed by QuEChERS with mechanically assisted vibrational shaking. No further clean-up steps were employed, and analysis was performed using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Trueness ranged between 78% and 158%, while precision ranged from 1.7% to 49.9% under within-laboratory reproducibility conditions. Beside the high degree of accuracy and sample throughput provided, the method can be applied to a large number of compounds currently not regulated, thus generating knowledge and for risk assessment purposes.

A rapid and convenient derivatization method for quantitation of short-chain fatty acids in human feces by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Short-chain fatty acids (SCFAs) are associated with intestinal microbiota and diseases in humans. SCFAs have a low response in mass spectrometry, and in order to increase sensitivity, reduce sample consumption, shorten analysis time, and simplify sample preparation steps, a derivatization method was developed. METHODS: We converted seven SCFAs into amide derivatives with 4-aminomethylquinoline. The reaction occurred for 20 min at room temperature. The analytes were separated on a reversed-phase C18 column and quantitated in the positive ion electrospray ionization mode using multiple reaction monitoring. Acetic acid-d4 was used as the stable-isotope-labeled surrogate analyte for acetic acid in the working solutions, while the other stable-isotope-labeled standards were used as internal standards (ISs). RESULTS: Method validation showed that the intra-day and inter-day precision of quantitation for the seven SCFAs over the whole concentration range was ≤3.8% (n = 6). The quantitation accuracy ranged from 85.5% to 104.3% (n = 6). Most important, the collected feces were vortexed immediately with ethanol. CONCLUSIONS: This study provides a new derivatization method for a precise, accurate, and rapid quantitation of SCFAs in human feces using ultra-performance liquid chromatography/tandem mass spectrometry. This method successfully determined the concentration of SCFAs in human feces and could assist in the exploration of intestinal microbiota and diseases.

Fast and sensitive UHPLC-MS/MS analysis of cannabinoids and their acid precursors in pharmaceutical preparations of medical cannabis and their metabolites in conventional and non-conventional biological matrices of treated individual.

Monitoring pharmacological active compounds in pharmaceutical preparations of medical cannabis and in conventional and non-conventional biological matrices of treated individuals use requires both a wide linear range and sensitive detection. We have developed and validated a fast and sensitive method using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for analysis of Δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), their acidic precursors Δ-9-tetrahydrocannabinolic acid A (THCA-A) and cannabidiolic acid (CBDA) and some major metabolites of THC such as 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC), Δ-9-THC-Glucuronide (THC-GLUC) and THC-COOH-Glucuronide (THC-COOH-GLUC) in conventional (whole blood and urine) and non-conventional (oral fluid and sweat) of individual treated with medical cannabis preparation. Specifically, THC, THCA-A, CBD and CBD-A were determined in cannabis decoction and oil prepared to treat individuals. The method used positive electrospray ionization (ESI) mode to reach the sensitivity needed to detect minimal amounts of analytes under investigations exposure with limits of quantification ranging from 0.2 to 0.5 ng per milliliter (ng/mL) or ng per patch in case of collected sweat. The validation results indicated this method was accurate (average inter/intra-day error, <10%), precise (inter/intra-day imprecision, <10%), and fast (10 min run time). In addition, time-consuming sample preparation was avoided applying dilute and shoot procedure, meeting the needs for potential large-scale population studies. The analysis of real samples demonstrated a pharmacokinetics of cannabinoids, their precursors and their metabolites dependent from quantity of carboxylated and decarboxylated compounds in pharmaceutical preparations.

Rapid quantification of tenofovir in umbilical cord plasma and amniotic fluid in hepatitis B mono-infected pregnant women during labor by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Tenofovir (TFV) is a first-line antiviral agent against hepatitis B virus (HBV) and is recommended for the prevention of mother-to-infant transmission of HBV. To study the distribution of TFV in umbilical cord plasma and amniotic fluid of HBV-infected pregnant women, a rapid and sensitive method for TFV determination was developed and validated. METHODS: The quantification method was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The analytes were separated on an Acquity UPLC HSS T3 column under gradient elution with methanol and 0.01% ammonia solution in 10 mM ammonium acetate/water. This is the first reported method for the determination of TFV using alkaline rather than acidic mobile phases. Linearity, accuracy, precision, limit of quantification, specificity and stability were assessed. RESULTS: Detection of TFV was achieved within 4 min. The calibration curves for TFV quantification showed excellent linearity in the range of 1-500 ng/mL. The intra- and interbatch precision and accuracy ranged from -4.35% to 6.92%. This method was successfully applied to determination of samples from 50 HBV mono-infected women undergoing tenofovir disoproxil fumarate therapy. The mean concentrations of TFV in the umbilical cord and amniotic fluid samples were 29.2 (4.6-86) and 470.9 (156-902) ng/mL, respectively, which showed a moderate positive correlation (r = 0.5299, P<0.001). CONCLUSIONS: A simple, rapid but sensitive bioanalytical method to determine TFV concentration in both umbilical cord plasma and amniotic fluid using LC/MS/MS was developed and applied to HBV-infected women during labor who were undergoing TDF therapy, which will help us understand the efficacy and safety of tenofovir during pregnancy.

Rapid and sensitive analytical procedure for biomonitoring of organophosphate pesticide metabolites in human urine samples using a vortex-assisted salt-induced liquid-liquid microextraction technique coupled with ultra-high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Organophosphate pesticides (OPPs) are the most commonly used insecticides around the world in various agricultural and domestic practices, and humans are frequently exposed to these hazardous insecticides that can lead to several chronic health effects. Therefore, a fast and sensitive analytical method is required for biomonitoring the markers of OPPs in humans for exposure estimation. In this study, a fast and sensitive analytical procedure was developed for the determination of the metabolites of OPPs in human urine samples. METHODS: Metabolites of OPPs were extracted from 2 mL of urine sample using a novel vortex-assisted salt-induced liquid-liquid microextraction (VA-SI-LLME) technique, and the preconcentrated metabolites were analyzed by ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS). Various factors affecting the efficiency of VA-SI-LLME were thoroughly investigated. RESULTS: The metabolites of OPPs exhibited very good linearity over the concentration range between 0.05 and 50 ng mL-1 with coefficient (r2 ) values ranging between 0.9986 and 0.9999. The method showed excellent sensitivity with detection limits ranging from 0.01 to 0.03 ng mL-1 and quantification limits from 0.03 to 0.05 ng mL-1 . The developed method was applied to the analysis of real samples and the recoveries ranged between 85.0 and 114.1% with related standard deviations <5%. CONCLUSIONS: The results showed the VA-SI-LLME/UHPLC/MS/MS method to be a simple, rapid, sensitive, and selective analytical procedure for the biomonitoring of the metabolites of OPPs in humans. This efficient and cost-effective analytical method could be a potential alternative method for the biomonitoring of the metabolites of pesticides in humans.

Rapid quantification of acetaminophen in plasma using solid-phase microextraction coupled with thermal desorption electrospray ionization mass spectrometry.

RATIONALE: Solid-phase microextraction coupled with thermal desorption electrospray ionization tandem mass spectrometry (SPME-TD-ESI-MS/MS) is proposed as a novel method for the rapid quantification of acetaminophen in plasma samples from a pharmacokinetics (PK) study. METHODS: Traces of acetaminophen were concentrated on commercial fused-silica fibers coated with a polar polyacrylate (PA) polymer using direct immersion SPME. No agitation, heating, addition of salt, or adjustment of the pH of the sample solution was applied during the extraction. Any acetaminophen absorbed on the SPME fibers was subsequently desorbed and detected by TD-ESI-MS/MS. RESULTS: Parameters of the absorption, sensitivity, reproducibility, and linearity for the SPME-TD-ESI-MS/MS method were evaluated. The time required to complete a TD-ESI-MS/MS analysis was less than 30 seconds. Matrix-matching calibration was performed to calculate the concentration of acetaminophen in the sample. A linear calibration curve with a concentration range of 100-10,000 ng/mL was constructed to calculate the quantity of acetaminophen. The SPME-TD-ESI-MS quantification results for acetaminophen in plasma were in good agreement with those obtained by the conventional LC/MS/MS method. CONCLUSIONS: With the proposed method, a 10-min SPME time was enough to achieve the lower limit of quantitation (i.e. 100 ng/mL) and for a complete PK profiling of acetaminophen. A shorter extraction time could be achieved by applying agitation, heating, adding salt, or adjusting the pH of the sample solution to enhance analyte absorption efficiency. The time required to detect acetaminophen on the SPME fiber was less than 30 s, allowing the rapid quantification of acetaminophen in plasma with good accuracy.

Simple and sensitive method for the analysis of 14 antituberculosis drugs using liquid chromatography/tandem mass spectrometry in human plasma.

Monitoring plasma concentration and adjusting doses of antituberculosis (TB) drugs are beneficial for improving responses to drug treatment and avoiding adverse drug reactions. A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed to measure the plasma concentrations of 14 anti-TB drugs: ethambutol, isoniazid, pyrazinamide, levofloxacin, gatifloxacin, moxifloxacin, prothionamide, linezolid, rifampin, rifapentine, rifabutin, cycloserine, p-aminosalicylic acid, and clofazimine. METHODS: Human plasma was precipitated by acetonitrile and was subsequently separated by an AQ-C18 column with a gradient elution. Drug concentrations were determined using multiple reaction monitoring in positive ion electrospray ionization mode. According to pharmacokinetic data of patients, the peak concentration ranges and the timing of blood collection were determined. RESULTS: Intra- and interday precision was < 14.8%. Linearity, accuracy, extraction recovery, and matrix effect were acceptable for each drug. The stability of the method satisfied different storage conditions. CONCLUSIONS: The method allowed the sensitive and reproducible determination of 14 frequently used anti-TB drugs which has already been of benefit for some TB patients.

Increasing clinical liquid chromatography/tandem mass spectrometry assay throughput using a full calibration curve generated by one injection from a single-tube calibrator.

Mass spectrometry (MS) generally delivers more accurate results than immunoassay (IA) for certain clinically relevant analytes, but IA is still the more prevalent methodology used by clinical laboratories because of barriers to MS adoption, such as lower throughput. Therefore, it is increasingly important to develop new strategies to increase LC/MS/MS throughput so that more accurate results can be delivered to patients and clinicians. METHODS: Throughput can be increased by reducing assay calibration time using a single-tube calibrator, a mix of isotopologues of the target analyte at different concentrations in a biological matrix, rather than a set of traditional, multiple-tube calibrators. One injection from a single-tube calibrator can generate a full calibration curve such that each calibration point is from the multiple reaction monitoring (MRM) signal corresponding to a specific isotopologue. RESULTS: In this study, a single-tube calibrator (five levels in one vial) and a set of multiple-tube calibrators (seven levels in seven vials) were used to measure the concentration of testosterone in 42 serum samples originally value assigned by the Centers for Disease Control and Prevention (CDC) reference method. The bias between the CDC reference method and the single-tube calibrator measurements and the multiple-tube calibrators measurements was +1.1% and - 5.5%, respectively. These results were within the CDC Hormone Standardization (HoSt) program bias acceptance criteria of ±6.4%. CONCLUSIONS: The results show that LC/MS/MS throughput can be increased using a single-tube calibrator because it reduces assay calibration time while delivering equivalent results to those generated using traditional, multiple-tube calibrators. The single-tube calibrator may also save cost to laboratories through reductions in consumable consumption, technician labor time, and inventory management, as well as to manufacturers because fewer vials would need to be manufactured, tested, stored, and shipped.

A sensitive and cost-effective high-performance liquid chromatography/tandem mass spectrometry (multiple reaction monitoring) method for the clinical measurement of serum hepcidin.

RATIONALE: Hepcidin is a peptide hormone that plays a central role in regulating iron metabolism. It is a potential biomarker for the diagnosis, monitoring and treatment of iron metabolism disorders. Serum hepcidin level can differ by 3 orders of magnitude depending on the patient's condition. Existing liquid chromatography/mass spectrometry (LC/MS) assays lack clinical sensitivity or require costly sample preparation steps. A simple, sensitive, robust and cost-effective assay for serum hepcidin quantitation in routine clinical laboratories is needed. METHODS: A high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable-isotope-labeled hepcidin as the internal standard. The method was validated according to CLSI-C62A guidelines. Calibrators were prepared with hepcidin-free serum. Clinical samples were separately processed and compared using solid-phase extraction (SPE) and acetonitrile (ACN) protein precipitation. RESULTS: The calibration curve was validated over the range of 0.1-100 nmol/L with R2  >0.99. Both the SPE and the ACN precipitation methods had excellent and comparable reproducibility. The intra-day and inter-day coefficients of variation (CVs) were <3% and <6%. There was 89% and 88% hepcidin recovery by SPE and ACN preparation. Measurement of secondary reference material using non-traceable calibrators yielded up to 30% positive bias, comparable with values obtained by an external comparator. Hepcidin was stable in serum at ambient temperature and at 4°C. The relative errors (REs) were ≤1.2% and ≤4.4%, respectively. The freeze-thaw (-70°C) stability after 3 cycles showed a relative error (RE) of ≤1.8%. The impact on hepcidin recovery due to hemolysis (4+), lipemia (4+) and Icterus (4+) was <3%. CONCLUSIONS: We have developed and validated a simple, sensitive, robust and cost-effective HPLC/MS/MS method for the quantitation of serum hepcidin. The method uses ACN protein precipitation for sample preparation and reversed-phase normal-flow HPLC. Sample preparation is inexpensive; it can be automated with a liquid handling system to allow high-throughput application.

Intensive optimization and evaluation of global DNA methylation quantification using LC-MS/MS.

DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS has emerged as an excellent method from the viewpoint of sensitivity, reproducibility, and cost. However, LC-MS/MS methods have limitations due to a lack of the stability and the standardization required for a laboratory assay. The present study aimed to establish a robust assay that guarantees highly accurate measurements of global DNA methylation levels. There are at least three facets of the developed method. The first is discovery of the solvent conditions to minimize sodium adducts. The second is improvement of separation of nucleosides by LC using the columns that had not been used in previous similar studies. The third is success in reduction of the uncertainty of the measurement results, which was achieved by the calibration using the ratio of mdC but not the absolute amount in the presence of internal standards. These facets represent the advantage over methods reported previously. Our developed method enables quantification of DNA methylation with a short time length (8 min) for one analysis as well as with the high reproducibility of measurements that is represented by the inter-day CV% being less than 5%. In addition, data obtained from measuring global DNA methylation levels in cultured cell lines, with or without pharmacological demethylation, support its use for biomedical research. This assay is expected to allow us to conduct initial screening of epigenetic alterations or aberration in a variety of cells.

A rapid UHPLC-MS/MS screening method for the detection of the addition of porcine blood plasma to emulsion-type pork sausages.

The adulteration of meat products by the undeclared addition of commercially available blood plasma powder is quite conceivable due to low costs, high protein contents (about 70%), and advantageous functional properties. This applies particularly to pork, which has the highest meat production rate in the European Union. Evidence of this type of food fraud has been rather difficult to identify due to the lack of appropriate analytical methods, especially when adding plasma to meat of the same animal species. Consequently, a rapid UHPLC-MS/MS method for the detection of porcine blood plasma in emulsion-type pork sausages was developed. After protein extraction and tryptic digestion in a quick and simple one-pot process, species-specific marker peptides for porcine blood cell proteins (four markers) and plasma proteins (12 markers) were measured by UHPLC-MS/MS. Emulsion-type pork sausages were produced from a variety of raw materials that differed in the age or sex of the slaughtered pigs. Sausages were spiked with 0.5, 1, 1.5, 2, 3, or 5% meat substitution by one of two plasma powders, or produced as corresponding blank samples, and subjected to different thermal treatments as full or semi-preserves. Four plasma peptides were identified for the overall sample that allowed detection down to 0.7% meat substitution from the sum of their peak areas, with 5% error probability for both false positives and negatives.

De novo structural determination of mannose oligosaccharides by using a logically derived sequence for tandem mass spectrometry.

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.

Rapid determination of isocitrate dehydrogenase mutation status of human gliomas by extraction nanoelectrospray using a miniature mass spectrometer.

Isocitrate dehydrogenase (IDH) I and II mutations in gliomas cause an abnormal accumulation of 2-hydroxyglutarate (2-HG) in these tumor cells. These mutations have potential prognostic value in that knowledge of the mutation status can lead to improved surgical resection. Information on mutation status obtained by immunohistochemistry or genomic analysis is not available during surgery. We report a rapid extraction nanoelectrospray ionization (nESI) method of determining 2-HG. This should allow the determination of IDH mutation status to be performed intraoperatively, within minutes, using a miniature mass spectrometer. This study demonstrates that the combination of tandem mass spectrometry with low-resolution mass spectrometry allows this analysis to be performed with confidence. Graphical Abstract.