Detection and Quantitation of Selected Food Allergens by Liquid Chromatography with Tandem Mass Spectrometry: First Action 2017.17.

BACKGROUND: In response to a need for accurate and reliable methods for food allergen regulatory compliance, a method for the detection and quantitation of whole egg, whole milk, peanut, and hazelnut in eight food matrices was developed and evaluated in a single-laboratory validation. The matrices include cookies, cookie dough, bread, breakfast cereal, salad dressing, ice cream, and red wine. OBJECTIVE: The method was compared with Standard Method Performance Requirements (SMPR) 2016.002 established by the AOAC Stakeholder Panel on Strategic Food Analytical Methods. METHODS: The method involves tryptic digestion of allergen proteins in food matrices incurred or spiked with allergen standards [reference materials (RMs), Standard RMs (SRMs), or in-house prepared standard] and uses labeled peptide internal standards. LC-tandem MS analysis of the signature tryptic peptides of the four allergens is performed using multiple reaction monitoring. RESULTS: For 10 allergen/matrix combinations, the method demonstrated adequate sensitivity with a minimum quantitation limit of 3 mg/kg for whole egg and 10 mg/kg for milk, peanut, and hazelnut allergens. Repeatability precision across 3 days of analyses was <17% with analytical range of 10-1000 mg/kg. Recovery from incurred and spiked matrix-matched standards varied from 60 to 118%. CONCLUSIONS: The method met the minimum performance requirements of SMPR 2016.002 for whole egg in cookies, bread, cookie dough, and salad dressing; whole milk in cookies and red wine; peanut in breakfast cereal; and hazelnut in cookies. HIGHLIGHTS: The ERP determined that the data presented met the SMPR and accordingly recommended the method to be granted First Action status. In September 2017, the Official Methods Board approved the method as First Action.

Simultaneous Analysis of Multiple Allergens in Food Products by LC-MS/MS.

There is currently no cure for food allergies, and sufferers can only rely on the correct labeling of foods to avoid allergens. Hence, it is important that analytical methods are sensitive and accurate enough to screen for the presence of multiple allergens in food products. In this study, we developed an LC-tandem MS method that is able to simultaneously screen or quantify the signature tryptic peptides of multiple allergen commodities. This method is capable of screening and identifying egg white, skim milk, peanut, soy, and tree nuts (almond, Brazil nut, cashew, hazelnut, pecan, pine nut, pistachio, and walnut) at a detection limit of 10 ppm in incurred bread and cookies. It was further tested for the quantitative analysis of whole-egg, whole-milk, peanut butter, and hazelnut commodities, which are incurred or spiked into selected food matrixes as defined in AOAC INTERNATIONAL Standard Method Performance Requirement (SMPR®) 2016.002. The method demonstrated excellent sensitivity with a Method quantitative limit of 3 ppm for whole egg and 10 ppm for the remaining three allergen commodities. It also demonstrated good recovery (60-119%) and repeatability (RSDr <20%), with an analytical range of 10-1000 ppm for each allergen commodity and was able to meet the minimum performance requirements of the SMPR.

First metabolic profile of XLR-11, a novel synthetic cannabinoid, obtained by using human hepatocytes and high-resolution mass spectrometry.

BACKGROUND: Since the mid-2000s synthetic cannabinoids have been abused as recreational drugs, prompting scheduling of these substances in many countries. To circumvent legislation, manufacturers constantly market new compounds; [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), the fluorinated UR-144 analog, is one of the most recent and widely abused drugs, and its use is now linked with acute kidney injury. Our goal was to investigate XLR-11 metabolism for identification of major urinary targets in analytical methods and to clarify the origin of metabolites when one or more parent synthetic cannabinoids can be the source. METHODS: We incubated 10 µmol/L XLR-11 with pooled human hepatocytes and sampled after 1 and 3 h. Samples were analyzed by high-resolution mass spectrometry with a TOF scan followed by information-dependent acquisition triggered product ion scans with dynamic background subtraction and mass defect filters. Scans were thoroughly data mined with different data processing algorithms (Metabolite Pilot 1.5). RESULTS: XLR-11 underwent phase I and II metabolism, producing more than 25 metabolites resulting from hydroxylation, carboxylation, hemiketal and hemiacetal formation, internal dehydration, and further glucuronidation of some oxidative metabolites. No sulfate or glutathione conjugation was observed. XLR-11 also was defluorinated, forming UR-144 metabolites. On the basis of mass spectrometry peak areas, we determined that the major metabolites were 2'-carboxy-XLR-11, UR-144 pentanoic acid, 5-hydroxy-UR-144, hydroxy-XLR-11 glucuronides, and 2'-carboxy-UR-144 pentanoic acid. Minor metabolites were combinations of the biotransformations mentioned above, often glucuronidated. CONCLUSIONS: These are the first data defining major urinary targets of XLR-11 metabolism that could document XLR-11 intake in forensic and clinical investigations.

First characterization of AKB-48 metabolism, a novel synthetic cannabinoid, using human hepatocytes and high-resolution mass spectrometry.

Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, new synthetic cannabinoids were robustly marketed. N-(1-Adamantyl)-1-pentylindazole-3-carboxamide (AKB-48), also known as APINACA, was recently observed in Japanese herbal smoking blends. The National Forensic Laboratory Information System registered 443 reports of AKB-48 cases in the USA from March 2010 to January 2013. In May 2013, the Drug Enforcement Administration listed AKB-48 as a Schedule I drug. Recently, AKB-48 was shown to have twice the CB1 receptor binding affinity than CB2. These pharmacological effects and the difficulty in detecting the parent compound in urine highlight the importance of metabolite identification for developing analytical methods for clinical and forensic investigations. Using human hepatocytes and TripleTOF mass spectrometry, we identified 17 novel phase I and II AKB-48 metabolites, products of monohydroxylation, dihydroxylation, or trihydroxylation on the aliphatic adamantane ring or N-pentyl side chain. Glucuronide conjugation of some mono- and dihydroxylated metabolites also occurred. Oxidation and dihydroxylation on the adamantane ring and N-pentyl side chain formed a ketone. More metabolites were identified after 3 h of incubation than at 1 h. For the first time, we present a AKB-48 metabolic scheme obtained from human hepatocytes and high-resolution mass spectrometry. These data are needed to develop analytical methods to identify AKB-48 consumption in clinical and forensic testing.

Inhibitory effect of the paraoxonase gene on the formation of rabbit coronary atherosclerosis.

OBJECTIVE: To observe the effect on the inhibition of coronary atherosclerosis hardening of the paraoxonase gene (PON-1) which transfected to the rabbit epicardial adipose tissue. METHODS: Rabbit coronary atherosclerosis model was established by high-fat feeding, liposome-encapsulated recombinant plasmid pEGFP-PON-1 50 µ L was injected to the rabbit pericardial cavity, and was harvested 4 weeks after transfection. RESULTS: The epicardial fat transfected PON-1 gene had effect on the high lipid level. It significantly increased expression of PON-1 in peripheral arterial vascular tissue (P <0.05); and significantly reduced total cholesterol and low-density lipoprotein cholesterol levels (P<0.05), and the thickness ratio of coronary artery intima/media (P <0.05). CONCLUSIONS: The injection of the PON-1 gene in the pericardial cavity can effectively suppress the formation of coronary atherosclerosis.

11-Nor-9-carboxy-∆9-tetrahydrocannabinol quantification in human oral fluid by liquid chromatography-tandem mass spectrometry.

Currently, ∆9-tetrahydrocannabinol (THC) is the analyte quantified for oral fluid cannabinoid monitoring. The potential for false-positive oral fluid cannabinoid results from passive exposure to THC-laden cannabis smoke raises concerns for this promising new monitoring technology. Oral fluid 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH) is proposed as a marker of cannabis intake since it is not present in cannabis smoke and was not measureable in oral fluid collected from subjects passively exposed to cannabis. THCCOOH concentrations are in the picogram per milliliter range in oral fluid and pose considerable analytical challenges. A liquid chromatography-tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying THCCOOH in 1 mL Quantisal-collected oral fluid. After solid phase extraction, chromatography was performed on a Kinetex C18 column with a gradient of 0.01% acetic acid in water and 0.01% acetic acid in methanol with a 0.5-mL/min flow rate. THCCOOH was monitored in negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. The THCCOOH linear range was 12-1,020 pg/mL (R(2) > 0.995). Mean extraction efficiencies and matrix effects evaluated at low and high quality control (QC) concentrations were 40.8-65.1 and -2.4-11.5%, respectively (n = 10). Analytical recoveries (bias) and total imprecision at low, mid, and high QCs were 85.0-113.3 and 6.6-8.4% coefficient of variation, respectively (n = 20). This is the first oral fluid THCCOOH LCMSMS triple quadrupole method not requiring derivatization to achieve a <15 pg/mL limit of quantification. The assay is applicable for the workplace, driving under the influence of drugs, drug treatment, and pain management testing.

Qualitative confirmation of 9 synthetic cannabinoids and 20 metabolites in human urine using LC-MS/MS and library search.

INTRODUCTION: Synthetic cannabinoids are an emerging illicit drug class. The variety of available substances is large and ever-changing, making it difficult for laboratories to remain current. We present a qualitative LC-MS/MS method identifying urinary metabolites of JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, RCS-4, and AM2201 and the parent compounds JWH-018, JWH-073, JWH-081, JWH-122, JWH-210, JWH-250, RCS-4, AM2201, and MAM2201. METHODS: After enzymatic hydrolysis, urinary proteins were precipitated with acetonitrile. Chromatography utilized a 10 min gradient on a Kinetex XB-C18 column with 0.1% formic acid in water and acetonitrile. Scheduled multiple reaction monitoring "survey scans" were followed by information-dependent acquisition-enhanced product ion scan experiments on an ABSciex 5500 QTRAP mass spectrometer. Analytes were identified by software-assisted library searching against reference spectra. RESULTS: The method was fully validated, including proof of selectivity (no exogenous or endogenous interferences were observed), assessment of matrix effects (95-122%) and recovery (53-95%), determination of limits of detection (0.5-10 ng/mL), carry-over studies (thresholds between 100 and 1000 ng/mL), and determination of autosampler stability (samples were stable for at least 3 days). Hydrolysis efficiency was thoroughly investigated for a wide range of glucuronides and for the reference standard, JWH-018 5-hydroxypentyl glucuronide.

High mobility group box 1 protein: possible pathogenic link to atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained dysrhythmia in clinical practice. The bulk of evidence suggests that inflammatory processes, oxidative stress and matrix metalloproteinase are associated with development of AF. However, these agents may be involved in high mobility group box 1 protein (HMGB1). We hypothesized that HMGB1 may be a possible pathogenic link to AF. A growing body of evidence supports these hypotheses. First, the level of serum HMGB1 is significantly increased in patients with AF including paroxysmal and persistent AF. Second, HMGB1 has been identified as a new pro-inflammatory cytokine in cardiovascular diseases, along with tumor necrosis factor (TNF)-α, interleukin (IL)-6, and C-reactive protein, and there is cross-talk between HMGB1 and inflammatory cytokines. Third, oxidative stress is involved in the release of the pro-inflammatory cytokine, HMGB1, indicating there is cross-talk between oxidative stress and inflammation, and oxidative stress may reinforce the effect of inflammation on the pathogenesis of AF and inflammation may play a more important role in the pathogenesis of AF. Fourth, HMGB1 can promote matrix metalloproteinase-9 upregulation and activation. Fifth, HMGB1 receptors (receptor for advanced glycation end products, Toll-like receptor-2,4) may mediate the atrial structural remodeling or be up-regulated in patients with non-valvular AF. These results suggest that HMGB1 may participate in the pathogenesis of AF and provide a potential target for pharmacological interruption of AF.

Determination of serum aldosterone by liquid chromatography and tandem mass spectrometry: a liquid-liquid extraction method for the ABSCIEX API-5000 mass spectrometry system.

AIMS: Accurate serum aldosterone determination is critical to the screening and diagnosis of primary aldosteronism, the localisation of aldosterone producing tumours, and the investigation of other disorders of the renin-angiotensin system. Mass spectrometry offers a means to overcome problems with method-dependent bias between competitive immunoassays for aldosterone. The authors have developed a simple, sensitive and precise liquid-liquid extraction aldosterone method for the ABSCIEX API-5000 liquid chromatography and tandem mass spectrometry (LC-MS/MS) system. METHODS: Using d7-aldosterone internal standard, 500 µl of sample is extracted with 2500 µl of methyl tertbutyl ether followed by dry-down, reconstitution and LC-MS/MS analysis in ESI negative mode. Method validation was undertaken using standard approaches and comparison made against a commercial radioimmunoassay. Accuracy was assessed using EQA material with assigned aldosterone concentrations. RESULTS: The assay was linear up to 3420 pmol/l (LOQ=50 pmol/l, LOD<22 pmol/l). Total CVs were ≤5% for concentrations ≥120 pmol/l and 10% at the LOQ. Mean accuracy was 98.5% against GCMS assigned material. CONCLUSION: The authors present a precise, sensitive and simple aldosterone method suitable for routine clinical use that requires no solid phase extraction or specialised ion sources.

Simultaneous screening of glutathione and cyanide adducts using precursor ion and neutral loss scans-dependent product ion spectral acquisition and data mining tools.

Drugs can be metabolically activated to soft and hard electrophiles, which are readily trapped by glutathione (GSH) and cyanide (CN), respectively. These adducts are often detected and structurally characterized using separate tandem mass spectrometry methods. We describe a new method for simultaneous screening of GSH and CN adducts using precursor ion (PI) and neutral loss (NL) scans-dependent product ion spectral acquisition and data mining tools on an triple quadrupole linear ion trap mass spectrometry. GSH, potassium cyanide, and their stable isotope labeled analogues were incubated with liver microsomes and a test compound. Negative PI scan of m/z 272 for detection of GSH adducts and positive NL scans of 27 and 29 Da for detection of CN adducts were conducted as survey scans to trigger acquisition of enhanced resolution (ER) spectrum and subsequent enhanced product ion (EPI) spectrum. Post-acquisition data mining of EPI data set using NL filters of 129 and 27 Da was then performed to reveal the GSH adducts and CN adducts, respectively. Isotope patterns and EPI spectra of the detected adducts were utilized for identification of their molecular weights and structures. The effectiveness of this method was evaluated by analyzing reactive metabolites of nefazodone formed from rat liver microsomes. In addition to known GSH- and CN-trapped reactive metabolites, several new CN adducts of nefazodone were identified. The results suggested that current approach is highly effective in the analysis of both soft and hard reactive metabolites and can be used as a high-throughput method in drug discovery.

Development of a robust and sensitive LC-MS/MS method for the determination of adenine in plasma of different species and its application to in vivo studies.

A simple, robust, and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the measurement of endogenous adenine in mouse, rat, cynomolgus monkey, and human plasma. A "surrogate analyte" strategy was adopted by employing [(13)C(U)]-adenine as the surrogate analyte. The plasma samples were processed by protein precipitation, and the extracted supernatant samples were subjected directly to LC-MS/MS analysis. The analysis was carried out in the negative ion detection mode using selected-reaction monitoring (SRM). The method achieved a lower limit of quantification (LLOQ) of 5.0nM with a signal-to-noise ratio of 10. The intra- and inter-day assay coefficients of variation (CV) were ≤6.67% in rat plasma, and the mean recoveries and matrix effects across species and at various concentrations ranged from 88.8% to 104.2% and 86.0% to 110.8%, respectively. Using this methodology, the endogenous concentration of adenine in plasma of four species was found to range from 8.7nM in human to 93.1nM in cynomolgus monkey plasma. The assay was further applied to both an adenine pharmacokinetic study and a pivotal pharmacodynamic study evaluating the plasma concentration of adenine after a dose of 5'-deoxy-5'-methylthioadenosine (MTA).

The in vitro metabolism of bupropion revisited: concentration dependent involvement of cytochrome P450 2C19.

The involvement of cytochrome P450 2B6 (CYP2B6) to the in vitro and in vivo metabolism of bupropion has been well studied. In these investigations we performed a detailed in vitro phenotyping study to characterize isoforms other than CYP2B6. A total of nine metabolites were identified (M1-M9) in the incubations with cDNA-expressed P450s (rhCYP) and human liver microsomes (HLM). Incubations in rhCYP identified CYP2B6 as the isoform responsible for the formation of hydroxybupropion (M3). CYP2C19 was involved in bupropion metabolism primarily through alternate hydroxylation pathways (M4-M6) with higher activity at lower substrate concentrations, near 1 microM. The results from HLM inhibition studies using CYP2B6 and CYP2C19 inhibitory antibodies indicated that CYP2B6 contributed to approximately 90% of M3 formation, and CYP2C19 contributed to approximately 70-90% of M4, M5, and M6 formation. Studies using single donor HLM with varying degrees of CYP2B6 and CYP2C19 activities showed a good relationship between M3 formation and CYP2B6 activity and M4/M5 formation and CYP2C19 activity. These results confirmed the principle role of CYP2B6 in hydroxybupropion formation, as a selective CYP2B6 probe. In addition, the new findings revealed that CYP2C19 also contributes to bupropion metabolism through alternate hydroxylation pathways.

[Effects of antiarrhythmic drug use on atrial fibrillation recurrence in atrial fibrillation patients post circumferential pulmonary vein ablation].

OBJECTIVE: This prospective and randomize-controlled trial was designed to investigate the effects of antiarrhythmic drug use (AADs) on atrial fibrillation (AF) recurrence in atrial fibrillation patients post circumferential pulmonary vein ablation (CAPV). METHODS: Seventy-four consecutive AF patients underwent CAPV (41 paroxysmal and 33 drug refractory AF) were randomly assigned to receive placebo (Group A) or AADs (Group B) for 3 months. Monthly standard electrocardiograms (ECG) and Holter monitoring were performed to assess AF recurrences during 17 - 28 months follow-up. RESULTS: CAPV was successful in all patients. The recurrence rate of AF in Group B was significantly lower than that in Group A at 3 months post CAPV (13.5% vs. 37.8%, P < 0.01) and similar thereafter (29.7% vs. 24.3% at 12 months and 8.1% vs. 8.1% at more than 12 months, all P > 0.05). There was also no significant difference in terms of total recurrence rate between the two groups (37.8% vs. 32.4%, P > 0.05). CONCLUSION: Post CAPV antiarrhythmic drug therapy could only decrease the early AF recurrence rate but was not effective for decreasing AF recurrence rate on later stage.