HS-SPME Gas Chromatography Approach for Underivatized Acrylamide Determination in Biscuits

Acrylamide (AA) is a food contaminant in thermally processed products that is object of tight control. A simple and easy-to-apply methodology for routine monitoring of AA levels in food products could allow producers to be players in the control of their own products. In this work, a simple methodology for AA quantification without derivatization was developed for biscuits, for which the benchmark levels recommended by EFSA are 350 µg/kg, and 150 µg/kg for biscuits for infants and young children. Headspace-solid phase microextraction (HS-SPME) was used in 120 mL screwed-cap vials with a carboxen/polydimetylsiloxane fiber, 4 g of biscuits, and 10 mL of water during 15 min at room temperature under stirring. The addition of 30 mL of propanol under stirring during 15 min at room temperature and 15 min at 60 °C was used to promote AA transfer to the headspace. The fiber exposure was 45 min. A gas chromatography-mass spectrometry analysis allowed to obtain an external calibration curve at m/z 71, with linearity R2 > 0.99 and precision RSD < 9%. The detection and quantification limits were 27.4 µg/kg and 91.5 µg/kg, respectively. The methodology was successfully used in biscuits with lower AA amount, where mitigation strategies (asparaginase or pectate) were applied.

Construction and Validation of a Nomogram for The Prediction of Overall Survival in Intrahepatic Cholangiocarcinoma

Objective: This study aimed to establishand validates a nomogram to predict the overall survival (OS) of patients with intrahepatic cholangiocarcinoma (ICC).Patients and methods: The ICC patients were collected from the Surveillance, Epidemiology, and End Results (SEER) database from 2004 to 2015. Then, the independent prognosis-related factors were obtained from the training set using the Cox regression model for the establishment of a nomogram.Results: We identified 3675 eligible patients with a median survival time of 9 months (0–153 months). According to multivariate analysis, age, sex, marital status, grade, T stage, N stage, M stage, surgery, chemotherapy and radiotherapy were identified as the factors to independently predictthe prognosis for ICC (all P<0.05). Thereafter, the above factors were incorporated for the construction of a nomogram. In comparison with the AJCC 8th TNM classification system and the SEER summary stage system, our constructed nomogram showed higher ability in discrimination, as revealed by the C-index (all P<0.001).Besides, the internal as well as external calibration curve analysis demonstrated that the predicted results were highly consistent with the actual ones. On the other hand, our nomogram outperformed the AJCC 8th TNM classification system and the SEER summary stage system in predicting the 3- and 5-year OS, as suggested by time-independent area under the curve (tAUC) values.Conclusion: Our constructed nomogram performs well, indicating its potential as an efficient approach to evaluate the prognosis of ICC patients.

20 Quantification of sBCMA in human plasma using a high-throughput mass spectrometry workflow for exploratory, CAP/CLIA or regulated studies

BackgroundBioanalytically validated methods are needed for reliable measurement of B cell maturation antigen (BCMA) in clinical samples. BCMA contributes to multiple myeloma (MM) pathophysiology and is targeted by various novel immunotherapies. Soluble BCMA (sBCMA) is a promising novel biomarker for disease monitoring and prediction. To address the need for sBCMA measurement, we developed a mass spectrometry-based assays for the quantitation of total sBCMA in plasma.MethodsImmunoaffinity enrichment of sBCMA from human plasma was performed on a Agilent AssayMap Bravo platform using streptavidin cartridges. Recovered sBCMA protein was digested to peptides using trypsin, spiked with a fixed level of stable isotope labeled (SIL) peptide standard, and analyzed by multiple-reaction-monitoring (MRM) mass spectrometry. The MRM assay targeted a BCMA-specific endogenous peptide used as a surrogate measure of the protein, as well as the corresponding spiked SIL peptide at known concentration. An 8-point external calibration curve was prepared by spiking varying amounts of recombinant BCMA and fixed amounts of SIL peptides in surrogate matrix. Endogenous sBCMA levels were determined by back-calculating against the curve. To assess the performance of the method, ≥ 2 precision and accuracy runs were performed. To assess the impact of ligand or drug binding on the developed assay, performance of the assay was tested using plasma spiked at a range of concentrations with rhAPRIL, rhBAFF or simulated mAb drug.ResultsThe developed assay allowed the quantitation of sBCMA from 1 to 1000 ng/mL. The precision and accuracy at different QC levels was within 20% CV and 20% bias. The presence of binding proteins (rhAPRIL, rhBAFF, simulated mAb) did not interfere with the measurement of sBCMA indicating that the assay measures total sBCMA.ConclusionsWe have developed an assay for the absolute quantitation of total sBCMA in human plasma. The assay can be analytically validated and deployed for clinical studies with a ~ 3-day turnaround time.

Application of 1H DOSY NMR in Measurement of Polystyrene Molecular Weights

In this work, we studied the applicability of diffusion ordered nuclear magnetic resonance spectroscopy (DOSY NMR) as an alternative method in determination of polystyrene molecular weight. DOSY NMR spectroscopy allows measuring the diffusion coefficient of molecule which directly depend on hydrodynamic radius and so on, average molar mass in weight (Mw). By using commercial polystyrene (PS) standards, an external calibration curve was established. Based on the excellent linear correlation between diffusion coefficient (logD) and molecular weight (logM), the molecular weight of polystyrene can be predicted using the following equation . The validation was done by comparing with the Mw value obtained by gel permeation chromatography within less than 5% deviation. From the diffusion coefficient, some property of polystyrene in solution, such as Flory coefficient and polymer-solvent interaction, were also studied. The Flory coefficient confirmed that chloroform is a good solvent for PS.

Metals in green coffee beans from major coffee-growing regions of Ethiopia

The metal contents of green coffee beans cultivated in major coffee growing zones of Ethiopia (Wollega, Sidamo, Harar, Bench Maji and Kafa) have been determined in representative samples of the five coffee varieties collected from Coffee Quality Inspection and Liquoring Center located in Addis Ababa, the capital city of Ethiopia. Different sample preparation procedures were tested by varying reagent volumes and types, digestion time, digestion temperature and amount of the sample to decompose the green coffee beans and solubilize the metals. The optimal procedure required 4 h to completely digest 0.5 g of green coffee beans with 5 mL HNO3 (70%) and 1.5 mL HClO4 (70%) at 270 oC. Concentrations of metals (Ca, Cd, Cr, Co, Cu, Fe, K, Mg, Mn, Ni, Pb and Zn) were determined by flame atomic absorption spectrometer employing a four point external calibration curve. The accuracy of the optimized procedure was evaluated by analyzing sample digests spiked with standard solutions. Recoveries of the spiked samples varied from 90 to 110% in green coffee beans. The metals levels observed in green coffee beans are comparable to literature reports. Cd was not detected in any of the five samples while Pb was detected at trace level in only one of the five samples. This indicated that the Ethiopian green coffee beans are free from the toxic metals. The Pearson correlation coefficients indicated strong to medium positive or negative correlation among the metals in the green coffee beans. The analysis of variance results at 95% confidence level suggested that there were significant difference in the concentrations of all the metals except K between the five sampling areas. Thus, this study has revealed variation of metal composition of green coffee beans with the geographical origin of the coffee verities. The variation in composition among coffee sample might be due the differences in mineral contents of the corresponding soils.

Accurate molecular weight determination of small molecules via DOSY-NMR by using external calibration curves with normalized diffusion coefficients

We describe a novel development of MW-determination by using an external calibration curve approach with normalized diffusion coefficients.

Quantitative Evaluation of Benzene, Toluene, and Xylene in the Larvae of Drosophila melanogaster by Solid-Phase Microextraction/Gas Chromatography/Mass Spectrometry for Potential Use in Toxicological Studies

A simple, rapid, and solvent-free method for quantitative determination of benzene, toluene, and Xylene in exposed Drosophila larvae was developed using headspace solid-phase microextraction (HS-SPME) coupled to GC/MS. Larvae fed on standard Drosophila food mixed with benzene, toluene, and Xylene for 48 h were homogenized in Milli-Q water. Extraction of benzene, toluene, and Xylene was performed at 65C for 30 min on the SPME fiber (silica-fused). Subsequently, the fiber was desorbed in the GC injection port, followed by GC/MS analysis in the selected-ion monitoring mode. An external calibration curve was used for the quantification of benzene, toluene, and Xylene in the exposed organism. Recoveries were in the range of 78-82% (intraday) and 76-81% (interday) in larvae, and 9196 (intraday) and 87-92% (interday) in the diet. LOD with an S/N of 3:1 and LOQ with an S/N of 10:1 were in the range of 0.010.023 and 0.0340.077 µg/L, respectively. Percent RSD values for benzene, toluene, and Xylene were in the range of 0.500.81 (intraday) and 0.891.23 (interday) for retention time, and 2.163.85 (intraday) and 2.994.95 (interday) for peak concentration, showing good repeatability. This method was sensitive enough to quantitate benzene, toluene, and Xylene in small exposed organisms like Drosophila larvae. The SPME/GC/MS method developed may have wider applications in various in vivo toxicological studies.

Quantification of Urinary Albumin by Using Protein Cleavage and LC-MS/MS

Background: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). Methods: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, 15N-labeled recombinant HSA (15NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides 42LVNEVTEFAK51 and 526QTALVELVK534 from albumin and 15NrHSA were referenced to the calibration curve. Results: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13–200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%–11.0% for the peptide 42LVNEVTEFAK51, and 1.9%–12.3% for the 526QTALVELVK534 peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%–14.8% for the 42LVNEVTEFAK51 peptide, and 6.4%–14.1% for the 526QTALVELVK534 peptide. For the 42LVNEVTEFAK51 peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R2 value of 0.97 and a regression line of y = 0.99x + 23.16. Conclusions: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a 15NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Simultaneous Quantification of Prostate-specific Antigen and Human Glandular Kallikrein 2 mRNA in Blood Samples from Patients with Prostate Cancer and Benign Disease

Background: Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease. Methods: PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1–pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 102–106 mRNA copies. Results: PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P &lt;0.0001, Mann–Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (≤5) were negative for both PSA and hK2 (P = 0.02, Mann–Whitney U-test). Conclusions: The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.

Headspace Gas Chromatographic Determination of Residual 1,3-Butadiene in Rubber-Modified Plastics and Its Migration from Plastic Containers into Selected Foods

A headspace gas chromatographic procedure has been developed for the determination of 1,3-butadiene in rubber-modified plastics and in some foods. Polymer solutions or foods are equilibrated in sealed vials at 90°C, and headspace samples are injected into a gas chromatograph. 1,3-Butadiene residues are measured using a flame ionization detector and are quantitated by the method of standard additions or an external calibration curve. Refrigerator tubs, vegetable oil bottles, chewing gum, and foods in contact with this type of packaging were analyzed. Limits of quantitation varied with the matrix, ranging from 2 ng/g (ppb) in chewing gum to 20 ng/g in polymers. 1,3-Butadiene was found in one polymer at 53 ng/g with an 8% coefficient of variation. The procedure yields "apparent" trace levels of 1,3-butadiene, and confirmation by a complementary technique is required.