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Concept: Detection limit

166

The most common technique used to detect ochratoxin A (OTA) in food matrices is based on extraction, clean-up, and chromatography detection. Different clean-up cartridges, such as immunoaffinity columns (IAC), molecular imprinting polymers (MIP), Mycosep™ 229, Mycospin™, and Oasis® HLB (Hydrophilic Lipophilic balance) as solid phase extraction were tested to optimize the purification for red wine, beer, roasted coffee and chili. Recovery, reproducibility, reproducibility, limit of detection (LOD) and limit of quantification (LOQ) were calculated for each clean-up method. IAC demonstrated to be suitable for OTA analysis in wine and beer with recovery rate >90%, as well as Mycosep™ for wine and chili. On the contrary, MIP columns were the most appropriate to clean up coffee. A total of 120 samples (30 wines, 30 beers, 30 roasted coffee, 30 chili) marketed in Italy were analyzed, by applying the developed clean-up methods. Twenty-seven out of 120 samples analyzed (22.7%: two wines, five beers, eight coffees, and 12 chili) resulted positive to OTA. A higher incidence of OTA was found in chili (40.0%) more than wine (6.6%), beers (16.6%) and coffee (26.6%). Moreover, OTA concentration in chili was the highest detected, reaching 47.8 µg/kg. Furthermore, three samples (2.5%), two wines and one chili, exceeded the European threshold.

Concepts: Oenology, Polyphenol, Harshad number, Alcoholic beverage, Detection limit, Measurement, Wine, Analytical chemistry

38

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

Concepts: Detection limit, Genotyping, DNA, Quantitative research, Biopsy, Single-nucleotide polymorphism, Measurement, Organ transplant

28

Nine recently described Aspergillus species and four known species in section Versicolores were tested for their ability to produce sterigmatocystin on two liquid media, Czapek w/20 % Sucrose Broth and Yeast Extract Broth grown in the dark for 1 week at 25 °C. Detection and quantification of ST were performed by reversed-phase liquid chromatography coupled with electrospray ionization ion trap mass spectrometry. Limit of detection was 3 ng/mL and limit of quantification was 10 ng/mL. Nine newly described Aspergillus species from various substrates, A. amoenus, A. creber, A. cvjetkovicii, A. fructus, A. jensenii, A. puulaauensis, A. subversicolor, A. tennesseensis and A. venenatus in section Versicolores were found to produce sterigmatocystin. Production was confirmed in recently collected isolates of A. protuberus and A. versicolor. A. austroafricanus and A. tabacinus did not produce sterigmatocystin.

Concepts: Chromatography, Detection limit, Taylor cone, Quadrupole ion trap, Electrospray ionization, Ion source, Mass spectrometry, Analytical chemistry

27

Abstract Background: The increasing importance of anti-Müllerian hormone (AMH) for the assessment of ovarian reserve requires accurate AMH measurements. There have been conflicting results about the reliability of currently existing manual AMH assays. Methods: Development of a high sensitive, fast and fully automated AMH assay on the Elecsys®/cobas e electrochemiluminescence immunoassay platform. Results: Elecsys® AMH is a monoclonal two-site assay used to measure AMH in 50 µL of serum or lithium heparin plasma in about 18 min. Its measuring range is from 0.01 to 23 ng/mL. The assay detects primarily 140 kDa total AMH (proAMH and AMHN,C). Standardization was against the Beckman AMH Gen II. Recovery against the highly cited Immunotech calibration was about 90%. Within-run imprecision coefficient of variation (CV) calculated on 10 serum samples were between 0.5% and 1.8%. Repeatability and intermediate precision calculated on 14 serum samples ranged from 2.6% to 1.7% and 2.9% to 2.1%, respectively. Limit of detection (LoD) [limit of quantitation (LoQ)] was 0.01 ng/mL (0.03 ng/mL). Percent recovery in dilution studies was <10% and after mixing of high and low AMH pools 94% to 103%. Elecsys® AMH, when compared to revised AMH Gen II (or Ansh Labs ultrasensitive AMH ELISA) using 57 female serum samples yielded a correlation coefficient of 0.98 (0.97) and a slope of 0.81 (0.73). There was no evidence for sample instability or variability. Samples stored at 20-25°C or 2-8°C up to 7 days showed no significant storage issues. Freeze-thaw cycles or sample storage at -20°C and -80°C for up to 9 months was without any effect on measured AMH. Conclusions: Availability of an automated Elecsys® AMH assay offers an attractive alternative to the current manual AMH assays.

Concepts: ELISPOT, Analytical chemistry, Metrology, Immunoassay, Assay, ELISA, Detection limit, Measurement

27

A sensitive and accurate method based on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of eight illegal synthetic dyes (Sudan (I-IV), Para Red, Rhodamine B, Chrysoidin and Auramine O) in chili products. A simple sample treatment procedure entailing the use of an extraction step with acetonitrile/H2O (9/1) without further cleanup was developed. HPLC was performed on a C18 column using a multistep gradient elution with 5mM ammonium acetate (pH 3.0 with formic acid) and methanol as the mobile phase. Mass spectral acquisition was done in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). Linear calibrations were obtained with correlation coefficients R(2)>0.99. Limit of detection (LOD) and limit of quantification (LOQ) for the studied dyes were in the ranges of 0.05-0.6μgkg(-1) and 0.3-3.0μgkg(-1) depending on matrices, respectively. The recoveries of the eight synthetic dyes in five matrices ranged from 70.5% to 119.2%. The intra- and inter-day precisions (RSDs) were between 2.3-15.8% and 5.7-15.6%, respectively. The applicability of the method to the determination of eight banned dyes in chili products was demonstrated.

Concepts: Detection limit, Rhodamine dyes, Rhodamine, Measurement, Liquid chromatography-mass spectrometry, Mass spectrometry, Analytical chemistry, Acetic acid

27

A simple, selective and stability-indicating high-pressure liquid chromatographic method was developed for the analysis of ribavirin. Chromatographic separation was achieved by using a CPS Hypersil cyano column (4.6 × 250 mm, 5 µm particle size) with isocratic elution of the mobile phase, which was composed of 50 mM phosphate buffer, adjusted at pH 4 with phosphoric acid. The mobile phase was pumped at a flow rate of 0.8 mL/min. The detector was set at 240 nm and quantification of the analyte was based on peak area measurement. The method was validated with respect to linearity, range, precision, accuracy, selectivity, robustness, limit of detection and limit of quantitation. The calibration curve was linear in the range of 5-200 µg/mL with correlation coefficient > 0.999. Ribavirin was subjected to forced degradation studies under two conditions: mild and extensive stress testing. These studies included the effects of hydrolysis (neutral, acidic and alkaline) and oxidation, photolysis and dry heat). The proposed method was proved to be stability-indicating by the resolution of the drug from its forced degradation products, making use of the diode array detector as a tool for confirmation of peak identity and purity. Moreover, the kinetics of alkaline degradation of ribavirin were investigated, an Arrhenius plot was constructed and the activation energy was calculated. The developed method was also extended to analyze ribavirin in capsules and in human plasma with good recovery values.

Concepts: Detection limit, Acid dissociation constant, Phosphoric acid, Acid, Measurement, PH, Chromatography, Analytical chemistry

27

BACKGROUND: The Siemens Immulite hCG assay detects all major hCG variants in serum. Currently, this assay is only FDA approved for qualitative measurement of hCG in urine. METHODS: Complete validation of the hCG assay in urine was performed on the Siemens Immulite 1000 immunoassay platform. Reference intervals were established for females <55 y, females ≥55 y, and males 20 - 70 y. RESULTS: The limit of quantitation was 2.0IU/l. The Immulite hCG assay was precise for measuring hCG in urine from pregnant patients with intra- and inter-assay imprecision of <11% CV. The assay was linear over a dynamic range of 2 - 2600IU/l and 2 - 3500IU/l for hCG and hCGβ respectively. The assay was non-linear for hCGβcf. No hook effect was observed at concentrations up to 1,200,000 pmol/l, for hCGβ or hCGβcf. The reference intervals were <2.0IU/l for males, <2.2IU/l for females <55 y, and <12.2IU/l for females ≥55 y. CONCLUSION: The Immulite 1000 hCG assay can accurately quantify hCG in urine.

Concepts: Qualitative data, Reproduction, Scientific method, Human chorionic gonadotropin, Detection limit, Qualitative research, Measurement

25

In this study, based on the electrostatic integrating radon monitor (EIRM) developed by Iida et al., a new type of EIRM with the allyl glycol carbonate (CR-39) as alpha-particle detector was developed for outdoor radon measurements. Besides using the CR-39 to replace the cellulose nitrate film as alpha-particle detector, the electrode and the setting place of the CR-39 were also optimally designed based on the simulation results of the electric field and the detection efficiency. The calibration factor of the new EIRM was estimated to be 0.136±0.002 tracks cm(-2) (Bq m(-3) h)(-1), with the lower detection limit of 0.6 Bq m(-3) for a 2-month exposure. Furthermore, both the battery and the dry agent were also replaced to protect the environment. The results of intercomparison and field experiments showed that the performances of the new EIRM were much better than the original one. It suggests that the new type of ERIM is more suitable for large-scale and long-term outdoor radon surveys.

Concepts: Measurement, Celluloid, Detection limit, Cotton, Michael Faraday, Natural environment, Cellulose, Nitrocellulose

24

A new reverse phase-liquid chromatography (RP-HPLC) method has been developed for simultaneous determination of entacapone and its pharmacopoeia impurities, in-house impurities and degradation impurities (total 17 analytes). Chromatographic separation was achieved on a C18 column (size: 250 × 4.6 mm; 5 µm particle size) at a flow rate of 1.0 mL/min with 210 nm detection. The mobile phase (MP) consists of 1.361 g of potassium di-hydrogen phosphate and 1.742 g of di-potassium phosphate in 1.0 L water, pH adjusted to 2.5 with ortho phosphoric acid (MP-A) and acetonitrile (MP-B) through gradient elution. The product was subjected to stress conditions such as acid, base, peroxide, thermal and photolytic degradation. Two new impurities above 2% level were observed and isolated through preparative HPLC and well characterized. However, no interference observed due to degradation impurities and entacapone and its EP impurities, in-house impurities. As part of the method validation, specificity, limit of detection, limit of quantitation (LOQ), linearity, accuracy, precision, robustness and ruggedness were determined. LOQ values were achieved between 0.01 and 0.04%. Good linearity (r(2) > 0.99) was obtained ranging from LOQ to 150%. Recovery was verified for all impurities at concentrations ranging from LOQ to 150%. Hence, a newly developed RP-HPLC method was capable for well separation of all analytes with acceptable resolution and tailing factor.

Concepts: Acid, Phosphoric acid, Gas chromatography, High performance liquid chromatography, Measurement, Detection limit, Chromatography, Analytical chemistry

24

Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids, among these are nornicotine, anatabine and anabasine. We developed and applied a 96 well plate-based capillary LC-tandem mass spectrometry method for the analysis of nornicotine, anatabine and anabasine in urine. The method was validated with regard to accuracy and precision. Anabasine was quantifiable to low levels with a limit of quantitation (LOQ) of 0.2 ng/ml even when nicotine, which is isobaric, is present at concentrations >2500-fold higher than anabasine. This attribute of the method is important since anatabine and anabasine in urine have been proposed as biomarkers of tobacco use for individuals using nicotine replacement therapies. In the present study, we analyzed the three minor tobacco alkaloids in urine from 827 smokers with a wide range of tobacco exposures. Nornicotine (LOQ 0.6 ng/ml) was detected in all samples and anatabine (LOQ, 0.15 ng/ml) and anabasine were detected in 97.7% of the samples. The median urinary concentrations of nornicotine, anatabine and anabasine were 98.9, 4.02 and 5.53 ng/ml. Total nicotine equivalents (TNE) were well correlated with anatabine (r(2) = 0.714) and anabasine (r(2) = 0.760). TNE was most highly correlated with nornicotine, which is also a metabolite of nicotine. Urine samples from a subset of subjects (n = 110) were analyzed for the presence of glucuronide conjugates by quantifying any increase in anatabine and anabasine concentrations after β-glucuronidase treatment. The median ratio of the glucuronidated to free anatabine was 0.74 (range, 0.1 to 10.9) and the median ratio of glucuronidated to free anabasine was 0.3 (range, 0.1 to 2.9). To our knowledge this is the largest population of smokers for whom the urinary concentrations of these three tobacco alkaloids has been reported.

Concepts: Measurement, Detection limit, Alkaloids, Anabasine, Tobacco, Analytical chemistry, Alkaloid, Nicotine