Direct analysis of melamine in complex matrices using a handheld mass spectrometer † ‡

A low temperature plasma ambient ionization source, coupled to a portable mass spectrometer (Mini 10.5), is used for the determination of melamine contamination in whole milk and related materials. Thermally assisted desorption and ionization of the analyte was achieved with the plasma probe. The small size, low power consumption and capability for direct sampling without pretreatment makes plasma ionization an appropriate ionization method for use with a handheld mass spectrometer. The standard discontinuous atmospheric pressure interface used to connect atmospheric pressure ion sources to mass spectrometers (Gao et al., Anal. Chem., 2008, 80, 4026-4032) was modified by using supplementary pumping to increase the ion transfer efficiency. Whole milk, fish, milk powder and other complex matrices spiked with melamine were placed on glass slides close to the vacuum inlet and analyzed without sample pretreatment. Quantitation in complex matrices was achieved using MS/MS of protonated melamine m/z 127 to yield the characteristic fragment ion of m/z 85. Analysis rates of two samples per minute, levels of melamine as low as 250 ng/mL in whole milk (below the regulatory level in the US of 1 ppm (1 mg/mL) or the European level of 2.5 ppm (mg/mL)), a linear dynamic range of 0.5-50 mg/mL and a relative standard deviation of ca. 7.6-16.2% were achieved. The importance of melamine to public health and the prior lack of a rapid, sensitive and yet highly specific field analysis method add to the relevance of this study. Introduction Melamine, a nitrogen-rich (66.7% by weight) industrial chemical, has been deliberately added into various foods to artificially increase the apparent protein content as judged by total nitrogen measurement (the Kjeldahl nitrogen determination 1 ). In 2007, pet food adulteration with melamine leading to kidney toxicity in cats and dogs was reported, and in September of 2008, melaminecontaminated milk resulted in kidney stones and renal failure in infants. 2-4 Melamine reacts with its metabolite cyanuric acid to form a poorly soluble stable complex which can precipitate in renal tubules and lead to kidney failure. 5 Instrumental methods for melamine analysis in food have been developed rapidly in response to the public health alarm: they include immunoassays, 6 capillary zone electrophoresis with diode array and mass spectrometry detection, 7,8 high performance liquid chromatography coupled with ultraviolet absorption 9,10 and with mass spectrometry, 11-15 gas chromatography/mass spectrometry 10,16 and ultra-performance liquid chromatography with tandem mass spectrometry. 17 However, all these methods require tedious sample preparation, for example, the US Food and Drug administration (FDA) has published a method for screening melamine in pet food using GC/MS, 18 which takes about 3 h for a single detection although by using parallel operations the time per sample can be decreased. The continuing need for rapid screening of melamine has led to the application of several of the new ambient ionization mass spectrometric techniques, specifically low temperature plasma (LTP), Based on a rectilinear ion trap (RIT) mass analyzer, 19 ) These ionization methods allow the direct mass spectrometric analysis of compounds present in condensed phase samples. In a prior short communication, we have reported LTP probe ionization for melamine screening using conventional benchtop mass spectrometers. 20 LTP is a particularly appropriate ionization method for use in conjunction with a miniature mass spectrometer, given the following characteristics: (1) direct sampling occurs without sample pretreatment (except for optional heating), (2) no solvents are used, (3) air can serve as the plasma support gas, (4) low power consumption ($3 W), (5) small size, and (6) rapid analysis. In the present paper, we report the characteristics of the LTP/miniature mass spectrometer combination for the detection and quantification of melamine. A modified discontinuous atmospheric pressure interface (DAPI) 47 was used to increase the ion transfer efficiency into the mass spectrometer. The interface includes a compact version of the LTP probe, a heater which focuses the heat and the plasma onto a 5 mm  5 mm surface area, and supplementary pumping to transfer the ions effectively into the ion trap. The LTP was used to desorb and ionize the analyte from a glass surface with thermal assistance (up to 200 C). Analysis speeds can reach the rate of two samples per minute. Results show that levels of melamine as low as 250 ng/mL (250 ppb) spiked into whole milk can be detected and that the linear dynamic range is 0.4-50 mg/mL. The detection limit is well below the US regulatory level of 1 ppm and the European level of 2.5 ppm. Experimental Chemical and reagents All the foods were randomly bought in local supermarkets and were used directly without further treatment. All chemicals, including melamine, cyanuric acid and methanol, were purchased from Sigma-Aldrich (USA) and used without purification. Synthetic urine was purchased from CST Technologies, Inc. (NY, USA). The deionized water used for preparing standard solutions was obtained using a Milli-Q purification system (Millipore, Bedford, MA, USA). Melamine in methanol and water (v:v 1:1) at a concentration of 1000 mg/mL served as stock solution. Spiked milk samples were made by diluting the melamine solution with whole milk using a dilution ratio of 1:20 (melamine solution:milk). For each measurement, 3 mL of solution was placed on a glass slide giving a sample spot of ca. 2 mm  2 mm, then the glass slide was placed directly under the heated LTP probe to execute the analysis. Fish meat was first ground, then 2 g of the fishpaste was mixed with selected amounts of melamine solution (500 mg/mL in methanol/water) to achieve different concentrations. The fishpaste was vortexed for 20 min to assure homogeneous mixing and allowed to stand for another 20 min before analysis (these steps were used in preparing a sample representative of contaminated fish, they are not needed for analysis). For each analysis, 5 mg fishpaste was used. All samples were prepared and measured at room temperature (15-25 C). LTP ionization source The LTP probe consists of a glass tube (o.d. 6.35 mm and i.d. 3.75 mm) with an axial grounded electrode (stainless steel; diameter, 1.57 mm) and an outer electrode (copper tape) wrapped onto the tube, as reported in a previous study. Miniature mass spectrometer and its ambient interface A previously described handheld rectilinear ion trap mass spectrometer (Mini 10.5) 25 was used for the experiments reported in this paper. To confirm the miniature mass spectrometer results, a parallel study was conducted using an LTQ (Thermo Fisher Scientific, Inc., San Jose, CA, USA). A miniature rough pump together with a miniature turbo pump was used to achieve an ultimate vacuum below 1  10 À5 Torr in the Mini 10.5. A twostage KNF Neuberger diaphragm pump (1091-N84.0-8.99) with a pumping speed of 5 L/min was used to provide a backing pressure below 2 Torr for the turbo pump. The latter was a 10 L/s Pfeiffer TPD 011 (Pfeiffer Vacuum Inc., Nashua, NH, USA) and it constituted the main vacuum pump of the system. All components of the Mini 10.5, including the electronics and vacuum systems, are assembled in an aluminium case, length 34 cm, width 22 cm and height 19 cm. The total weight of the instrument is 10 kg. A discontinuous DAPI 47 interface is used to transfer the ions created by the LTP source into the vacuum chamber of the Mini 10.5 for detection. The discontinuous interface acts as a mechanical switch, which opens the ion introduction channel briefly (10-30 ms) and then closes it during the subsequent periods of each scan cycle (ion cooling, mass analysis, ion clearance and reset). The pressure inside the vacuum chamber increases significantly (up to 10 mTorr) when the channel is open for ion (and accompanying air/sample vapor) introduction. It is appropriate to shut off all high voltages and maintain only a low RF voltage during this period. After ion introduction, the channel is closed to allow the pressure to decrease over a period of time (300-500 ms) until it reaches a value (normally 1 mTorr) that allows further ion manipulation and mass analysis. At this point, the high voltage is turned on and the RF is scanned to perform mass analysis. Each cycle takes around 1 s and all mass spectra were averaged over 3 cycles and are reported with background This journal is ª The Royal Society of Chemistry 2010 View Article Online subtraction. For this study an improved version of the interface (described in the next section) was employed. This optimized interface Results and discussion LTP and Mini 10.5 interface The LTP ambient ionization source can be directly combined with a benchtop mass spectrometer. The interface between LTP and Mini 10.5 was improved further in this study by using an extra pumping system (shown in This journal is ª The Royal Society of Chemistry 2010 Analyst, 2010, 135, 705-711 | 707 View Article Online Neuberger, Trenton, NJ, USA) was used to provide an air flow rate of 2.5 L/min toward the vacuum inlet. Ions created by the LTP were sucked into the quarter inch capillary (c1 in Direct detection of melamine in complex matrices With the LTP/Mini 10.5 described above Direct detection of melamine in milk. Soon after the 2008 melamine scandal, 2-4 several groups developed rapid melamine detection methods for complex samples using benchtop mass spectrometers operated with various ambient ionization methods, including LTP, 19,20 EESI, 21 DART 22 and DAPCI. 23 The first study to detect melamine using a miniature mass spectrometer (Mini 10.5) is presented in the present study. Organic milk spiked with melamine to a concentration of 5 mg/mL (sample dilution and operational details described in the Experimental section) gave a mass spectrum using the LTP/Mini 10.5 from which the presence of melamine could be detected 20 Direct detection of melamine in milk powder. Detection of melamine in milk powder could become a quality control activity in dairy plants. Due to the unique advantages of the LTP source, View Article Online i.e. no direct contact with the sample, low carrier gas flow rate and direct analyte desorption from the sample, the LTP/Mini 10.5 procedure can be used to detect melamine directly in milk powder at a concentration of 5 mg/g Direct detection of melamine in urine. If, unfortunately, the above food safety measures are unsuccessful and melaminecontaminated products do get into the food market, rapid and direct analysis of body fluid (such as urine) is important for clinical diagnosis. Given the complexity of the urine matrix, standard methods such as ESI and APCI are not suited to direct melamine detection. Although matrix assisted laser desorption ionization (MALDI) has recently been used to detect melamine in urine, 48 addition of R-cyano-4-hydroxycinnamic acid is still needed. The methods reported in this paper allow direct detection of melamine in urine using a portable mass spectrometer. As illustrated in Optimization and analytical performance Optimization experiments were conducted to decrease the LOD and increase the linear dynamic range of the LTP/Mini 10.5 combination 20 Similar results were obtained for the LTP/Mini 10.5 system and, based on the data shown in 25 The notch of the waveform for isolation of protonated melamine was set to cover 138-142 kHz corresponding to an isolation window of m/z 127 AE 2. The frequency of the AC for CID was set to 140 kHz to optimize the fragment ion abundance. The amplitudes of the AC for both isolation and activation were optimized and set at 6 V and 0.4 V Under these optimized conditions, the linear dynamic range for determination of melamine in whole milk is between 0.4 and 50 mg/mL with a LOD of 250 ng/mL (see Supplementary Materials ‡). The detection limit is far below the regulatory level in the US of 1 ppm or the European level of 2.5 mg/kg or 2.5 ppm. As noted in the European regulations, ''.the level of 2.5 mg/kg is the appropriate level to distinguish between the unavoidable background presence of melamine and unacceptable adulteration.''. 49 The analytical performance of the LTP/Mini 10.5 for all matrices tested in the present work is summarized i

Progression of coronary artery atherosclerosis in rheumatoid arthritis: comparison with participants from the Multi-Ethnic Study of Atherosclerosis

Introduction: In cross-sectional studies, patients with rheumatoid arthritis (RA) have higher coronary artery calcium (CAC) than controls. However, their rate of progression of CAC and the predictors of CAC progression have heretofore remained unknown. Methods: Incidence and progression of CAC were compared in 155 patients with RA and 835 control participants. The association of demographic characteristics, traditional cardiovascular risk factors, RA disease characteristics and selected inflammatory markers with incidence and progression of CAC were evaluated. Results: The incidence rate of newly detected CAC was 8.2/100 person-years in RA and 7.3/100 person-years in non-RA control subjects [IRR 1.1 (0.7-1.8)]. RA patients who developed newly detectable CAC were older (59±7 vs. 55±6 years old, p=0.03), had higher triglyceride levels (137±86 vs. 97±60 mg/dL, p=0.03), and higher systolic blood pressure (129±17 vs. 117±15 mm Hg, p=0.01) compared to those who did not develop incident CAC. Differences in blood pressure and triglyceride levels remained significant after adjustment for age (p<=0.05). RA patients with any CAC at baseline had a median rate of yearly progression of 21 (7–62) compared to 21 (5–70) Agatston units in controls. No statistical differences between RA progressors and RA non-progressors were observed for inflammatory markers or for RA disease characteristics. Conclusions: The incidence and progression of CAC did not differ between RA and non-RA participants. In patients with RA, incident CAC was associated with older age, higher triglyceride levels, and higher blood pressure, but not with inflammatory markers or RA disease characteristics

Unique and redundant functions of NKp46+ ILC3s in models of intestinal inflammation

Group 3 ILCs (ILC3s) are innate sources of IL-22 and IL-17 and include lymphoid tissue-inducer (LTi)-like and NKp46(+) subsets. Both depend on RORγt and aryl hydrocarbon receptor, but NKp46(+)ILC3s also require Notch and T-bet for their development and are transcriptionally distinct. The extent to which these subsets have unique functions, especially in the context of T cell– and B cell–sufficient mice, remains largely unclear. To investigate the specific function of NKp46(+)ILC3s among other ILC3 subsets and T cells, we generated mice selectively lacking NKp46(+)ILC3s or all ILC3s and crossed them to T cell–deficient mice, thus maintaining B cells in all mice. In mice lacking T cells, NKp46(+)ILC3s were sufficient to promote inflammatory monocyte accumulation in the anti-CD40 innate colitis model through marked production of GM-CSF. In T cell–competent mice, lack of NKp46(+)ILCs had no impact on control of intestinal C. rodentium infection, whereas lack of all ILC3s partially impaired bacterial control. Thus, NKp46(+)ILC3s have a unique capacity to promote inflammation through GM-CSF–induced accumulation of inflammatory monocytes, but are superseded by LTi-like ILC3s and T cells in controlling intestinal bacterial infection

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Large-scale exome-wide association analysis identifies loci for White Blood Cell Traits and Pleiotropy with Immune-Mediated Diseases

White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3 UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake