Glow flow ionization mass spectrometry of small molecules. A comparison of a glow flow ionization source ('GlowFlow') with electrospray ionization and atmospheric pressure chemical ionization.

RATIONALE: Ionization by atmospheric pressure gas discharge has been employed for a long time in mass spectrometry. Inductively coupled plasma mass spectrometry is an exemplar, and widely used for elemental analysis. The technique has less uptake in organic mass spectrometry. We describe a simple source design that can be readily implemented in most atmospheric pressure ionization (API) systems and compare its performance with that of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). METHODS: An in-house designed helium gas discharge source (referred to as 'GlowFlow') was used on a Xevo G2-S time-of-flight mass spectrometer. The GlowFlow source was transferred to a compatible Xevo TQ-S triple-quadrupole mass spectrometer using an ultrahigh-performance liquid chromatograph inlet. Its performance was compared to that of Waters ESI and APCI sources. RESULTS: Preliminary results of GlowFlow on the Swansea instrument are presented to establish context and include analysis of low-molecular-mass polymers, benzoic acid and cinnamic acid. Comparison of performance on the Xevo TQ-S triple-quadrupole mass spectrometer involved three test mixtures. The method limits of detection (six-mix) for positive-ion GlowFlow source were between 0.03 and 10.00 pg with good linear response over two to four orders of magnitude and values of R2 > 0.98. The GlowFlow ionization source provided a signal intensity that was an order of magnitude greater than that of ESI for an atmospheric pressure gas chromatography standard mix and ionized several compounds that ESI could not. CONCLUSIONS: The current GlowFlow design is relatively simple to retrofit to most API systems due to its small size. The sensitivity of the GlowFlow design is typically an order of magnitude less than that of ESI in positive-ion mode, but similar in sensitivity in negative-ion mode and comparable to that of APCI.

Convolutional Neural Network for Second Metacarpal Radiographic Osteoporosis Screening.

PURPOSE: Osteoporosis and osteopenia are extremely common and can lead to fragility fractures. The purpose of this study was to determine whether a computer learning system could classify whether a hand radiograph demonstrated osteoporosis based on the second metacarpal cortical percentage. METHODS: We used the second metacarpal cortical percentage as the osteoporosis predictor. A total of 4,000 posteroanterior (PA) radiographs of the hand were standardized through laterality correction, vertical alignment correction, segmentation, proxy osteoporosis predictor, and full pipeline. Laterality was classified using a LeNet convolutional neural network (CNN). Vertical alignment classification used 2,000 PA x-rays to determine vertical alignment of the second metacarpal. We employed segmentation to determine which pixels belong to the second metacarpal from 1,000 PA x-rays using the FSN-8 CNN. The full pipeline was tested on 265 previously unseen PA x-rays. RESULTS: Laterality classification accuracy was 99.62%, with a specificity of 100% and sensitivity of 99.3%. Rotation of the hand within 10° of vertical was accurate in 93.2% of films. Segmentation was 94.8% accurate. Proxy osteoporosis predictor was 88.4% accurate. Full pipeline accuracy was 93.9%. In the testing data set, the CNN had a sensitivity of 82.4% and specificity of 95.7%. In the balanced data set, 6 of 39 osteoporotic films were classified as nonosteoporotic; sensitivity was 82.4% and specificity, 94.3%. CONCLUSIONS: We have created a series of CNN that can accurately identify osteoporosis from non-osteoporosis. Furthermore, our CNN is able to make adjustments to images based on laterality and vertical alignment. CLINICAL RELEVANCE: Convolutional neural network and computer learning can be used as an adjunct to dual-energy x-ray absorptiometry scans or to screen and make appropriate referrals for further workup in patients with suspected osteoporosis.

Acoustic Mist Ionization Platform for Direct and Contactless Ultrahigh-Throughput Mass Spectrometry Analysis of Liquid Samples.

Mass spectrometry (MS) has many advantages as a quantitative detection technology for applications within drug discovery. However, current methods of liquid sample introduction to a detector are slow and limit the use of mass spectrometry for kinetic and high-throughput applications. We present the development of an acoustic mist ionization (AMI) interface capable of contactless nanoliter-scale "infusion" of up to three individual samples per second into the mass detector. Installing simple plate handling automation allowed us to reach a throughput of 100 000 samples per day on a single mass spectrometer. We applied AMI-MS to identify inhibitors of a human histone deacetylase from AstraZeneca's collection of 2 million small molecules and measured their half-maximal inhibitory concentration. The speed, sensitivity, simplicity, robustness, and consumption of nanoliter volumes of sample suggest that this technology will have a major impact across many areas of basic and applied research.

Decreased Bacterial Adherence, Biofilm Formation, and Tissue Reactivity of Barbed Monofilament Suture in an In Vivo Contaminated Wound Model.

BACKGROUND: Monofilament and barbed monofilament sutures have been shown in in vitro models to have less bacterial adherence than braided suture. This study evaluates bacterial adherence to suture materials and tissue reactivity with an in vivo contaminated wound mouse model. METHODS: Staphylococcus aureus was used to create an in vivo contaminated wound model at 2 amounts (106 colony-forming units [CFU] and 108 CFU) using a mouse air pouch. Three types of commonly used absorbable suture were evaluated: braided, monofilament, and barbed monofilament. Bacterial suture adherence was evaluated with suture culture, a photon-capturing camera system, and scanning electron microscopy. Tissue reactivity was assessed through histology and protein expression. RESULTS: The braided suture group with the high amount of S aureus exhibited frank purulence and air pouch hypertrophy in all 8 mice. A significant difference was found between suture groups inoculated with 108 CFU (P < .05) as measured by bacterial culture concentration using the optical density method. The braided suture hosted more bacteria than either monofilament (P < .005) or barbed monofilament suture (P < .005). No difference was appreciated between the monofilament and barbed monofilament groups. Kruskal-Wallis test demonstrated a significant difference between groups in regard to levels of tumor necrosis factor-α (P < .05) and interleukin-1 (P < .05). CONCLUSION: Our in vivo contaminated wound model demonstrated that barbed monofilament suture performed similarly to monofilament suture and better than braided suture in terms of bacterial adherence, biofilm formation, and tissue reactivity.

Intracompartmental Pressure Monitoring Using a Handheld Pressure Monitoring System.

INTRODUCTION: We describe the correct technique for measuring compartment pressure with a handheld device to diagnose compartment syndrome. STEP 1 DEVICE PREPARATION: Proper preparation of the handheld pressure monitoring device (Stryker Surgical, Kalamazoo, Michigan) is critical to ensure that the device performs appropriately. STEP 2 IDENTIFICATION OF THE COMPARTMENT OF INTEREST: The needle must be placed in the proper location to appropriately measure a compartment's pressure. STEP 3 INJECTION: Inject saline solution from the pressure monitoring device to clear any soft tissue from the side port on the needle that could result in inaccurate pressure measurements. STEP 4 STABILIZATION AND PRESSURE READING: The pressure must reach a stable state before it is recorded; different pressure thresholds for decompression have been recommended in the literature. STEP 5 REPEAT MEASUREMENTS: As mistakes can be made with any single measurement, accuracy may be improved by repeating steps 1 through 4 and averaging the results. STEP 6 ADDITIONAL COMPARTMENTS: After the reading is obtained, move on to any additional compartment(s) that need to be evaluated, repeating the steps listed above. RESULTS: The handheld intracompartmental monitoring device with a side-ported needle has been shown to be extremely accurate in the laboratory.IndicationsContraindicationsPitfalls & Challenges.

Mice lacking pten in osteoblasts have improved intramembranous and late endochondral fracture healing.

The failure of an osseous fracture to heal (development of a non-union) is a common and debilitating clinical problem. Mice lacking the tumor suppressor Pten in osteoblasts have dramatic and progressive increases in bone volume and density throughout life. Since fracture healing is a recapitulation of bone development, we investigated the process of fracture healing in mice lacking Pten in osteoblasts (Ocn-cre(tg/+;)Pten(flox/flox) ). Mid-diaphyseal femoral fractures induced in wild-type and Ocn-cre(tg/+;)Pten(flox/flox) mice were studied via micro-computed tomography (µCT) scans, biomechanical testing, histological and histomorphometric analysis, and protein expression analysis. Ocn-cre(tg/+;)Pten(flox/flox) mice had significantly stiffer and stronger intact bones relative to controls in all cohorts. They also had significantly stiffer healing bones at day 28 post-fracture (PF) and significantly stronger healing bones at days 14, 21, and 28 PF. At day 7 PF, the proximal and distal ends of the Pten mutant calluses were more ossified. By day 28 PF, Pten mutants had larger and more mineralized calluses. Pten mutants had improved intramembranous bone formation during healing originating from the periosteum. They also had improved endochondral bone formation later in the healing process, after mature osteoblasts are present in the callus. Our results indicate that the inhibition of Pten can improve fracture healing and that the local or short-term use of commercially available Pten-inhibiting agents may have clinical application for enhancing fracture healing.

Multi-center evaluation of a commercial Kit for tacrolimus determination by LC/MS/MS.

OBJECTIVES: A multi-center evaluation (3 sites) of the LC/MS/MS MassTrak tacrolimus Immunosuppressants Kit (Kit) was undertaken. DESIGN AND METHODS: Ten aspects of the analytical performance of the Kit were investigated based on FDA and CLSI guidelines. RESULTS: The linear analytical range of the procedure was between 0.68 and 31.7ng/mL. Within-run and total imprecision were <6% and <8% (n=240), respectively. Recoveries of tacrolimus added to clinical samples that contained between 5 and 10ng/mL of tacrolimus before addition were 99, 102 and 105% at 5.0, 10 and 20ng/mL, respectively. Comparison of in-house and Kit procedures in samples from liver (n=50-58) or kidney (n=50 or 51) transplant recipients yielded method mean biases between -2.0 and +10.7% at 5 and 15ng/mL. CONCLUSIONS: This evaluation indicates that the Kit is suitable for the monitoring of tacrolimus in kidney and liver transplant recipients.

Plasma free metanephrine measurement using automated online solid-phase extraction HPLC tandem mass spectrometry.

BACKGROUND: Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range. METHODS: We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode. RESULTS: Total run-time including sample cleanup was 8 min. Intra- and interassay analytical variation (CV) varied from 2.0% to 4.7% and 1.6% to 13.5%, respectively, whereas biological intra- and interday variation ranged from 9.4% to 45.0% and 8.4% to 23.2%. Linearity in the 0 to 20 nmol/L calibration range was excellent (R(2) > 0.99). For all compounds, recoveries ranged from 74.5% to 99.6%, and detection limits were <0.10 nmol/L. Reference intervals for 120 healthy adults were 0.07 to 0.33 nmol/L (MN), 0.23 to 1.07 nmol/L (NMN), and <0.17 nmol/L (3-methoxytyramine). CONCLUSIONS: This automated high-throughput XLC-MS/MS method for the measurement of plasma free MNs is precise and linear, with short analysis time and low variable costs. The method is attractive for routine diagnosis of pheochromocytoma because of its high analytical sensitivity, the analytical power of MS/MS, and the high diagnostic accuracy of free MNs.

Simultaneous analysis of gamma-hydroxybutyric acid and its precursors in urine using liquid chromatography-tandem mass spectrometry.

We have developed a rapid method that enables the simultaneous analysis of gamma-hydroxybutyrate (GHB) and its precursors, i.e. gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in urine. The method comprised a simple dilution of the urine sample, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation was achieved using an Atlantis dC18 column, eluted with a mixture of formic acid and methanol. The method was linear from 1-80 mg/L for GHB and 1,4-BD and from 1-50 mg/L for GBL. The limit of quantification was 1 mg/L for all analytes. The procedure, which has a total analysis time (including sample preparation) of less than 12 min, was fully validated and applied to the analysis of 182 authentic urine samples; the results were correlated with a previously published GC-MS procedure and revealed a low prevalence of GHB-positive samples. Since no commercial immunoassay is available for the routine screening of GHB, this simple and rapid method should prove useful to meet the current increased demand for the measurement of GHB and its precursors.

Simultaneous and rapid analysis of cyclosporin A and creatinine in finger prick blood samples using liquid chromatography tandem mass spectrometry and its application in C2 monitoring.

A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of cyclosporin A (CsA) and creatinine using capillary blood has been developed. Venous and capillary blood samples were taken predose and at C2 from 65 heart and lung transplant recipients (65 x 4 samples). For comparisons, serum creatinine and blood CsA concentrations were measured by the Jaffe and EMIT methods, respectively, using an Olympus AU600 analyzer. For the LC-MS/MS assay, samples were prepared in a 96 x 700-microL well block by adding 10 microL of blood (or serum) to 40 microL of 0.1 mol/L zinc sulphate solution containing deuterated creatinine internal standard. Proteins were precipitated by adding 100 microL acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 5 microL of the supernatant was injected into the LC-MS/MS system. A Waters 2795 high-performance liquid chromatography (HPLC) system was used to elute a C18 cartridge (3 mm x 4 mm) at 0.6 mL/min with a step gradient of 50-100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. The column was maintained at 55 degrees C, and the retention times were creatinine, 0.4 minutes; ascomycin, 0.98 minutes; and CsA, 1.2 minutes. Cycle time was 2.5 minutes, injection to injection. The analytes were monitored using a Quattro microtandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: creatinine, m/z 114>44; d3-creatinine (IS), m/z 117>47; ascomycin (IS), m/z 809>756; and CsA, m/z 1,220>1,203. Assay characteristics were CsA intraassay CV, 3.6-3.0% (33-1,500 microg/L); CsA interassay CV, 6.7-2.5% (10-5,000 microg/L); LC-MS/MS capillary [CsA] = 0.99 x LC-MS/MS venous [CsA] - 4.2, R = 0.98; and LC-MS/MS venous [CsA] = 0.93 x EMIT venous [CsA] + 2.9, R = 0.98. Creatinine intraassay CV, 6.6-2.5% (20-720 micromol/L); interassay CV, 5.7-3.3% (80-590 micromol/L); LC-MS/MS capillary [creatinine] = 0.99 Jaffe plasma [creatinine] -42.6, R = 0.87. Total time for the preparation and analysis of 30 samples was approximately 2 hours. This assay will provide a flexible, robust, and cost-effective solution for the monitoring of CsA and creatinine in transplant recipients with potential applications in pediatric medicine and pharmacokinetic studies, in which frequent sampling is required.

Rapid liquid chromatography-tandem mass spectrometry method for routine analysis of cyclosporin A over an extended concentration range.

BACKGROUND: Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical range. The consequence of this is that low CsA concentrations that may be clinically significant are difficult to measure and that high concentrations require sample dilution, which introduces error and increases cost. More specific assays, such as HPLC, do not have the required turnaround times for busy transplant clinics. METHODS: CsA was measured in whole blood from 180 cardiac and lung transplant recipients by a liquid chromatography--tandem mass spectrometry (MS) assay, and the results were compared with the Dade Behring Emit assay. Proteins were precipitated with acetonitrile containing ascomycin as internal standard. We used isocratic elution on a Supelco CN column (33 x 3.0 mm; 3- microm bead size) with a mobile phase of 65% aqueous acetonitrile containing ammonium acetate (2 mmol/L) and formic acid (1 g/L), at a flow rate of 0.5 mL/min, with a sample injection volume of 6 microL. We used positive-ion electrospray MS to monitor the ammonium adducts of the compounds of interest decomposing under controlled conditions to the most dominant fragments of the individual molecules. Calibration curves used linear least-squares regression with 1/x weighting. RESULTS: Maximum sensitivity was obtained by monitoring fragmentation of the ammonium adducts m/z 1220-->m/z 1203 for CsA and m/z 809-->m/z 765 for ascomycin. Sample throughput, including preparation time, was 30 samples in 1.5 h with an injection-to-injection cycle time of 1.5 min. The calibration curve was linear to 5000 microg/L, with a detection limit of 0.03 microg/L and a limit of quantification of 1 microg/L. Regression analysis [tandem MS method (y) and Emit assay (x)] yielded a slope of 1.09 (+/- 0.03), an intercept of 6.2 (+/- 4.5) microg/L, and S(y/x) = 27 microg/L. CONCLUSIONS: Tandem MS assay is a realistic alternative to immunoassay for the routine monitoring of CsA in transplant recipients. Its wide dynamic range has utility for pharmacokinetic studies of CsA.

Reconstructive surgery for the last 20 years

Os avanços e refinamentos nas técnicas de reconstruçäo em cirurgia de cabeça e pescoço têm sido marcantes nos últimos 20 anos. As técnicas atuais reduziram drasticamente o período de hospitalizaçäo e morbidez e melhoraram os resultados cosméticos e funcionais. Os princípios básicos de recontruçäo ensinados pelos nossos antecessores näo mudaram, mas a miríade de opçöes de reconstruçäo disponíveis na atualidade necessita se amplamente conhecida, para que o cirurgiäo possa empregar a técnica mais apropriada para cada situaçäo em particular