Quantum Cascade Laser Based Infrared Spectroscopy: A New Paradigm for Protein Secondary Structure Measurement.

Mid-infrared spectroscopy is one of the major analytical techniques employed for measurements of protein structure in solution. Traditional Fourier Transform-Infrared (FT-IR) measurement is limited by its blackbody light source that is inherently spatially incoherent and has low optical power output. This limitation is pronounced when working with proteins in aqueous solutions. Strong absorbance of water in protein amide I region 1600-1700 cm-1 restricts light path length to <10 µm and imposes significant experimental challenges in sample and flow cell handling. Emerging laser spectroscopic techniques use high-power coherent laser as light source that overcomes the limitation in FT-IR measurement. In this study, we employed an innovative infrared spectrometer that uses quantum cascade laser (QCL) as light source. Continuous infrared radiation from this laser source can be swiftly swept within the amide I region (1600-1700 cm-1) and amide II region (1500-1600 cm-1), which makes this technique ideal for protein secondary structure study. Protein solutions as low as 0.5 mg/mL were measured rapidly without any sample preparation. Infrared spectra of model proteins were thus collected, and a chemometric model based on partial least squares regression was developed to quantify α-helix and ß-strand motifs in protein secondary structure. The model was applied to measurement of the native secondary structure of commercial therapeutic proteins and bovine serum albumin (BSA) and in thermal degradation studies.

Clean Chemistry for Elemental Impurities Analysis of Pharmaceuticals in Compliance with USP 232.

United States Pharmacopeia updated its 100 years old metal analysis method with inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). These sensitive instruments require that sample preparation be at least as sophisticated as the instrumentation used in the analysis. Sample contamination during sample preparation has to be controlled to an acceptable level given the low detection limit of these instruments and the ubiquitous presence of elements. This article focused on sample contamination during sample preparation. Contaminations from environment, reagents, and lab apparatus were investigated for their impact on trace element analysis. Advice on clean lab practice was offered to the pharmaceutical industry in regard to contamination control in elemental analysis labs at a time when the industry is preparing for compliance with elemental impurities in drug products.

Measurement of Moisture Content in Pharmaceutical Tablets by Handheld Near-Infrared Spectrometer: Adopting Quality by Design Approach to Analytical Method Lifecycle Management.

Control of moisture content in pharmaceutical solids (raw materials and solid dosage forms) is a challenge to pharmaceutical development and manufacturing. Pharmaceutical solids come in several forms and presentations requiring different, and often lengthy, sample preparation methods for moisture determination. Rapid screening of samples for their moisture content calls for an analytical method that can provide in-situ measurement with no or minimal sample preparation. We presented a near-infrared (NIR) spectroscopic method for rapid and non-destructive measurement of moisture content in a pharmaceutical tablet product. A handheld NIR spectrometer was selected for the quantitative measurement because of its ease of use, low cost, and high signals selective to water absorption in the NIR spectral range. Analytical quality by design (QbD) principles were explored during method design, qualification, and continued performance verification to increase robustness and promote continuous improvement of the analytical procedure. The International Council for harmonization (ICH) Q2 validation criteria were followed for validation of its linearity, range, accuracy, repeatability, intermediate precision, and method robustness. Limit of detection and limit of quantitation were also estimated based on the multivariate nature of the method. Practical considerations were also given to method transfer and a lifecycle approach to implementation of the method.

[Investigating the toxicity characteristics of Nassarius Sp. in Ningbo City].

OBJECTIVE: To study the type, distribution, the growth and decline of toxins in Nassarius Sp. and the source of toxins to acquire the basis for the control of Nassarius Sp. poisonings. METHODS: The toxicity of Nassarius Sp. was detected by mouse bioassay. The saxitoxin (STX), gonyau toxin (GTX), and tetrodo toxin (TTX) were detected by ELISA and HPLC. RESULTS: Sixty-three poisonous Nassarius Sp. were identified in 127 samples collected from long-term monitoring sites. The detection rate was 49.6%. The detection rate of poisonous Nassarius Sp. was different in varies habitats (P < 0.01). The toxicity of Nassarius Sp. reached a peak in 1991 and hit the rock bottom in 1988. The rate of carrying toxins and the toxicity of detected toxins (STX, GTX and TTX) in Nassarius semiplicatus, Nassa succincta A. Adams and Niotha livescens were high. CONCLUSION: The toxins of Nassarius Sp. in Ningbo City were composed of TTX, STX or GTX, or both TTX and STX. The surveillance proved that some toxins present in Nassarius Sp. in Ningbo City. Poisoning could be caused by eating Nassarius Sp.

Limits of recognition for binary and ternary vapor mixtures determined with multitransducer arrays.

The discrimination of simple vapor mixtures from their components with polymer-coated multitransducer (MT) arrays as a function of the absolute and relative concentrations of those components is explored. The data set consists of calibrated responses to 11 organic vapors from arrays of 5 or 8 microsensors culled from a group of 5 cantilever, 5 capacitor, and 5 calorimeter transducers coated with 1 of 5 different sorptive-polymer films. Monte Carlo methods are applied to simulate error-enhanced composite responses to all possible binary and ternary mixtures of the 11 vapors, and principal component regression models are established for estimating expected rates of recognition as a function of mixture composition. The limit of recognition (LOR), defined as the maximum recognizable mixture composition range, is used as the metric of performance. With the optimal 8-sensor MT array, 19 binary and 3 ternary mixtures could be identified (i.e., discriminated from their components) with <5% error. The binary-mixture LORs are shown to decrease with increases in the baseline noise levels and random sensitivity variations of the sensors, as well as the similarity of the vapors. Importantly, most of the binary LOR contours are significantly asymmetric with respect to composition, and none of the mixtures could be recognized with <5% error at component relative concentration ratios exceeding 20:1. Discrimination of ternary mixtures from their components and binary subcomponent mixtures is possible only if the relative concentration ratio between any two of the components is <5:1. In comparing binary LORs for the best five-sensor single-transducer (ST) array to those of the best five-sensor MT array, the latter were larger in nearly all cases. The implications of these results are considered in the context of using such arrays as detectors in microanalytical systems with upstream chromatographic modules.

Investigation of spectral interferences on the accuracy of broadband CW-NIRS tissue SO(2) determination.

An accurate SO(2) prediction method for using broadband continuous-wave diffuse reflectance near infrared (NIR) spectroscopy is proposed. The method fitted the NIR spectra to a Taylor expansion attenuation model, and used the simulated annealing method to initialize the nonlinear least squares fit. This paper investigated the effect of potential spectral interferences that are likely to be encountered in clinical use, on SO(2) prediction accuracy. The factors include the concentration of hemoglobin in blood, the volume of blood and volume of water in the tissue under the sensor, reduced scattering coefficient, µ(s)', of the muscle, fat thickness and the source-detector spacing. The SO(2) prediction method was evaluated on simulated muscle spectra as well as on dual-dye phantoms which simulate the absorbance of oxygenated and deoxygenated hemoglobin.

[Epidemiological and etiological characteristics of typhoid and paratyphoid fever in Ningbo during 1988 - 2007].

OBJECTIVE: To study the epidemiological and etiological characteristics of typhoid and paratyphoid fever in high epidemic areas. METHODS: Reported data on typhoid and paratyphoid fever during 1988-2007 in Ningbo were analyzed epidemiologically. Shellfish from the market was collected for laboratory testing and Salmonella typhi strains collected from the patients were also studied. RESULTS: Number of reported cases on both typhoid and paratyphoid fever was 19 404 with 7 deaths, from 1988 to 2007. The annual mean incidence was 17.68 per one hundred thousand with the fatality rate as 0.36 per thousand. Most cases were among adults aged 20-50 years and an obvious regional distribution was observed with high incidence seen in winter and spring. Since 1990s, the advantage strain had changed from Salmonella typhi to Salmonella paratyphi A. Etiologic studies showed that raw Anadara subcrenata and oyster were the main risk factors. One Salmonella paratyphi A strain was detected in both Anadara subcrenata and oysters collected from the market, which contained TEM-1 drug resistance gene. PFGE genotyping showed that PFGE-X2 was the strain which causing pandemic in Ningbo. CONCLUSION: Eating contaminated raw shellfish like oysters and hairy clams was the primary risk factor, responsible for the outbreaks. Salmonella paratyphi A was the advantages pandemic strain in Ningbo. Strategies as supervision on personal hygiene and health education should be strengthened.

Evaluation of multitransducer arrays for the determination of organic vapor mixtures.

A study of vapor recognition and quantification by polymer-coated multitransducer (MT) arrays is described. The primary data set consists of experimentally derived sensitivities for 11 organic vapors obtained from 15 microsensors comprising five cantilever, capacitor, and calorimeter devices coated with five different sorptive-polymer films. These are used in Monte Carlo simulations coupled with principal component regression models to assess expected performance. Recognition rates for individual vapors and for vapor mixtures of up to four components are estimated for single-transducer (ST) arrays of up to five sensors and MT arrays of up to 15 sensors. Recognition rates are not significantly improved by including more than five sensors in an MT array for any specific analysis, regardless of difficulty. Optimal MT arrays consistently outperform optimal ST arrays of similar size, and with judiciously selected 5-sensor MT arrays, one-third of all possible ternary vapor mixtures are reliably discriminated from their individual components and binary component mixtures, whereas none are reliably determined with any of the ST arrays. Quaternary mixtures could not be analyzed effectively with any of the arrays. A "universal" MT array consisting of eight sensors is defined, which provides the best possible performance for all analytical scenarios. Accurate quantification is predicted for correctly identified vapors.

Beta-haemolytic group A streptococci emm75 carrying altered pyrogenic exotoxin A linked to scarlet fever in adults.

OBJECTIVE: To determine the etiological cause of a food-borne outbreak of scarlet fever in adults. METHODS: Swabs from the throats of the patients and asymptomatic control were cultured on blood agar plates individually. Biochemical identification of all isolates was performed with a VITEX automated system. Antibiotic susceptibility was examined by using the Kirby-Bauer disc diffusion method. emm gene and extracellular pyrogenic exotoxins of each isolate were amplified by using polymerase chain reaction and subjected to DNA sequencing. Sequence differences between the isolated and the highly similar reference sequences were compared on BLAST. Bioinformatics was used to predict protein structures. RESULTS: Beta-haemolytic group A streptococci (GAS) emm75 were identified from 10 of 13 available patients. The isolates were susceptible to penicillin, ampicillin, vancomycin, cefatriaxone, ofloxacin, linezolid and quinupristin. All of the isolates carried pyrogenic exotoxin A (speA) and cysteine protease (speB). Isolated speA was phylogenetically different from 30 highly similar references on BLAST. Differences in the primary sequence of the deduced protein were 14.37-20.12% between the speA and each of 11 references. Secondary protein structure of the speA was different from the references at the N-terminal. CONCLUSIONS: GAS emm75 encoding altered speA was responsible for the food-borne outbreak of scarlet fever in adults.

[Analysis of beta-lactams-resistance genes in Pseudomonas aeruginosa in burn ward].

OBJECTIVE: To investigate the resistance genes and antibiotic resistance patterns against beta-lactams in Pseudomonas aeruginosa prevalent in burn ward. METHODS: K-B method was performed to test bacterial resistance patterns against 9 species of beta-lactams in Pseudomonas aeruginosa isolated from wounds and dressings of the patient in burn wards. Seven species of resistance genes against beta-lactams were detected with PCR. Tazobactam-inhibited piperacillin resistance test was performed to study whether the above strains produce extended spectrum beta-lactams. RESULTS: All 12 strains of bacteria with resistance genes detected were resistant to penicillin and cephalosporins (100%), among them 11 were resistant to all antibiotics. Tazobactam-inhibited piperacillin resistance test demonstrated that all strains with resistance genes were ESBLs. CONCLUSION: High incidence of beta-lactams resistance genes is found in Pseudomonas aeruginosa isolated from burn ward, and they have close relationship with the occurrence of multiple drug-resistance.

Chamber evaluation of a portable GC with tunable retention and microsensor-array detection for indoor air quality monitoring.

The evaluation of a novel prototype instrument designed for on-site determinations of VOC mixtures found in indoor working environments is described. The instrument contains a miniature multi-stage preconcentrator, a dual-column separation module with pressure-tunable retention capabilities, and an integrated array of three polymer-coated surface acoustic wave sensors. It was challenged with dynamic test-atmospheres of a set of 15 common indoor air contaminants at parts-per-billion concentrations within a stainless-steel chamber under a range of conditions. Vapours were reliably identified at a known level of confidence by combining column retention times with sensor-array response patterns and applying a multivariate statistical test of pattern fidelity for the chromatographically resolved vapours. Estimates of vapour concentrations fell within 7% on average of those determined by EPA Method TO-17, and limits of detection ranged from 0.2 to 28 ppb at 25 degrees C for 1 L samples collected and analyzed in <12 min. No significant humidity effects were observed (0-90% RH). Increasing the chamber temperature from 25 to 30 degrees C reduced the retention times of the more volatile analytes which, in turn, demanded alterations in the scheduling of column-junction-point pressure (flow) modulations performed during the analysis. Reductions in sensor sensitivities with increasing temperature were predictable and similar among the sensors in the array such that most response patterns were not altered significantly. Short-term fluctuations in concentration were accurately tracked by the instrument. Results indicate that this type of instrument could provide routine, semi-autonomous, near-real-time, multi-vapour monitoring in support of efforts to assess air quality in office environments.