Peak Height Sensitivity Research Articles

Developmental Validation of the Microreader 23HS Plex ID System: A Novel Supplementary Non-CODIS STR Multiplex Assay for Forensic Application.

A novel supplementary non-CODIS STR multiplex assay designated as the "Microreader 23HS Plex ID System" was developed. The Microreader 23HS Plex ID System enables simultaneous profiling of 23 STR loci and the amelogenin locus. The majority of these loci are non-CODIS STRs (D4S2408, D9S2157, D20S161, D3S2459, D18S1364, D13S305, D1S2142, D19S400, D6S1017, D7S1517, D2S1776, D2S1360, D3S1744, D16S3391, D3S1545, D11S4463, D20S85, D1S549, D10S2325, D21S2055), with the exception of three CODIS STRs (D2S441, D12S391, and D22S1045). Followed the recommendations of Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese validation standards, a comprehensive set of validation studies were conducted, encompassing PCR conditions, stutter ratio and peak height balance, sensitivity, precision and accuracy, reproducibility, species specificity, inhibition, as well as mixture testing. The results demonstrated that the Microreader 23HS Plex ID System is a reliable and robust assay, with well-balanced peak heights, high precision and accuracy, species specificity, and resistance to common inhibitors. The sensitivity of the assay was determined to be 0.125ng of template DNA. In mixture study, all minor alleles were detected in two-sample mixtures across various ratios (1:19, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 4:1, 9:1, and 19:1). In population study, a total of 500 unrelated individuals of Han ethnicity from East China were genotyped. The allele frequencies and forensic population genetic parameters were calculated, with a cumulative random match probability of 7.757×10-27, and a total power of discrimination exceeding 0.999,999,999,999,999,999,999,999,99. In conclusion, the Microreader 23HS Plex ID System shows promise as a valuable supplementary tool for forensic applications, particularly in addressing complex kinship testing and challenges posed by STR mutation.

Improving HPLC post-column derivatisation assays via In-Column Derivatisation

In column derivatisation (ICD) has been applied to antioxidants in food and agricultural matrices. ICD’s ability to reduce the post-column reaction loop volume is critical to investigate chromatographically before future applications are established. Due to the large number of post-column derivatisation (PCD) assays, this is out of the scope of this study to cover in one article. Hence, in this communication, we strictly focused on implementing ICD and its ability to reduce the post-column volume associated to the PCD’s reaction loop. Specifically, reversed phase (RP) separations where we take three different reaction schemes: (i) amino acids derivatised with ninhydrin, (ii) amino acids derivatised with o-phthalaldehyde (OPA), and (iii) sulfonamides derivatised with fluorescamine to exemplify three different case scenarios. This communication can be used as a practical guide for analysts. In the order of the workflow’s largest reduction in the reaction loop volume: (i) the amino acids derivatised with OPA, the ICD enabled the post-column volume to be reduced significantly from 500 µL down to 80 µL. This drastic reduction resulted in the chromatographic gain in performance – number of theoretical plates (N) (55–167 %) and peak height sensitivity (35–181 %). For the sulfonamides derivatised with fluorescamine, the ICD setup reduced the post-column volume from 500 µL down to 200 µL, and demonstrated significant improvements in efficiency – N (33–324 %), and peak height sensitivity (4–33 %). The amino acids derivatised with ninhydrin, the PCD reaction loop could not be decreased, and ICD demonstrated decreased signal to noise (S/N).

Developmental validation of a novel 8-dye fluorescent typing system with 59 Y-STRs and 3 Y-indels for forensic applications.

An 8-dye fluorescence-labeling forensic Y-chromosomal short tandem repeats (Y-STRs) kit, the 62-plex Y-STR multiplex amplification system, was developed and optimized. The system was validated by testing PCR conditions, stutter ratios (SR) and peak height ratios, sensitivity, mixture samples, precision and accuracy, species-specificity, and inhibition studies according to the Scientific Working Group on DNA Analysis Methods guidelines. PCR-based studies showed that the recommended PCR conditions were optimized for this kit. In the sensitivity study, a full profile was obtained from template DNA with a quantity of u125pg. Consistent profiles were obtained from three different laboratories. The SRs in all loci were less than 15%, and nice balance and suitable average peak height were shown. No peaks were detected in the profiles of common animal species and microorganisms. In the male-male mixture studies, all loci were observed at a ratio of 1:8, and in the male-female mixture study, all alleles could be profiled at a ratio of 1:500 if the male DNA inputs were ≥0.5ng/µL. An inhibitor study demonstrated that the kit had varying degrees of resistance to the presence of common inhibitors. Population study demonstrated the 62-plex Y-STR Kit improved the power of discrimination in unrelated Chinese Han males (n=192). When haplotype diversity was 1, the probability of discrimination power of the 62-plex Y-STR Kit was 0.9948, which is suitable for forensic investigations. The results show that the developed 8-dye fluorescence labeling 62 loci system is sensitive, robust, convenient, and highly informative for forensic applications.

DiveNet: Dive Action Localization and Physical Pose Parameter Extraction for High Performance Training

The tremendous progress of deep convolution neural networks has shown promising results on the classification of various sports activities. However, the accurate localization of a particular sports event or activity in a continuous video stream is still a challenging problem. The accurate detection of sports actions enables the comparison of different performances, objectively. In this work, we propose the DiveNet action localization module to detect the springboard diving sports action in an unconstrained environment. We used Temporal Convolution Network (TCN) over a backbone feature extractor to localize diving actions, with low latency. We estimate the divers center of mass (COM) trajectory and the peak dive height using the temporal demarcations provided by the action localization step via the projectile motion formula. In addition, we train a DiveNet pose regression network, which extends the Unipose architecture with direct physical parameter estimation, i.e COM and 2D joint keypoints. We propose a new homography computation method between the diving motion plane and the image-view for each dive. This enables the representation of physical parameters in metric scale, without any calibration. We release the first publicly available diving sports video dataset, recorded at 60 Hz with a static camera setup for different springboard heights. DiveNet action localization achieves an accuracy of 95% with a single frame latency (< 25 ms). The DiveNet pose regression model shows competitive results around 70% PCK on different diving pose datasets. We achieve COM accuracy of 6 pixels, dive peak height sensitivity of 20 cm and mean joint angle errors around 10 degrees.

Internal validation of the DNAscan/ANDE™ Rapid DNA Analysis™ platform and its associated PowerPlex® 16 high content DNA biochip cassette for use as an expert system with reference buccal swabs

Rapid DNA platforms are fully integrated systems capable of producing and analyzing short tandem repeat (STR) profiles from reference sample buccal swabs in less than two hours. The technology requires minimal user interaction and experience making it possible for high quality profiles to be generated outside an accredited laboratory. The automated production of point of collection reference STR profiles could eliminate the time delay for shipment and analysis of arrestee samples at centralized laboratories. Furthermore, point of collection analysis would allow searching against profiles from unsolved crimes during the normal booking process once the infrastructure to immediately search the Combined DNA Index System (CODIS) database from the booking station is established. The DNAscan/ANDE™ Rapid DNA Analysis™ System developed by Network Biosystems was evaluated for robustness and reliability in the production of high quality reference STR profiles for database enrollment and searching applications. A total of 193 reference samples were assessed for concordance of the CODIS 13 loci. Studies to evaluate contamination, reproducibility, precision, stutter, peak height ratio, noise and sensitivity were also performed. The system proved to be robust, consistent and dependable. Results indicated an overall success rate of 75% for the 13 CODIS core loci and more importantly no incorrect calls were identified. The DNAscan/ANDE™ could be confidently used without human interaction in both laboratory and non-laboratory settings to generate reference profiles.

Broadband Mid-Infrared Stand-Off Reflection-Absorption Spectroscopy Using a Pulsed External Cavity Quantum Cascade Laser.

Broadband mid-infrared molecular spectroscopy is essential for detection and identification of many chemicals and materials. In this report, we present stand-off mid-infrared spectra of 1,3,5-trinitro-1,3,5-triazine or cyclotrimethylene trinitramine (RDX) residues on a stainless-steel surface measured by a broadband external cavity quantum cascade laser (QCL) system. The pulsed QCL is continuously scanned over 800 cm-1 in the molecular fingerprint region and the amplitude of the reflection signal is measured by either a boxcar-averager-based scheme or a lock-in-amplifier-based scheme with 1 MHz and 100 kHz quartz crystal oscillators. The main background noise is due to the laser source instability and is around 0.1% of normalized intensity. The direct absorption spectra have linewidth resolution around 0.1 cm-1 and peak height sensitivity around 10-2 due to baseline interference fringes. Stand-off detection of 5-50 µg/cm2 of RDX trace adsorbed on a stainless steel surface at the distance of 5 m is presented.

Two‐step stacking by sweeping and micelle to solvent stacking using a long‐chain cationic ionic liquid surfactant

Coupling of long-chain ionic liquid (LCIL)-based sweeping and micelle to solvent stacking (MSS) in CZE for anionic compounds was proposed. N-Cetyl-N-methylpyrrolidinium bromide (C16MPYBr) was used as a novel cationic surfactant. The capillary column was conditioned with poly(1-vinyl-3-butylimidazolium) bromide, a kind of polymeric ionic liquid, to obtain the anodic electroosmotic flow (EOF). There is a micellar solution (MS) zone which is prepared with C16MPYBr before the sample zone. The micelles penetrated into the sample zone, swept and transported the analytes toward the micelle to solvent boundary (MSSB). Meanwhile, a sufficient amount of methanol in the background solution (BGS) resulted in the reversal of effective electrophoretic mobility of analytes and completed the MSS. Under optimal conditions, good linearity (0.9988–0.9999) was obtained for model analytes in a wide linear range with limits of detection (LODs) from 0.025 to 0.25 mg/L. The intraday and interday repeatabilities (%RSD, n=5, 10) were acceptable in the range from 2.12 to 7.29%. 34 and 25 times increases in peak area sensitivity for benzoic acid (BA) and 2-nitrophenol (2-NP) and 60 times increase in peak height sensitivity for 4-chlorophenol (4-CP) were obtained. The proposed method is applied to analyze two spiked environmental water samples obtaining satisfactory recoveries.

Two-step stacking in capillary zone electrophoresis featuring sweeping and micelle to solvent stacking: II. Organic anions

Two-step stacking of organic anions by sweeping and micelle to solvent stacking (MSS) using cationic cetyltrimethylammonium micelles in co-electroosmotic flow (co-EOF) capillary zone electrophoresis (CZE) is described. The co-EOF condition where the direction of the EOF is the same as the test anions was satisfied by positive dynamic coating of a fused silica capillary with hexadimethrine bromide. The strategy was as follows. After conditioning the capillary with the background solution (BGS), a micellar solution (MS) was injected before the sample solution (S). The BGS, MS and S have similar conductivities. Voltage was applied at negative polarity. The analytes in the micelle-free S zone were swept by micelles from the MS. The swept analytes were brought by the micelles to the MSS boundary where the second stacking step was induced by the presence of organic solvent in the BGS. Finally was the separation of concentrated analytes by CZE. The effect of electrolyte concentration in the S, injection time of the MS and the S and surfactant concentration in the MS were studied. A 20–29, 17–33 and 18–21 times increase in peak height sensitivity was obtained for the test hypolipidaemic drugs (gemfibrozil, fluvastatin and atorvastatin), non-steroidal anti-inflammatory drugs (diflunisal, naproxen, ketoprofen, indoprofen and indomethacin), and herbicides (mecoprop and fenoprop), respectively. The LODs (S/N=3) were from 0.05 to 0.55μg/mL. The intraday and interday repeatabilities (%RSD, n=12) in terms of retention time, corrected peak area, and peak heights was less than 3.6, 8.9, and 10.8%, respectively. The application of sweeping and MSS in co-EOF CZE together with a simple extraction procedure to a waste water sample spiked with the test herbicides was also demonstrated.

Neutral analyte focusing by micelle collapse in partial‐filling MEKC with UV and ESI‐MS detection

Analyte focusing by micelle collapse is investigated as an on-line sample preconcentration technique in partial-filling MEKC with detection using UV and ESI-MS. Enhancement in peak height sensitivity of more than a 100 was obtained for model alkylphenylketones and dialkylphthalates using analyte focusing by micelle collapse-partial-filling MEKC with UV or ESI-MS detection.

Determination of lead by hydride generation atom trapping flame atomic absorption spectrometry

An atom trapping approach is described for the determination of lead by hydride generation flame atomic absorption spectrometry. Lead hydride (PbH4), generated on-line by reacting lead in hydrochloric acid–potassium ferricyanide medium with sodium borohydride (NaBH4), was trapped on the interior walls of a slotted T-tube under highly oxidizing flame conditions. Atomization was achieved by aspirating 50 µL of methyl isobutyl ketone (MIBK) to the flame. Optimization of the experimental parameters for the generation and collection of lead hydride was described to achieve the highest peak height sensitivity. The detection limits were 0.075, 0.047 and 0.028 µg L−1 for 2.6, 5.2 and 7.8 mL of blank solution (n = 13), respectively. Calibration was linear up to 3.0 µg L−1 for a 30-s trapping period. The relative standard deviation was between 2 and 4%. The method was successfully applied to the determination of Pb in NIST certified reference materials, San Joaquin Soil (SRM 2709) and Apple Leaves (SRM 1515).

Trace anion determination in concentrated hydrofluoric acid solutions by two-dimensional ion chromatography: II. Method performance study with a hydroxide eluent and a low noise suppressor

The two-dimensional ion exclusion chromatography/ion chromatography (ICE–IC) approach is considered to be the method of choice for the determination of trace anions in concentrated hydrofluoric acid (48–50%, w/w). In order to achieve lower detection limits, this method was for the first time used with electrolytically generated and purified hydroxide eluents in combination with a low noise electrochemical suppressor. Compared to carbonate based eluents, the achieved gain in peak height sensitivity for chloride, sulfate, nitrate and phosphate is a factor of 6, 7, 16 and 13, respectively. The instrumental detection limits, based on the background noise, are 2, 0.2, 0.4 and 1.4 μg/kg HF 50% (w/w) for the same anions. Their method detection limits, calculated according to SEMI, are all within the 6–10 μg/kg HF 50% (w/w) range and thus at least 10 times lower than the current Tier C grade requirements. The chromatographic run time could be shortened with some 10 min by the use of a relatively fast high-capacity hydroxide selective anion exchange column.

Determination of lead in dialysis concentrates using flow injection hydride generation atomic absorption spectrometry

Flow injection hydride generation atomic absorption spectrometry (FI-HGAAS) was used for determination of lead in dialysis concentrates. The parameters such as acidity, concentration of oxidising and reducing agents and argon gas flow rate were investigated to reach the best peak height sensitivity. No significant background signal was observed at high salt concentrations. The detection limit, concentration giving a signal equal to three times standard deviation of the blank signal, was 0.7 ng ml −1 for a 500 μl injection volume. Precision of the measurements at the 20 ng ml −1 level was 3.7% R.S.D. The dialysis concentrates analysed by FI-HGAAS were found to have 10–70 ng ml −1 of lead. The same samples were analysed by ETAAS after removing the matrix using solid phase extraction with Chelex 100. The results were in agreement with those obtained by FI-HGAAS.

Improved resolution using symmetrically shifted pulses.

An approach to Hadamard phase encode the two halves of the F(1) dimension of a gHSQC experiment is presented. The phase encoding is achieved by excitation sculpting of the F(1) dimension using symmetrically shifted pulses. This approach (IMPRESS-improved resolution using symmetrically shifted pulses) increases the resolution of the F(1) dimension by exploiting spectral folding, but the folding is coded in the fashion of a Hadamard H(2) matrix. Editing of the IMPRESS spectra during processing sorts out spectral crowding which is a typical consequence of F(1) spectral folding. It is shown that for the same total experiment time, the IMPRESS-gHSQC experiment provides narrower peaks along the F(1) dimension compared to the normal gHSQC experiment. As a consequence of decreased linewidth, the peak height (sensitivity) is also increased.

Enhancement of Signal-to-Noise Ratios in Capillary Gas Chromatography by Using a Longitudinally Modulated Cryogenic System

A Longitudinally Modulated Cryogenic System (LMCS) was evaluated for its use in detection enhancement in capillary gas chromatography. The mechanism for chromatographic re-elution for the LMCS is substantially different to other cryogenic devices. The cooled region of the capillary column in which chromatographic bands may be focused is heated by the surrounding oven temperature either by moving the trap along the column, or by moving the column out of the trap. By continually modulating the LMCS at the detector end of the capillary column, signal-to-noise ratios of routine chromatograms can be readily increased by a factor of ten, thus enhancing chromatographic detection. Base widths of peaks, which are often about 2–3 s or more can be easily reduced to 0.3 s when the LMCS is employed in the detection enhancement mode, thus offering a simple avenue to improved peak height sensitivity in capillary gas chromatography.

Determination of Phosphorus Content in Steels Using Flow Injection into an Argon-Water Carrier for Inductively Coupled Plasma-Mass Spectrometry.

This paper describes a methodology for the determination of phosphorus in steels using argon-water carrier, in flow injection with Inductively Coupled Plasma Mass Spectrometry. 31P is a monoisotopic element and its determination, at very low levels, is very difficult due to contamination problems and interference inherent in the plasma and solvents. These unduly raise the background being necessary a sample pretreatment step to achieve the required contents. To dissolve the samples in this study, a microwave digestion system was applied to minimize the sample preparation time, the quantities of reagents and the risk of contamination. A mercury cathode electrolysis was used for performing a rapid matrix removal/analyte enrichment. The use of an argon-water stream as carrier yielded an enhancement in sensitivity and the detection limit was improved by a factor of approximately 2.5 when compared with that experienced using flow injection into an all-water carrier. Optimization of the operating conditions for the argon-water flow injection and for Inductively Coupled Plasma Mass Spectrometer, were selected with respect to the peak height sensitivity and were found to be dependent on the nebulizer gas flow-rate, the radio frequency power, the sample injection volume and the carrier flow-rate. The detection limit based on a sample rate of 0.02 gm/-1 was 0.4 μg·g-1 and the relative standard deviations at 100 and 10 μg·g-1 P solution were 2.0 and 15 % respectively. Data are presented for 3 Standard Reference Materials: JSS 003-2, (from Japanese Iron and Steel Certified Reference Materials); and ECRMs 285-1 and 278-1 (from Bureau of Analysed Samples LTD).

The use of nafion dryer tubes for moisture removal in flow injection chemical vapor generation atomic absorption spectrometry

A significant problem with flow injection chemical vapor generation atomic absorption spectrometry is a loss of sensitivity and blockage of transfer lines due to excessive moisture transported to the atom cell or lodged in the transfer line. A Nafion dryer tube was used to remove moisture from the wet carrier gas stream. The hygroscopic Nafion membrane removed water at 1.7 mg/min at an efficiency of 95 ± 4% when SnCl 2 was used as the reductant. When 0.4% (m/v) borohydride was used as the reductant, 2.3 mg/min of water was removed at an efficiency of 91 ± 3%. No measurable change in precision was observed and a 5% reduction in peak height sensitivity (vs. PTFE) was seen for mercury when using the Nafion transfer line. At 60°C the Nafion dryer removed 4.9 mg/min of water vapor at an efficiency of 93 ± 3%. The loss of mercury vapor through the Nafion membrane was no more than 0.04%. Only a 3% reduction in peak height sensitivity was observed for the determination of arsenic and selenium. Detection limits for mercury, calculated from calibration data, were 77 ppt, 20 ppt and 150 ppt for the PTFE tube, the model MD-250 dryer and the model MD-125 dryer, respectively.

Univariate optimization of segmented-flow injection for inductively coupled plasma mass spectrometry

Through optimization of the operating conditions for the segmented air–water carrier, the peak height sensitivity was found to be dependent on the nebulizer gas flow rate and the r.f. power but largely independent of the sample injection volume, the carrier flow rate, and the ratio of air to water in the carrier. Under-optimized operating conditions, performing flow injection (Fl) into the air bubbles of a segmented air–water carrier resulted in similar or improved detection limits for all analytes, except Se, when compared with those obtained using continuous nebulization. This was true even if only 0.1 cm3 injections were made. Changing the segmenting gas from air to Ar, N2 or H2, did not result in appreciable changes in analyte signals or positions of maximum ionization with in the plasma, nor did it improve the tolerance of inductively coupled plasma mass spectrometry to the effect caused by 0.01 and 0.1 mol dm–3 Na compared with that experienced using Fl into an all-water carrier.

Examination of separation efficiencies of mercury vapour for different gas–liquid separators in flow injection cold vapour atomic absorption spectrometry with amalgam preconcentration

A comparison has been made of the separation efficiency of three designs of gas–liquid separator when used in a flow injection (Fl) manifold for the determination of Hg by cold vapour atomic absorption spectrometry. The manifold used with each device was separately optimized for maximum sensitivity. This involved studies of the effects of reagent flow rates, argon purge gas flow rate, injection time and post-injection purge time. A significant difference, with respect to both peak height and integrated signal sensitivity (by a factor of approximately 3) between the performance of a miniature design and that of two larger volume designs was obtained. No significant differences in precisions were observed. For the miniature design, the use of either tetrahydroborate or tin(II) reductant was investigated. No difference in peak height sensitivity was found, but the integrated signal sensitivity for the tetrahydroborate was 36% lower. The efficiency of separation was measured by comparison of the signal obtained from a known mass of Hg vapour, introduced via an amalgam preconcentration unit, and the signal obtained from a known mass of Hg in solution, introduced via the Fl manifold and amalgam preconcentration unit. The efficiencies were found to be 101 ± 4% and 103 ± 6% for peak height and integrated signal, respectively.

The performance of a new laboratory-constructed cathodic-sputtering atomizer in atomic absorption spectrometry

The performance characteristics of a laboratory-constructed cathodic sputtering atomizer for atomic absorption spectrometry have been evaluated. The new atomizer allows for the independent control of argon flow rate and pressure, whereas the only cathodic-sputtering atomizer commercially available for atomic absorption spectrometry (Atomsource, manufactured by Analyte Corporation) does not. Our results follow the model of consecutive, first-order reactions: ▪ where A is the analyte atom that has not yet entered the analysis volume of the atomizer, B is the analyte atom that absorbs and C is the analyte atom that is removed from the analysis volume, and k 1, and k 2 are first order rate constants. The importance of independent control of argon flow rate and pressure is shown by the increase in the k 1 k 2 ratio for Ag for the new atomizer compared to that for the Atomsource. The peak height sensitivity of the new atomizer is up to a factor of 5 better than that of the Atomsource, which does not allow independent control of argon flow rate and pressure. Also, the value of k 2 for the new atomizer has been decreased, the result is a peak area sensitivity for the new atomizer that is 1–2 orders of magnitude better than that of the Atomsource. A parametric evaluation of the new atomizer has shown that most of the improvement in peak area sensitivity results from the increased residence time of the analyte atoms. A comparison of the sensitivity of the new atomizer with the sensitivity of the Atomsource has been made using both peak height and peak area absorbance.

Capacitive-Discharge Circuit for Controlled Heating of Anisotropic Graphite Tubes. Part II: Characterization for Graphite Furnace Atomic Absorption

Performance data from a recently described capacitive-discharge heating system are presented. The capacitive-discharge heating circuit, which used a 0.15 F capacitor bank, is shown to provide reproducible heating of a small anisotropic graphite tube (5 mm o.d. × 9 mm long, 1 mm wall thickness) at heating rates of up to 65°C per ms to a final temperature of 2500°C. The performance and resistance of graphite from two manufacturers are compared. The effect of the capacitor bank voltage on the heating rate and maximum achievable temperature is studied. Photographic and optical pyrometric evidence of the circumferential temperature anisotropy is presented. Pyrolytically coated anisotropic graphite tubes were investigated. Preliminary investigations using Pb indicated that sample deposition on the unbroken lamellar graphitic planes provides about 1.5 times the peak-area and peak-height sensitivity obtained from sample deposition across the lamellar planes.