Quantitative analysis of hindered amine light stabilizers by CZE with UV detection and quadrupole TOF mass spectrometric detection

The current work describes the development of a CZE method with quadrupole QTOF‐MS detection and UV detection for the quantitation of Cyasorb 3529, a common hindered amine light stabilizer (HALS), in polymer materials. Analysis of real polymer samples revealed that the oligomer composition of Cyasorb 3529 changes during processing, a fact hampering the development of a straightforward method for quantitation based on calibration with a Cyasorb 3529 standard. To overcome this obstacle in‐depth investigations of the oligomer composition of this HALS using QTOF‐MS and QTOF‐MS/MS had to be performed whereby 22 new oligomer structures, in addition to the ten structures already described, were identified. Finally, a CZE method for quantitative analysis of this HALS was developed starting with a comprehensive characterization of a Cyasorb 3529 standard using CZE‐QTOF‐MS, subsequently allowing the correct assignment of most Cyasorb 3529 oligomers in an electropherogram with UV detection. Employing the latter detection technique and hexamethyl‐melamine as internal standard, peak areas obtained for the melamine could be correlated with those from the triazine ring, the UV‐absorbing unit present in the HALS. This approach finally allowed proper quantitation of the single oligomers of Cyasorb 3529, an imperative for the quantitative assessment of this HALS in real polymer samples.     

Determination of low-level residual ethylene oxide by using solid-phase microextraction and gas chromatography

Current methods of analysis for ethylene oxide (EO) in medical devices include headspace and simulated-use extractions followed by gas chromatography with either a packed or a capillary column. The quantitation limits are about 0.5-1.0 microg/g for a packed column and about 0.1-0.2 microg/g for a capillary column. The current allowable levels of EO on medical devices sterilized with EO gas as outlined in International Organization for Standardization (ISO) 10993-7 may be significantly reduced from current levels by applying the ISO Draft International Standard 10993-17 method for establishing allowable limits. This may require EO test methods with detection and quantitation limits that are much lower than those of the currently available methods. This paper describes a new method that was developed for the determination of low-level EO by solid-phase microextraction using the direct-immersion method. Factors such as temperature and stirring were found to affect absorption efficiency and absorption time. A low extraction temperature (about 6 degrees C) was found to be more efficient than room-temperature extraction. Stirring was found to reduce absorption time by about 50%. Under these conditions, detection and quantitation limits of 0.002 and 0.009 microg/g, respectively, were obtained by using a capillary column. As a result, this method makes compliance with lower EO limits feasible.     

Analysis of methoxypyrazines in wine using headspace solid phase microextraction with isotope dilution and comprehensive two-dimensional gas chromatography

This study reports an optimized headspace-solid phase microextraction (HS-SPME) method for the determination of methoxypyrazines in wine. Analysis was performed by using comprehensive two-dimensional gas chromatography with novel detection capabilities, including nitrogen phosphorus detection (GC x GC-NPD) and time-of-flight mass spectrometry (GC x GC-TOFMS). In the latter, stable isotope dilution was performed for the quantitation of 2-methoxy-3-(2-methylpropyl) pyrazine (IBMP), using labelled 2-(2H3)methoxy-3-(2-methylpropyl)pyrazine (d3-IBMP) as the internal standard, and resolution of the two analogues was facilitated using the deconvolution capabilities of the TOFMS. This research represents the first report of HS-SPME with isotope dilution and GC x GC-TOFMS (GC x GC-IDTOFMS). Analysis by GC x GC-NPD enabled detection limits of 0.5 ng/L for the quantitation of IBMP, which was superior to that obtained using GC x GC-IDTOFMS (1.95 ng/L). Nevertheless, both methods were adequately sensitive for real wine analysis, yielding highly comparable IBMP concentrations of 26.1 and 27.8 ng/L, respectively, from a Sauvignon blanc wine. The complexity of the real wine headspace was simplified as a result of selective detection using GC x GC-NPD and, in the case of GC x GC-IDTOFMS, the use of extracted ion chromatograms (EICs).     

Use of a capillary electrophoresis instrument with laser-induced fluorescence detection for DNA quantitation. Comparison of YO-PRO-1 and PicoGreen assays

Highly selective and sensitive assays are required for detection and quantitation of the small masses of DNA typically encountered in clinical and forensic settings. High detection sensitivity is achieved using fluorescent labeling dyes and detection techniques such as spectrofluorometers, microplate readers and cytometers. This work describes the use of a laser-induced fluorescence (LIF) detector in conjunction with a commercial capillary electrophoresis instrument for DNA quantitation. PicoGreen and YO-PRO-1, two fluorescent DNA labeling dyes, were used to assess the potential of the system for routine DNA analysis. Linearity, reproducibility, sensitivity, limits of detection and quantitation, and sample stability were examined for the two assays. The LIF detector response was found to be linear (R2 > 0.999) and reproducible (RSD < 9%) in both cases. The PicoGreen assay displayed lower limits of detection and quantitation (20 pg and 60 pg, respectively) than the YO-PRO-1 assay (60 pg and 260 pg, respectively). Although a small variation in fluorescence was observed for the DNA/dye complexes over time, quantitation was not significantly affected and the solutions were found to be relatively stable for 80 min. The advantages of the technique include a 4- to 40-fold reduction in the volume of sample required compared to traditional assays, a 2- to 20-fold reduction in the volume of reagents consumed, fast and automated analysis, and low cost (no specific instrumentation required).     

磷脂组学研究中的分析检测技术

磷脂是细胞膜的主要组分,有非常重要的生理功能,与人类健康、疾病密切相关。因此,有必要对生物体内磷脂化合物进行全面的分析,深入研究磷脂在疾病发生发展过程中的生物功能。磷脂组学是通过全面的定性与定量分析磷脂化合物解决现有生物学问题的研究模式。磷脂分析检测技术是实现磷脂组学研究的关键,包括样品前处理、分离、定性与定量分析和组学数据挖掘等。本文综述了磷脂组学研究中的分析检测技术,为发展快速、稳定、可靠、高灵敏、高分辨、高通量的磷脂检测平台技术提供参考。磷脂组学与其他组学的整合研究将促进生命分析化学的进一步发展。     

Quantitation of Cu+‐catalyzed Decomposition of S‐Nitrosoglutathione Using Saville and Electrochemical Detection: a Pronounced Effect of Glutathione and Copper Concentrations

S‐nitrosothiols (RSNOs) are composed of nitric oxide (NO) bound to the sulfhydryl group of amino acids of peptides or proteins. There is a great interest for their quantitation in biological fluids as they have a crucial impact on physiological and pathophysiological events. Most analytical methodologies for quantitation of RSNOs are based on their decomposition followed by the detection of the released NO. In order to obtain the optimal sensitivity for each detection method, the total decomposition of RSNOs is highly desired. The decomposition of RSNOs can be obtained by using catalytically active metal ions, such as Cu⁺, obtained from CuSO₄ in presence of a reducing agent such as glutathione (GSH) that is naturally present in biological environment. In this work, we have re‐investigated the decomposition of S‐nitrosoglutathione (GSNO) which is the most abundant in vivo low molecular weight RSNO, with a special emphasis on the effect of CuSO₄, GSH, and GSNO concentrations and of their ratio. To this aim, GSNO decomposition optimization was performed by both indirect (Griess assay) and direct (real time electrochemical detection of NO at NO‐microsensor) quantitation methods. Our results show that the ratio between CuSO₄, GSH and GSNO should be adjusted to tune the highest decomposition rate of GSNO and the most efficient electrochemical detection of released NO; also it shows the deleterious effect of very high GSH concentration on the detection of GSNO.     

Investigation of diazo‐derivatization of bisphenol A and its applicability for quantitation in food safety inspections using high‐performance liquid chromatography

Bisphenol A (BPA) is a common additive in food packaging materials, and many methods have been discovered to limit BPA for the sake of food safety. This study aims to find a more sensitive quantification method for BPA. It was determined that a diazo derivative of BPA could be obtained when reacted with a stable and inexpensive reagent, p‐methylaniline, exhibiting an increased response in the ultraviolet spectrum than that of nonreacted BPA. Based on this discovery, an accurate method of BPA quantitation was established using a precolumn derivatization HPLC method. The results of this study showed the low limit of detection and the limit of quantitation for BPA were 3.6 and 10.9 pg/mL on‐column, respectively. This method is proven to have much higher sensitivity and a lower limit of detection than direct HPLC determination methods. This method is robust, easily handled, sensitive and cost‐effective for the quantitation of BPA. In addition, the broad application of this method to determine BPA content in plastic bottled drinking water, distilled water, tap water, milk, Sprite and grape juice was demonstrated in this paper.     

Stir bar sorptive extraction of volatile compounds in vinegar: validation study and comparison with solid phase microextraction

Stir bar sorptive extraction was evaluated for analysing volatiles in vinegar. The procedure developed shows detection and quantitation limits, and linear ranges adequate for analysing this type of compounds. The accuracy obtained was close to 100%, with repeatability values lower than 13%. The extraction efficiency is inversely affected by the acetic acid content. Although the absolute areas decrease, the compound area/internal standard area ratio remains constant, so for quantitative analysis, the acetic acid concentration does not affect the analytical data. The method was compared with a previous SPME method. Similar performance characteristics were obtained for both methodologies, with lower detection and quantitation limits and better repeatability reproducibility values for SBSE. Both analytical methods were used to analyse a variety of vinegars. The results obtained from both methods were in agreement.     

Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps

The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events.     

A study of the relation between the limit of detection and the limit of quantitation in inductively coupled plasma spectrochemistry

The limit of quantitation based on a repeatability threshold concept is compared with the limit of detection in induction coupled plasma atomic emission spectrometry (ICP-AES) and induction coupled plasma mass spectrometry (ICP-MS). A 5%-based limit of quantitation would normally correspond to 10 times the 3-σ based limit of detection. However, because of a possible lack of linearity of the calibration graph at low concentrations, some additional noise not taken into consideration and the possible use of time-correlated multichannel detection, this ratio of 10 cannot be used in every case. It is suggested that a suitable way of determining the limit of quantitation is to establish the plot of the percentage relative standard deviation (RSD) of the net signal as a function of the concentration in a range from the limit of detection to 50 times this limit.     

Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody. Upfront, on-chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult-to-detect deamidation and glycation events at the intact protein level. In addition, on-chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on-chip electrospray tip and directly identified by in-line icIEF-MS using a connected quadrupole time-of-flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C-terminal lysine, N-terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi-attribute characterization within a 15-min run time.     

High-performance liquid chromatography coupled with chemiluminescence nitrogen detection for the study of ethoxyquin antioxidant and related organic bases

The Chemiluminescent Nitrogen Detector (CLND) for use with high-performance liquid chromatography (HPLC) allows for the low-level detection of nitrogen-containing compounds with simple quantitation. The nitrogen selective detector's equimolar response (i.e., equal response for nitrogen independent of its chemical environment) allows for any nitrogen-containing compound to be quantitated as long as the number of nitrogens are known. The HPLC-CLND provides a new detection method for analytes that are not available in large quantities or have unknown chemical or physical characteristics such as oxidation products, metabolites, or impurities. Ethoxyquin is a primary antioxidant that is used to preserve many food products and animal feeds. HPLC-CLND is used in the study of the oxidation products of ethoxyquin because limited quantities of these compounds are available and subsequent calibration curves are difficult to maintain. HPLC-CLND as a new method of detection has been evaluated for its equimolarity of response, linear range, limit of detection, and limit of quantitation.     

Analysis of protein phosphorylation by mass spectrometry

Phosphorylation is one of the most frequently occurring post-translational modifications in proteins. In eukaryotic cells, protein phosphorylation on serine, threonine and tyrosine residues plays a crucial role as a modulator of protein function. A comprehensive analysis of protein phosphorylation involves the identification of the phosphoproteins, the exact localization of the residues that are phosphorylated and the quantitation of phosphorylation. In this short review we will summarize and discuss the methodologies currently available for the analysis and full characterization of phosphoproteins with special attention at mass spectrometry-based techniques. In particular, we will discuss affinity-based purification of phosphopeptides coupled to MALDI-TOF analysis, their detection using mass mapping and precursor ion scan, identification of modified sites by MS/MS and quantitation analysis     

A new method for the determination of biogenic amines in cheese by LC with evaporative light scattering detector

This paper presents a new LC method with evaporative light scattering detection (ELSD), for the separation and determination of the biogenic amines (histamine, spermidine, spermine, tyramine, putrescine and β-phenylethylamine) which are commonly present in cheese, as their presence and relative amounts give useful information about freshness, level of maturing, quality of storage and cheese authentication. The LC-ELSD method is validated by comparison of the results with those obtained through LC-UV determination, based on a pre-column dansyl chloride derivatisation step. The obtained data demonstrate that both methods can be interchangeably used for biogenic amines determination in cheese. The new LC-ELSD method shows good precision and permits to achieve, for standard solutions, limit of detection (LOD) values ranging from 1.4 to 3.6 mg L⁻¹ and limit of quantitation (LOQ) values ranging from 3.6 to 9.3 mg L⁻¹. The whole methodology, comprehensive of the homogenization-extraction process and LC-ELSD analysis, has been applied in the analysis of a typical Calabria (Southern Italy) POD cheese, known as Caciocavallo Silano. The most aboundant amine found was histamine, followed, in decreasing order, by tyramine, spermine, putrescine, β-phenylethylamine and spermidine, for a total amount of 127 mg kg⁻¹. This value does not represent a possible risk for consumer health, according to the toxicity levels reported in literature and regarded as acceptable.     

Simultaneous measurements of signal and background in inductively coupled plasma atomic emission spectrometry: effects on precision,limit of detection and limit of quantitation

Use of simultaneous measurements of the line intensity and the off-peak background intensity results in a significant improvement of repeatability at low concentrations, the limit of detection and the limit of quantitation in inductively coupled plasma atomic emission spectrometry. Simultaneous measurements have been performed using an echellebased dispersive system equipped with a custom segmented-array charge coupled device detector. Correlation between background signals, and shape of the curve of the RSD of the net signal as a function of the concentration is studied from both a theoretical and practical aspect. Concepts of limit of detection and limit of quantitation are discussed both in terms of RSD of the net signal and correlation. Results have been obtained using the Ba II 455 nm line in the visible region and the Ni II 231 nm line in the UV region. Improvements in the limit of detection and limit of quantitation are up to a factor of 10 and are in good agreement with theoretical values. The main limitation of the procedure is related to the accuracy of the background correction.     

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have been shown to be present in blood by transfusion experiments, but based on the amount of infectivity found in these types of experiments, the amount of misfolded prion protein in blood is estimated to be only 30 to 625 amol/mL. More sensitive detection of prions in brain would allow earlier detection of disease and assure a safer food supply. We studied quantitation of the prion protein by use of nanoscale liquid chromatography coupled to a tandem mass spectrometer using the multiple reaction monitoring mode of operation. We developed a method based on the detection of VVEQMCTTQYQK obtained by reduction, alkylation, and digestion with trypsin of the prion protein. Detection of VVEQMCTTQYQK was more sensitive than for the derivative with phenylisothiocyanate (PITC) because of decreased ionization efficiency of the PITC-derivatized peptides. The VVEQMCTTQYQK method has a LOD of 20 to 30 amol for pure standards. Proof of principle is demonstrated by quantitation of the amount of PrP 27-30 in the brains of terminally ill Syrian hamsters.     

An interlaboratory study of quantitation procedures for the analysis of water and wastewater for organo-chlorine pesticides by gas chromatography–electron capture detector

An interlaboratory study was conducted to assess two widely used procedures for estimating quantitation levels. Six laboratories participated in the analysis of artificially prepared water samples for organo-chlorine compounds by liquid-liquid extraction followed by gas chromatography–electron capture detector using USEPA Method 608. The study consisted of three phases, including six months of results from analyte free samples, the replicate analysis of fortified samples at a single concentration by the laboratory, and finally the analysis of blind fortified samples prepared by a third party. Estimated detection and quantitation limits (Currie's L_C and L_Q and USEPA's MDL and ML) were determined for each laboratory-method-analyte combination and then compared to the observed detection and quantitation limits. The overwhelming majority of analyte free samples had a reported value of zero. As a result, observed quantitation and detection limits were frequently zero. When they were not zero, the observed quantitation limits were sometimes less than the observed detection limits and when they were not, there was no observed fixed ratio between the quantitation and detection limits. The variability between days of analysis and the use of noise reducing techniques proved to be a significant source of the observed non-normal distribution of results from distilled water samples with a concentration of zero. Conventional procedures and their underlying analytical and statistical assumptions did not provide useful predictions of laboratory quantitation based upon the results of this study. Rather than one time statistical determinations, ongoing verification of quantitation limits may be a better approach.     

The quality of our drinking water: aluminium determination with an acoustic wave sensor

A new methodology based on an inexpensive aluminium acoustic wave sensor is presented. Although the aluminium sensor has already been reported, and the composition of the selective membrane is known, the low detection limits required for the analysis of drinking water, demanded the inclusion of a preconcentration stage, as well as an optimization of the sensor. The necessary coating amount was established, as well as the best preconcentration protocol, in terms of oxidation of organic matter and aluminium elution from the Chelex-100. The methodology developed with the acoustic wave sensor allowed aluminium quantitation above 0.07 mg L(-1). Several water samples from Portugal were analysed using the acoustic wave sensor, as well as by UV-vis spectrophotometry. Results obtained with both methodologies were not statistically different (alpha=0.05), both in terms of accuracy and precision. This new methodology proved to be adequate for aluminium quantitation in drinking water and showed to be faster and less reagent consuming than the UV spectrophotometric methodology.     

A unique charge-coupled device/xenon arc lamp based imaging system for the accurate detection and quantitation of multicolour fluorescence

In recent years the use of fluorescent dyes in biological applications has dramatically increased. The continual improvement in the capabilities of these fluorescent dyes demands increasingly sensitive detection systems that provide accurate quantitation over a wide linear dynamic range. In the field of proteomics, the detection, quantitation and identification of very low abundance proteins are of extreme importance in understanding cellular processes. Therefore, the instrumentation used to acquire an image of such samples, for spot picking and identification by mass spectrometry, must be sensitive enough to be able, not only, to maximise the sensitivity and dynamic range of the staining dyes but, as importantly, adapt to the ever changing portfolio of fluorescent dyes as they become available. Just as the available fluorescent probes are improving and evolving so are the users application requirements. Therefore, the instrumentation chosen must be flexible to address and adapt to those changing needs. As a result, a highly competitive market for the supply and production of such dyes and the instrumentation for their detection and quantitation have emerged. The instrumentation currently available is based on either laser/photomultiplier tube (PMT) scanning or lamp/charge-coupled device (CCD) based mechanisms. This review briefly discusses the advantages and disadvantages of both System types for fluorescence imaging, gives a technical overview of CCD technology and describes in detail a unique xenon/are lamp CCD based instrument, from PerkinElmer Life Sciences. The Wallac-1442 ARTHUR is unique in its ability to scan both large areas at high resolution and give accurate selectable excitation over the whole of the UV/visible range. It operates by filtering both the excitation and emission wavelengths, providing optimal and accurate measurement and quantitation of virtually any available dye and allows excellent spectral resolution between different fluorophores. This flexibility and excitation accuracy is key to multicolour applications and future adaptation of the instrument to address the application requirements and newly emerging dyes.