Suppression of peptide sample losses in autosampler vials.

Protein or peptide sample losses could accompany all steps of the proteomic analysis workflow. We focused on suppression of sample adsorptive losses during sample storage in autosampler vials. We examined suppression capabilities of six different sample injection solutions and seven types of autosampler vial surfaces using a model sample (tryptic digest of six proteins, 1 fmol per protein). While the vial material did not play an essential role, the choice of appropriate composition of sample injection solution reduced adsorptive losses substantially. The combination of a polypropylene vial and solution of poly(ethylene glycol) (PEG) (0.001%) or a mixture of high concentrated urea and thiourea (6 M and 1 M) as injection solutions (both acidified with formic acid (FA) (0.1%)) provided the best results in terms of number of significantly identified peptides (p < 0.05). These conclusions were confirmed by analyses of a real sample with intermediate complexity (in-gel digest from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)). Addition of PEG into the real sample solution proved to prevent higher losses, concerning mainly hydrophobic peptides, during up to 48 h storage in the autosampler in comparison with a formic acid solution and even with a solution of highly concentrated urea and thiourea. Using PEG for several months was not accompanied by any adverse effect to the liquid chromatography system.

High-resolution quantitative metabolome analysis of urine by automated flow injection NMR.

Metabolism is essential to understand human health. To characterize human metabolism, a high-resolution read-out of the metabolic status under various physiological conditions, either in health or disease, is needed. Metabolomics offers an unprecedented approach for generating system-specific biochemical definitions of a human phenotype through the capture of a variety of metabolites in a single measurement. The emergence of large cohorts in clinical studies increases the demand of technologies able to analyze a large number of measurements, in an automated fashion, in the most robust way. NMR is an established metabolomics tool for obtaining metabolic phenotypes. Here, we describe the analysis of NMR-based urinary profiles for metabolic studies, challenged to a large human study (3007 samples). This method includes the acquisition of nuclear Overhauser effect spectroscopy one-dimensional and J-resolved two-dimensional (J-Res-2D) (1)H NMR spectra obtained on a 600 MHz spectrometer, equipped with a 120 µL flow probe, coupled to a flow-injection analysis system, in full automation under the control of a sampler manager. Samples were acquired at a throughput of ~20 (or 40 when J-Res-2D is included) min/sample. The associated technical analysis error over the full series of analysis is 12%, which demonstrates the robustness of the method. With the aim to describe an overall metabolomics workflow, the quantification of 36 metabolites, mainly related to central carbon metabolism and gut microbial host cometabolism, was obtained, as well as multivariate data analysis of the full spectral profiles. The metabolic read-outs generated using our analytical workflow can therefore be considered for further pathway modeling and/or biological interpretation.

Fast and sensitive flow-injection mass spectrometry metabolomics by analyzing sample-specific ion distributions.

Mass spectrometry based metabolomics is a widely used approach in biomedical research. However, current methods coupling mass spectrometry with chromatography are time-consuming and not suitable for high-throughput analysis of thousands of samples. An alternative approach is flow-injection mass spectrometry (FI-MS) in which samples are directly injected to the ionization source. Here, we show that the sensitivity of Orbitrap FI-MS metabolomics methods is limited by ion competition effect. We describe an approach for overcoming this effect by analyzing the distribution of ion m/z values and computationally determining a series of optimal scan ranges. This enables reproducible detection of ~9,000 and ~10,000 m/z features in metabolomics and lipidomics analysis of serum samples, respectively, with a sample scan time of ~15 s and duty time of ~30 s; a ~50% increase versus current spectral-stitching FI-MS. This approach facilitates high-throughput metabolomics for a variety of applications, including biomarker discovery and functional genomics screens.

Critical evaluation of novel dynamic flow-through methods for automatic sequential BCR extraction of trace metals in fly ash.

Two novel dynamic extraction approaches, the so-called sequential injection microcolumn extraction and sequential injection stirred-flow chamber extraction, based on the implementation of a sample-containing container as an external extraction reactor in a sequential injection network, are for the first time, optimized and critically appraised for fractionation assays. The three steps of the original Community Bureau of Reference (BCR) sequential extraction scheme have been performed in both automated dynamic fractionation systems to evaluate the extractability of Cr, Cu, Ni, Pb, and Zn in a standard reference material of coal fly ash (NIST 1633b). In order to find the experimental conditions with the greatest influence on metal leachability in dynamic BCR fractionation, a full-factorial design was applied, in which the solid sample weight (100-500 mg) and the extraction flow rate (3.0-6.0 mL min(-1)) were selected as experimental factors. Identical cumulative extractabilities were found in both sequential injection (SI)-based methods for most of assayed trace elements regardless of the extraction conditions selected, revealing that both dynamic fractionation systems, as opposed to conventional steady-state BCR extraction, are not operationally defined within the selected range of experimental conditions. Besides, the proposed automated SI assemblies offer a significant saving of operational time with respect to classical BCR test, that is, 3.3 h versus 48 h, for complete fractionation with minimum analyst involvement.

Modulation of mobile phase composition in flow-injection/sequential-injection chromatography exploiting multisyringe flow analysis.

In this paper, a time-based multicommutated flow system is proposed for appropriate selection and modulation of mobile phase composition in flow-injection (FI)/sequential-injection (SI) chromatography. The novel flow assembly involves the on-line coupling of a short monolithic reversed-phase chromatographic column with a multisyringe flow injection set-up furnished with a set of solenoid valves. The proposed hyphenated technique was applied to the simultaneous spectrophotometric determination of thiamine (B(1)), pyridoxine (B(6)) and cyanocobalamin (B(12)) which were taken as model analytes. The separation method capitalizes on a dual isocratic elution protocol involving the use of a single forward stroke of the multisyringe pump for initial delivery of 50 mmol L(-1) ammonium acetate (pH 7.0) for 2.4 min followed by 50 mmol L(-1) ammonium acetate-methanol (80:20, v/v) for 6.4 min at 0.5 mL min(-1) and room temperature. Detection was performed at the maximum wavelength for each target vitamin-280 nm for B(1), 325 nm for B(6), and 360 nm for B(12). A first-order, two-level full-factorial design was utilized to ascertain the significant variables influencing the chromatographic separation and the magnitude of the interaction effects. The experimental design method revealed that resolution of the target vitamins is highly dependent on the pH, percentage of organic modifier, and their second-order interaction. The multisyringe flow-injection-based monolithic column separation method, which should be viewed as an expeditious and cost-effective alternative to the high-performance liquid chromatography counterpart, was applied to the separation and determination of B(1), B(6), and B(12) in different pharmaceutical dosage forms in less than 9 min. Statistical comparison of the results from the proposed procedure with those from the HPLC method endorsed by the US Pharmacopeia revealed there were no significant differences at the 95% confidence level.

A protocol for studying the interaction between small-molecular drug and DNA using microdialysis sampling integrated with chemiluminescent detection.

It is of great significance to understand how drug molecules interact with DNA, which is one of the most important aspects of biological investigations in drug discovery at molecular level. Herein, with the model of ractopamine and calf thymus DNA (ct-DNA), a protocol using microdialysis (MD) sampling integrated with flow injection (FI)-chemiluminescent (CL) detection was developed for studying the interaction between small-molecular drug and DNA. After incubating ractopamine with ct-DNA, unbound ractopamine was on-line sampled using a MD probe, followed by being introduced into a FI-CL system for quantitation. The detected concentrations of unbound ractopamine were calibrated with the recovery of the MD probe, and then treated with Klotz analysis and Scatchard analysis to acquire the binding parameters. The MD probe exhibited a mean recovery of 27.3% for ractopamine sampling under the optimal conditions. The binding constants obtained by Klotz analysis and Scatchard analysis were 3.8 × 106 M-1 and 3.9 × 106 M-1, respectively, showing negligible difference. Ractopamine was estimated to have only one type of binding site on ct-DNA. The obtained results demonstrated that the protocol using on-line MD sampling integrated with FI-CL detection is a simple and reliable technique platform for studying the interaction between small-molecular drug and DNA.

On-line recalibration of spectral measurements using metabolite injections and dynamic orthogonal projection.

Spectrometers are enjoying increasing popularity in bioprocess monitoring due to their non-invasiveness and in situ sterilizability. Their on-line applicability and high measurement frequency create an interesting opportunity for process control and optimization tasks. However, building and maintaining a robust calibration model for the on-line estimation of key variables of interest (e.g., concentrations of selected metabolites) is time consuming and costly. One of the main drawbacks of using infrared (IR) spectrometers on-line is that IR spectra are compromised by both long-term drifts and short-term sudden shifts due to instrumental effects or process shifts that might be unseen during calibration. The effect of instrumental drifts can normally be reduced by referencing the measurements against a background solution, but this option is difficult to implement for single-beam instruments due to sterility issues. In this work, in order to maintain the robustness of calibration models for single-beam IR and to increase resistance to process and instrumental drifts, planned spikes of small amounts of analytes were injected periodically into the monitored medium. The corresponding measured difference spectra were scaled-up and used as reference measurements for updating the calibration model in real time based on dynamic orthogonal projection (DOP). Applying this technique led to a noticeable decrease in the standard error of prediction of metabolite concentrations monitored during an anaerobic fermentation of the yeast Saccharomyces cerevisiae.

Photo-induced chemiluminometric determination of Karbutilate in a continuous-flow multicommutation assembly.

The present paper deals with the chemiluminescent determination of the herbicide Karbutilate on the basis of its previous photodegradation by using a low-pressure Hg lamp as UV source in a continuous-flow multicommutation assembly (a solenoid valves set). The pesticide solution was segmented by a solenoid valve and sequentially alternated with segments of the 0.001 mol l(-1) of NaOH solution, the suitable media for the formation of photo-fragments; then it passes through the photo-reactor and was lead to the flow-cell after being divided in small segments which were sequentially alternated with the oxidizing system; 2 x 10(-5) mol l(-1) of potassium permanganate in 0.2% pyrophosphoric acid. The studied calibration range, from 0.1 microg l(-1) to 65 mg l(-1), resulted in a linear behaviour over the range 20 microg l(-1)-20 mg l(-1) and fitting the linear equation: I=(1180+/-30)C+(15+/-5) with the correlation coefficient 0.9998. The limit of detection was 10 microg l(-1) and the sample throughput 17 h(-1). After testing the influence of a large series of potential interfering species, the method was applied to water and human urine samples.

A simple and rapid flow injection chemiluminescence determination of cysteine with Ru(phen)3(2+)-Ce(IV) system.

A new chemiluminescence system was developed for the determination of cysteine by flow injection system. This method is based on the reaction of L-cysteine with Ru(phen)3(2+) and Ce(IV) to produce chemiluminescence. The calibration curve was linear over the range 8.0x10(-7) to 4.0x10(-5) and 4.0x10(-5) to 1.0x10(-3) M with a detection limit of 7.0x10(-7) M (S/N=3). The relative standard deviation of 4.0x10(-6) M cysteine was found 3.5% (n=10). The influence of potential interfering substances was studied. The proposed method was successfully applied for the flow injection determination of cysteine in the real samples with minimum sampling rate of 90 sample/h.

Total polyphenols content in white wines on a microfluidic flow injection analyzer with embedded optical fibers.

Absorbance detection in food microdevices has not been thoroughly used due to low levels of sensitivity in measurements. Thus, it is necessary to develop microfluidic methods for improving photometric detection. For this purpose, a simple coupled-optical-fiber-polydimethylsiloxane (PDMS) microdevice was developed, to quantify polyphenols content in white wine employing the Folin-Ciocalteu reaction method. A 6V and 10W halogen lamp with an optical path length of 7mm between optical fibers, which were placed into the microchip, using guides at the outlet of the flow, increased the level of sensitivity during detection. The linear range was from 0.03mmol/L to 0.18mmol/L. Thus, the corresponding equation was: Abs=4.00(±0.16) [tannic acid]+0.17(±0.017). Intra-laboratory repeatability and reproducibility percentages were 2.95% and 6.84%, respectively. Such results were compared to those obtained from applying the conventional flow-injection analysis method, based on the same type of reaction. The relative error between methods was less than 13%.

On-line monitoring of blend uniformity in continuous drug product manufacturing process--The impact of powder flow rate and the choice of spectrometer: Dispersive vs. FT.

One of the commonly acknowledged issues in continuous manufacturing of drug products is how to provide a representative sampling on flowing powder to assure its blend uniformity. An investigation was conducted to improve understanding on the impact of powder flow rate under different continuous manufacturing conditions and the impact of optical parameters (such as resolution, co-adds, and integration time) on NIR spectral quality with respect to a dispersive and a Fourier transform instrument. A partial least squares (PLS)-based spectral pretreatment was found useful to tackle the impact of different flow rates on NIR spectral signals. Multivariate figures of merit (FOM) were used to evaluate performances across different instruments and optical settings and discover the advantageous selectivity and sensitivity on the Fourier transform than the dispersive instrument regardless of the use of co-adds.

Development of a micro-fluidic manifold for copper monitoring utilising chemiluminescence detection.

The progressive development of a micro-fluidic manifold for the chemiluminescent detection of copper in water samples, based on the measurement of light emitted from the Cu(ii) catalysed oxidation of 1,10-phenanthroline by hydrogen peroxide, is reported. Micro-fluidic manifolds were designed and manufactured from polymethylmethacrylate (PMMA) using three micro-fabrication techniques, namely hot embossing, laser ablation and direct micro-milling. The final laser ablated design incorporated a reagent mixing channel of dimensions 7.3 cm in length and 250 x 250 microm in width and depth (triangular cross section), and a detection channel of 2.1 cm in length and 250 x 250 microm in width and depth (total approx. volume of between 16 to 22 microL). Optimised reagents conditions were found to be 0.07 mM 1,10-phenanthroline, containing 0.10 M cetyltrimethylammonium bromide and 0.075 M sodium hydroxide (reagent 1 delivered at 0.025 mL min(-1)) and 5% hydrogen peroxide (reagent 2 delivered at 0.025 mL min(-1)). The sample stream was mixed with reagent 1 in the mixing channel and subsequently mixed with reagent 2 at the start of the detection channel. The laser ablated manifold was found to give a linear response (R(2) = 0.998) over the concentration ranges 0-150 microg L(-1) and be reproducible (% RSD = 3.4 for five repeat injections of a 75 microg L(-1) std). Detection limits for Cu(ii) were found to be 20 microg L(-1). Selectivity was investigated using a copper selective mini-chelating column, which showed common cations found in drinking waters did not cause interference with the detection of Cu(ii). Finally the optimised system was successfully used for trace Cu(ii) determinations in a standard reference freshwater sample (SRM 1640).

Enzymatic determination of bicarbonate in serum by flow injection analysis.

An automated method for the determination of bicarbonate in human serum based on the enzymatic reaction between the analyte and phospho(enol)pyruvate (PEP) in the presence of PEP carboxylase is proposed. The analytical reaction is coupled with a derivatization reaction in which the NADH consumed is fluorimetrically monitored (lambda ex = 340 nm, lambda em = 460 nm). A stopped-flow/flow-injection approach is used in which the enzymes (PEP carboxylase and malate dehydrogenase) are immobilized on controlled-pore glass. The linear determination range is between 25 and 300 mmol/l (r2 = 0.9973). The %C.V. for the within- and between-run studies, performed at three concentration levels, ranges between 1.0 and 3.6% and the sampling frequency is 20 per h.

Sensitive fluorimetric method based on sequential injection analysis technique used for dissolution studies and quality control of prazosin hydrochloride in tablets.

This report introduces a fully automated flow system for drug-dissolution studies based on the coupling of the sequential injection analysis (SIA) technique with a conventional dissolution apparatus. The methodology described was used for monitoring of dissolution profiles of prazosin hydrochloride (PRH) in pharmaceutical formulation. The very sensitive fluorimetric detection of PRH was performed at lambda(ex)=244 nm (lambda(em)>or=389 nm). Under the optimal conditions, the calibration curve was linear over the range 0.02-2.43 mg x l(-1) of PRH with R.S.D. 1.89, 1.23, and 1.80% (n=10) when determining 0.02, 1.22, and 2.43 mg x l(-1) of PRH in standard solutions, respectively. Equation of the calibration curve was calculated giving the following values: F=4.108 c-3.9 (n=6), r=0.9996. Detection limit was calculated 0.007 mg x l(-1) of PRH. The dissolution test of Deprazolin tablets was programmed for 60 min, with a continuous sampling rate of 70 h(-1) under conditions required by USP 26. Results obtained by SIA technique compared well with HPLC standard method.

Determination of sulfate in natural and residual waters by turbidimetric flow-injection analysis.

A turbidimetric flow-injection system was developed for the determination of sulfate in natural and residual water samples, with no previous treatment, using spectrophotometric detection. The precipitating agent, 7.0% (w/v) barium chloride solution prepared in 0.10% (w/v) polyvinyl alcohol, was added by using the merging-zones approach. A 100 mg/L sulfate solution in 0.07M nitric acid was mixed with the sample before it entered the injection loop to improve the detection limit, provide in-line pH adjustment, and prevent the interference of some anionic species. The relative standard deviations of the results were between 1.4 and 3.0% and were in agreement with results obtained by the reference method. Samples within a linear concentration range of 10-120 mg SO4(2-)/L can be analyzed at a rate of 40/h. The detection limit is 5 mg SO4(2-)/L.

Semiautomatic flow-injection method for determination of volatile acidity in wines.

A flow-injection (FI) method based on analytical pervaporation was assessed for its routine use in the determination of volatile acidity in winery laboratories. The new method was compared with both the official method and the Mathieu method, which is most often used in Spanish wineries, by testing 30 different wines, including young and aged, and sweet and dry wines from Montilla-Moriles appellation d'origine. The robustness of the new method was established, and then all 3 methods were studied in terms of range of linearity and regression of the calibration curve, repeatability, reproducibility, sensitivity, detection and quantitation limits (LOD and LOQ, respectively), and time of analysis. The FI method surpassed the Mathieu method in reproducibility and both the Mathieu and official methods in LOD and LOQ and sensitivity; it also required less personnel involvement and shorter analysis time. The statistical criteria established by the Office International de la Vigne et du Vin were applied to the data and the results obtained indicated that the differences between the analytical parameters of the 3 methods are not significant and can be applied indistinctly. The correlation of the methods was studied by taking them 2 by 2, and the corresponding equations, coefficients, and deviations confirmed the statistical results. Thus, the new method can be used in winery laboratories with clear advantages over its 2 counterparts (the routine and official methods).

Evaluation of flow injection analysis for determination of urea in sheep's and cow's milk.

Difficulties in measuring the urea content in sheep's milk often occur with spectral photometry due to the high protein and fat concentrations of the milk. In this study an enzymatic flow procedure (QuickChem 8000 Ion Analyser, Lachat Instruments, Milwaukee, USA) to determine the urea content in ovine and bovine milk was evaluated. Urea content is determined by the Berthelot reaction after splitting it enzymatically with urease. The free ammonia diffuses through a teflon membrane into a stream of reagent solutions. Detection takes place by means of a reaction between the ammonium ions with hypochlorite and salicylate producing a green colour, which is measured spectrometrically in a flow meter at 660 nm. By using a diffusion cell chemical deproteinisation of milk is not necessary and capacity is high. The assessed procedure exhibited high accuracy and precision and reached a sample capacity of 55 samples an hour. Storage of the milk samples for several days as well as chemical preservation with bronopol had no effect on the measurement procedure. Due to the complexity of the apparatus and the costs associated therewith, the device proves less suitable for routine diagnostics but rather serves as a reference method for the measurement of urea concentration in milk.

Application of hot platinum microelectrodes for determination of flavonoids in flow injection analysis and capillary electrophoresis.

The determination of quercetin and rutin by flow injection analysis (FIA) and capillary electrophoresis (CE) using electrochemical detection was described. These flavonoids were determined at normal (unheated) and hot platinum microelectrodes using cyclic voltammetry. When quercetin or rutin is reaching the platinum electrode, a change of the current in the region of the platinum oxide formation is observed. Integration of the current changes in this in this region creates analytical signals in the form of peaks. An increase of temperature to about 76°C in a small zone adjacent to the microelectrode causes an increase of the analytical signal by more than 6 times under FIA conditions. This method enables the use of hot microelectrodes as detectors in HPLC or CE. In CE the improvement of the analytical signal at hot microelectrodes is smaller than in FIA and increase only 1.3-3.4 times. Heated microelectrodes were used for analysis of the flavonoids in natural samples of the plant (extract of sea buckthorn) and a pharmaceutical preparation (Cerutin).

Determination of acetaldehyde in saliva by gas-diffusion flow injection analysis.

The consumption of ethanol is known to increase the likelihood of oral cancer. In addition, there has been a growing concern about possible association between long term use of ethanol-containing mouthwashes and oral cancer. Acetaldehyde, known to be a carcinogen, is the first metabolite of ethanol and it can be produced in the oral cavity after consumption or exposure to ethanol. This paper reports on the development of a gas-diffusion flow injection method for the online determination of salivary acetaldehyde by its colour reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and ferric chloride. Acetaldehyde samples and standards (80 µL) were injected into the donor stream containing NaCl from which acetaldehyde diffused through the hydrophobic Teflon membrane of the gas-diffusion cell into the acceptor stream containing the two reagents mentioned above. The resultant intense green coloured dye was monitored spectrophotometrically at 600 nm. Under the optimum working conditions the method is characterized by a sampling rate of 9h(-1), a linear calibration range of 0.5-15 mg L(-1) (absorbance=5.40×10(-2) [acetaldehyde, mg L(-1)], R(2)=0.998), a relative standard deviation (RSD) of 1.90% (n=10, acetaldehyde concentration of 2.5 mg L(-1)), and a limit of detection (LOD) of 12.3 µg L(-1). The LOD and sampling rate of the proposed method are superior to those of the conventional gas chromatographic (GC) method (LOD=93.0 µg L(-1) and sampling rate=4 h(-1)). The reliability of the proposed method was illustrated by the fact that spiked with acetaldehyde saliva samples yielded excellent recoveries (96.6-101.9%), comparable to those obtained by GC (96.4-102.3%) and there was no statistically significant difference at the 95% confidence level between the two methods when non-spiked saliva samples were analysed.

Serum alkaline phosphatase assay with paired emitter detector diode.

A simple multicommutated flow system based on optoelectronic detector, three valves and peristaltic pump only has been developed for photometric determination of alkaline phosphatase activity in human serum. A miniaturized, compact flow-through detector dedicated to selective photometric detection of product formed in the course of the enzyme assay has been constructed using two paired light emitting diodes. The proposed analytical procedure based on kinetic methodology of enzyme activity detection and stopped-flow methodology of two-point measurements eliminates interferences caused by intense color of real samples and impurities present in commercial reagents. After optimization the system allows reproducible, mechanized analysis of human serum in relatively short time (8-9 samples per hour). Volume of serum required for single determination is 0.05mL only. The system validated with real clinical samples is useful for determination of enzyme activity in human serum at physiological and pathological levels.