Minimal Detectable Amounts Research Articles

Development of Cyanine 813@Imidazole-Based Doped Supported Devices for Divalent Metal Ions Detection

A NIR cyanine@imidazole derivative Cy1 was synthesized and evaluated as a metal ion sensor in solution. Cy1 was shown to be very sensitive to all metal ions tested, presenting a blue shift in the absorption from 668 nm to 633 nm, followed by a change in colour from pale green to blue with Zn2+, Cd2+, Co2+, Ni2+ and Hg2+ ions. Despite the blue shift in the absorption, a decrease at 633 nm (with a colour change from pale green to colourless), as well as a quenching in the emission intensity at 785 nm were observed for Cu2+ ions. The results show the formation of sandwich complexes of two ligands per metal ion with the highest association constant observed for Cu2+ (Log Kass.abs = 14.76 ± 0.09; Log Kass.emis. = 14.79 ± 0.06). The minimal detectable amounts were found to be 31 nM and 37 nM, with a naked eye detection of 2.9 ppm and 2.1 ppm for Hg2+ and Cu2+ ions, respectively. These results prompted us to explore the applicability of Cy1 by its combination with nanomaterials. Thus, Cy1@ doped MNs and Cy1@ doped PMMA nanoparticles were synthesized. Both nanosystems were shown to be very sensitive to Cu2+ ions in water, allowing a naked-eye detection of at least 1 ppm for Cy1@ doped MNs and 7 ppm for Cy1@ doped PMMA. This colourimetric response is an easy and inexpensive way to assess the presence of metals in aqueous media with no need for further instrumentation.

Minimal Detectable Amounts of Exogenous DNA after Systemic Cell Engraftment

In cellular therapy models, the establishment of accurate methods to track the homing, cell fate and survival are fundamental to ensure safety and efficacy. The PCR is used to detect donor cells DNA in xenotransplant models. Five different concentrations of EGFP+ DNA from C57/BL6 mouse muscle tissue: 1,000 pg/μL, 500 pg/μL, 100 pg/μL, 10 pg/μL e 1 pg/μL were used to contaminate 10 ng/μL of DNA Wistar rat. Polymerase chain reaction was performed using 2 μL of each sample. EGFP+DNA were detected in samples containing 2,000 pg, 1,000 pg, 200 pg and 20 pg. Samples with 2 pg EGFP+DNA did not show signal. Here we demonstrate an alternative to proving the presence or confirm the absence, of exogenous DNA in organs and tissues. The concentration curve for cell migration detection by real time PCR will allow the use in systemically transplanted cells in animal models subjected to cell therapy.

Ultrasensitive quantification of endogenous brassinosteroids in milligram fresh plant with a quaternary ammonium derivatization reagent by pipette-tip solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometry

Determination of endogenous brassinosteroids (BRs) in limited sample amount is vital to elucidating their tissue- and even local tissue-specific signaling pathway and physiological effects on plant growth and development. In this work, an ultra-sensitive quantification method was established for endogenous BRs in milligram fresh plant by using pipette-tip solid-phase extraction coupled with ultra-performance liquid chromatography tandem mass spectrometry (PT-SPE-UPLC-MS/MS), in which a quaternary ammonium phenyl boronic acid, 4-borono-N,N,N-trimethylbenzenaminium iodide (BTBA) was first developed for chemical derivatization of BRs. Due to the cationic quaternary ammonium group of BTBA, the ionization efficiencies of the BRs chelates with BTBA (BTBA-BRs) were enhanced by 1190–448785 times, which is the highest response enhancement factor among all derivatization reagents reported for BRs. In addition, PT-SPE packed with C18 sorbent was first used for purifying BRs from plant extracts, so the required sample amount was minimized, and recoveries higher than 91% were achieved. Under the optimized conditions, the minimal detectable amounts (MDA) of five target BRs were in the range of 27–94 amol, and the correlation coefficients (R2) were >0.9985 over four orders of magnitude. The relative recoveries of 75.8–104.9% were obtained with the intra- and inter-day relative standard deviations (RSDs) less than 18.7% and 19.6%, respectively. Finally, three BRs were successfully quantified in only 5mg fresh rice plant samples, and 24-epiBL can even be detected in only 0.5mg FW rice leaf segments. It is the first time that the BRs content in sub-milligram fresh plant sample has been quantified.

Analysis of ecstasy tablets using capillary electrophoresis with capacitively coupled contactless conductivity detection.

A method for the identification of 3,4-methylenedioxymethamphetamine (MDMA) and meta-chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4) D). Sample extraction, separation, and detection of "Ecstasy" tablets were performed in <10min without sample derivatization. The separation electrolyte was 20mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE-C(4) D and compared against routine gas chromatography-mass spectrometry (GC-MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90sec. The advantages of using C(4) D instead of traditional CE-UV methods for in-field analysis are also discussed.

Steady-state Kinetic Mechanism of PDK1

PDK1 catalyzes phosphorylation of Thr in the conserved activation loop region of a number of its downstream AGC kinase family members. In addition to the consensus sequence at the site of phosphorylation, a number of PDK1 substrates contain a PIF sequence (PDK1-interacting fragment), which binds and activates the kinase domain of PDK1 (PDK1(deltaPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(deltaPH)-catalyzed phosphorylation of PDK1-Tide (Tide), which contains an extended "PIF" sequence C-terminal to the consensus sequence for PDK1 phosphorylation. In two-substrate kinetics, a large degree of negative binding synergism was observed to occur on formation of the active ternary complex (alphaKd(ATP) = 40 microM and alphaKd(Tide) = 80 microM) from individual transitory binary complexes (Kd(ATP) = 0.6 microM and Kd(Tide) = 1 microM). On varying ATP concentrations, the ADP product and the (T/E)-PDK1-Tide product analog (p'Tide) behaved as competitive and noncompetitive inhibitors, respectively; on varying Tide concentrations, ADP and p'Tide behaved as noncompetitive and competitive inhibitors, respectively. Also, negative binding synergism was associated with formation of dead-end inhibited ternary complexes. Time progress curves in pre-steady-state studies under "saturating" or kcat conditions showed (i) no burst or lag phenomena, (ii) no change in reaction velocity when adenosine 5'-O-(thiotriphosphate) was used as a phosphate donor, and (iii) no change in reaction velocity on increasing relative microviscosity (0 < or = eta/eta0 < or = 3). Taken together, PDK1-catalyzed trans-phosphorylation of PDK1-Tide approximates a Rapid Equilibrium Random Bi Bi system, where motions in the central ternary complex are largely rate-determining.

Evaluation of minimum detectable amounts of iodine in biological samples by neutron activation analysis

Evaluation of minimal detectable amounts (MDA) of iodine in various or specified irradiation and measurement conditions is described. Interfering contributions due to Na and Cl to the spectral background as well as the dead time of detection system were calculated in details based on an experimental approach. The MDA of iodine for some important biological samples with various Na and Cl concentrations can be evaluated and predicted according to the specified irradiation and measurement arrangements to meet the requirement of analytical purposes.

Use of dyes as indirect detection probes for the high-sensitivity determination of anions by capillary electrophoresis

High sensitivity for indirect detection was achieved by utilising highly absorbing species as the displaced co-ion (or probe). Two highly absorbing dyes, bromocresol green and indigo-tetrasulfonate, were investigated as potential probes in the determination of small organic and inorganic anions. The concentration of these probes was kept as low as possible to ensure the signal-to-noise ratios were reasonable and the background absorbance was within the linear range of the detector. Four different protocols for buffering the electrolyte with such low probe concentrations were investigated. Buffering with agents that introduce co-anions [acetate or 2-(cyclohexylamino)ethanesulfonic acid (CHES) buffers] proved unsuitable as detection sensitivities were diminished due to competitive displacement by the analytes and system peaks were also induced. Buffering without introduction of co-anions was achieved using the buffering base, diethanolamine, or the use of ampholytes, lysine and glutamic acid. For separations performed with these two buffering approaches, migration time reproducibilities were less than 1% R.S.D. for most analytes. Minimal detectable amounts were in the low attomol region (1·10 −18 mol), corresponding to sub-μ M vacuum injected solution concentrations. These were an order of magnitude lower than the general detection limit reported for indirect photometric detection, and were comparable with detection limits achieved with indirect fluorescence detection. Finally, the detection limits were further improved by approximately three times for anions analysed with indigo-tetrasulfonate as the probe when a Z cell was employed as the detection cell.

An Enzyme Immunoassay for Rat Growth Hormone: Validation and Application to the Determination of Plasma Levels and in Vitro Release

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r=0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n=24) and 14% (n=9) respectively. Minimal detectable amounts of rGH were 0,6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.

Instrumental Requirements for Nanoscale Liquid Chromatography

Nanoscale liquid chromatography (nano-LC), with packed columns of typically 75 μm i.d. × 15 cm length, packed with C18, 5 μm of stationary phase, and optimal flow rates of 180 nL/min, can be considered as a miniaturized version of conventional HPLC. Using the down-scaling factor, which corresponds to the ratio of the column diameter in square, (d(conv)/d(micro))(2), excellent agreement between the theoretically calculated values and the values obtained using the down-scaling factor (∼3800) has been observed. This factor was applied to all system components, including flow rate, injection and detection volumes, and connecting capillaries. Down-scaling of a conventional HPLC system to a nano-LC system is easy to realize in practice and involves using a microflow processor for nanoflow delivery (50-500 nL/min), a longitudinal nanoflow cell (≤3 nL), a microinjection valve (≤ 20 nL), low-dispersion connecting tubing, and zero dead volume connections. Excellent retention time reproducibility was measured with RSD values of ±0.1% for isocratic and ±0.2% for gradient elution. Plates counts of more than 100 000/m indicate the excellent performance of the entire nano-LC system. With minimal detectable amounts of proteins in the low femtomole and subfemtomole ranges (e.g., 520 amol for bovine serum albumin), high mass sensitivity was found, making nano-LC attractive for the microcharacterization of valuable and/or minute proteinaceous samples. Coupling nano-LC with concomitant mass spectrometry using nanoscale ion spray or electrospray interfaces looks very promising and is obviously the next step for future work.

Highly Sensitive, Specific Enzyme-Linked Immunosorbent Assay of Neopterin and Biopterin in Biological Samples

An enzyme immunosorbent assay of neopterin and biopterin on a polystyrene microtiter plate has been developed. A conjugate of neopterin or biopterin to bovine serum albumin was used to raise a specific antiserum against neopterin or biopterin in rabbits. An incubation mixture of the antiserum and samples prepared from human serum underwent another antigen-antibody reaction with the hapten fixed on the microtiter plate. The amount of antibody bound to the fixed hapten, which is inverse to the amount of hapten in the sample, was determined by using anti-rabbit IgG-horseradish peroxidase conjugate in a usual manner by measuring absorbance at 490 nm after reaction with o-phenylenediamine and hydrogen peroxide. The minimal detectable amounts of neopterin and biopterin were approximately 0.1 pmol. The specificity of the assay was so high that the assay system for neopterin completely distinguished it from biopterin, as judged from the cross-reaction of 0.002%, and vice versa. The amounts of neopterin and biopterin in human serum determined by the present method agreed well with those determined by high-performance liquid chromatography. We used the present method to determine the concentrations of neopterin in serum from healthy control subjects and patients with cancers and systemic lupus erythematosus; the results were consistent with literature data.

Purity assay of sodium mercaptododecaborate by high-performance liquid chromatography

A simple high-performance liqiud chromatographic (HPLC) method for purity monitoring and determination of [B 12H 11SH] 2− ( I)_ and its oxidation impurities [B 12H 11SSB 12H 11] 4− ( II) and [B 12H 11S(O)SB 12H 11] 4− ( III is presented. The method is based on the use of the hydroxyethylmethacrylate sorbent Separon HEMA-BIO 300 and 100 m M sodium percholorate in 0.01 M phosphate buffer as mobile phase. Common HPLC equipment with direct spectrophotometric detection in the range 200–210 nm was used throughout. The minimal detectable amounts of the sodium salts of anions I, II and III at 204 nm were 8.6·10 −8, 8.8·10 −9 and 6.4·10 −8 g, respectively. The application of the method to the study of the stability of the sodium salt of I in solution revealed a strong positive effect of trace amounts of Cu + in enhancing the Na 2B 12H 11SH oxidation rate.

Purification and immunological quantification of rat liver lysosomal glycosidases

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.

Electrochemical, fluorescence, and UV detection for HPLC analysis of various cysteine derivatives

AbstractElectrochemical (ECD), fluorescence (FLD), and UV spectrophotometric (UVD) detection were used to monitor various S‐alk(en)yl‐L‐cysteines and their corresponding sulfoxide isomers following pre‐column derivatization with o‐phthaldialdehyde‐tert‐butylthiol and separation by reversed‐phase HPLC. Recording of hydrodynamic voltammograms, FLD stop‐flow scanning, and on‐line captured UV spectra were methods used for establishing optimal detector settings which were defined as a compromise between favorable selectivity and high sensitivity. Optimal detector settings were found at: (A) 750 mV vs. Ag/AgCl for ECD; (B) excitation at 230 nm and emission at 420 nm for FLD; and (C) 337 nm for UVD. Various aspects of detector practicability such as selectivity, baseline disturbances due to excessive reagent, scanning possibilities, as well as detection limits were evaluated and compared. Minimal detectable amounts of the compounds were in the range of 130‐160 fmol for ECD, and 2.5‐3.5 pmol and 13‐16 pmol for FLD and UVD. In addition, the possibilities and benefits of detector coupling were examined.

Replication of the scrapie agent in hamsters infected intracerebrally confirms the pathogenesis of an amyloid-inducing virosis.

Following intracerebral infection of hamsters with scrapie agent replication started with or without a very short lag phase. Infectivity titres increased exponentially within 35 to 40 days post-infection to a maximum level of 3 x 10(9) LD50 per brain and then remained constant until death. Minimal detectable amounts of scrapie-associated fibrils (SAF) appeared at 42 days and reached high levels 56 days after inoculation. The first clinical symptoms were diagnosed at about 65 days and animals died after 85 to 95 days. These data confirm earlier results in which peripheral infection first revealed agent replication, then SAF formation and finally clinical disease. Unconventional virus diseases, therefore, can best be described as virus-induced, organ-specific amyloidoses.

Gas-liquid chromatography of nucleosides : Derivatization and chromatography

The aims of this investigation were to establish the optimum reaction conditions for silylation of nucleosides with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and to investigate the chromatographic properties of the following nucleosides: adenosine, guanosine, cytidine, thymine, inosine, xanthosine, and uridine. Closed tube silylations were performed with a 1000 molar excess of BSTFA at 25, 75, 120, 150, and 175° for 15, 30, 60, 120 and 240 min. The optimal time and temperature for derivatization were found to be 150° and 15 min. Using these reaction conditions, samples were then silylated with 50, 100, 200, 500, and 1000 molar excess of BSTFA; a molar excess of 225 was best. The stability of the nucleoside derivatives on standing at room temperature for 1–7 days was investigated. Quantitative gas-liquid chromatography of trimethylsilyl (TMS) nucleosides can be performed if samples are analyzed within 48 h, after which time the relative weight response for the nucleosides decreased somewhat. Chromatographic column studies were made using various liquid phases and supports. With methylsiloxanes as the liquid phase, Supelcoport as support was found to be superior. Resolution of TMS guanosine from TMS cytidine was attempted at different column lengths using 3% (w/w) SE-30 on Supelcoport, different loadings (%, w/w) of SE-30 on Supelcoport, and different polarity liquid phases, 4% (w/w) OV-11 or 3% (w/w) OV-17 or 4% (w/w) Dexsil-300 on Supelcoport. A complete separation of the six ribonucleosides including guanosine and cytidine was obtained with 1 m × 4 mm I.D. glass columns of 4% (w/w) OV-11 and 3% (w/w) OV-17 on 100–120 mesh Supelcoport. It was observed that with 2′-deoxycytidine, one obtains the TMS cytosine peak (retention temperature 150°) plus another peak with a retention temperature of 120°, under all derivatization conditions with BSTFA and bis(trimethylsilyl)acetamide. Similar formation of bases from other 2′-deoxyribonucleosides occurred when the molar excess of BSTFA was greater than 500. The minimal detectable amounts obtained for all the nucleosides ranged from 5 to 10 ng injected with a signal-to-noise ratio of 3. The relative standard deviations for all nucleosides and deoxynucleosides ranged from 1.2 to 4.8% on different methylsiloxanes on Supelcoport columns.

Analysis of phenylethylamines in biological tissues by gas-liquid chromatography with electron-capture detection

2,4-Dinitrophenyl derivatives of phenylethylamines have been applied to the sensitive quantitative analysis of phenylethylamines in biological tissues by gas chromatography and electron-capture detection. The minimal detectable amounts are about 0.1 ng. A rapid and specific cleanup procedure allows the injection of 10% of all the amines extracted from 200 mg tissues, so that as little as 20 ng/gm tissue can be determined. Amines with or without hydroxyl groups can be determined as the DNP-derivatives with equal sensitivity. The corresponding 3,5-dinitro-4-trifluoromethyl derivatives were also prepared and their chromatographic behavior is reported.

Gas‐liquid partition chromatography

AbstractThe identification and determination of different aliphatic substances by means of a suitable system of gas chromatography is described.Gas‐chromatographic separations of different series of carboxylic acid esters from propionic‐ up to stearic acid ester, alcohols from pentanol up to hexadecanol, and aldehydes from pentanal up to decanal are described.Silicone grease or paraffin wax is used as the liquid phase with celite as a carrier. The separations have been carried out at temperatures up to 230° C, which made the use of atmospheric pressure at the outlet possible. Hence the fractions separated can be conveniently collected. A quantitative determination of milligram amounts in mixtures of methyl esters is described.The minimal detectable amounts as estimated for methyl propionate are of the order of micrograms.