Determination of Thiols by Fluorescence using Au@Ag Nanoclusters as Probes

A simple and label-free fluorescent assay for the sensitive determination of biological thiols was developed using Au@Ag nanoclusters. The sensing approach was based on the strong affinity of thiols to silver on the surface of the nanoclusters. In the presence of thiol-containing amino acids, the fluorescence of the Au@Ag nanoclusters was quenched due to the formation of a non-fluorescent coordination complex via the robust Ag-S bond, which allowed the determination of thiol-containing amino acids in a very simple and rapid way. Under the optimal conditions, an excellent linear relationship was present due to quenching of the Au@Ag nanoclusters over cysteine concentrations between 20 nM and 80 µM with a low detection limit of 5.87 nM. Glutathione was determined between 2 µM and 70 µM with a detection limit of 1.01 µM. In addition, the results reveal that the fluorescent assay has excellent selectivity toward thiol-containing amino acids compared to non-thiol containing amino acids. Moreover, the assay was successfully used to determine cysteine in human plasma, and thus Au@Ag nanoclusters are a suitable fluorescent probe for biological applications.     

Salt‐assisted acetonitrile extraction and HPLC‐QTOF‐MS/MS detection for residues of multiple classes of pesticides in human serum samples

Detecting pesticide residues in human serum is a challenging process. In this study we developed and validated a method for the extraction and recovery of residues of multiple classes of pesticides from serum using one reagent. Salt‐assisted acetonitrile extraction and high‐performance liquid chromatography with quadrupole time of flight tandem mass spectrometry were used to quantitate 34 pesticides classified in nine groups of chemicals in human serum samples, which are frequently detected in food. The recoveries for 33 of analyzed pesticides ranged from 86 to 112% with relative standard deviations below 15%. The limits of quantitation and linearity of 31 of the pesticides were 1 µg/L and >0.990, respectively. The lower limit of quantitation has been reported in the literature particularly for multi‐classes pesticide mixtures in human serum. The salt–acetonitrile reagent was allowed to achieve good recoveries and detection limits, which could be attributed to salt altering the solvent polarity, preferentially collecting the organic phase in the solution, and promoting the extraction. The developed method was applied for two organophosphate pesticide metabolites, diethylphosphate and 3,5,6‐trichloro‐2‐pyridinol, in serum from rats that were fed a nonlethal quantity of chlorpyrifos. The concentrations of these two were 252.18 ± 15.47 and 0.63 ± 0.23 µg/L, respectively.     

Rapid and sensitive LC‐MS method for pharmacokinetic study of vinorelbine in rats

A rapid and sensitive LC‐electrospray ionization‐MS method was developed for determining vinorelbine in rat plasma. A 100 µL plasma sample was treated using a protein precipitation procedure and was chromatographed within 4 min using an Inertsil ODS‐3 C₁₈ (2.1 × 50 mm, 5 µm) column. The selected ion monitoring ions [M + H]⁺ were m/z 779 and m/z 811 for vinorelbine and vinblastine (internal standard), respectively. The method validation showed that the calibration curve for vinorelbine was linear over a concentration range of 1–1000 ng/mL with lower limit of quantification at 1 ng/mL. The method has been successfully applied to pharmacokinetics in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrochemical detection of amino acids based on cucurbit[7]uril-mediated three-dimensional gold nanoassemblies

In this paper, cucurbit[7]uril (CB[7])-mediated three-dimensional gold nanoassemblies were successfully prepared to increase the loaded amount of CB[7] and enhance the electrochemical detection of amino acids. Particle sizes of gold nanoparticles (AuNPs) significantly affect stability and detection sensitivity of nanoassemblies. The volume of gold nanoassemblies first increased and then decreased with the increase of CB[7] concentration. The 3D gold nanoassemblies composed of 16 nm AuNPs and 100 µmol/L CB[7] had excellent stability and maximum volume, exhibiting more sensitive detection for a variety of amino acids. And the detection limits of aromatic amino acids are lower in virtue of the higher binding constant between aromatic amino acids and CB[7]. This study will develop and deepen our understanding of molecular recognition in amino acids detection.     

Silver‐109‐based laser desorption/ionization mass spectrometry method for detection and quantification of amino acids

A new methodology applicable for both high‐resolution laser desorption/ionization mass spectrometry and mass spectrometry imaging of amino acids is presented. The matrix‐assisted laser desorption ionization‐type target containing monoisotopic cationic ¹⁰⁹Ag nanoparticles (¹⁰⁹AgNPs) was used for rapid mass spectrometry measurements of 11 amino acids of different chemical properties. Amino acids were directly tested in 100,000‐fold concentration change conditions ranging from 100 μg/mL to 1 ng/mL which equates to 50 ng to 500 fg of amino acid per measurement spot. Limit of detection values obtained suggest that presented method/target system is among the fastest and most sensitive ones in laser mass spectrometry. Mass spectrometry imaging of spots of human blood plasma spiked with amino acids showed their surface distribution allowing optimization of quantitative measurements.     

Developing a novel packed in‐tube solid‐phase extraction method for determination ∆9‐tetrahydrocannabinol in biological samples and cannabis leaves

A novel plate‐like nano‐sorbent based on copper/cobalt/chromium layered double hydroxide was synthesized by a simple coprecipitation method. The synthesized nanoparticels were introduced into a stainless steel cartridge using a dry packing method. Then, the packed cartridge was introduced as a novel on‐line “packed in‐tube” configuration and followed by high performance liquid chromatography for the determination of trace amounts of ^?9‐tetrahydrocannabinol from biological samples and cannabis leaves. The as‐prepared sorbent exhibited long lifetime, good chemical stability, and high anion‐exchange capacity. Several important factors affecting the extraction efficiency, such as extraction and desorption times, pH of the sample solution and flow rates of the sample and eluent solutions, were investigated and optimized. Under optimized conditions, this method showed good linearity for ^?9‐tetrahydrocannabinol in the ranges of 0.09–500, 0.3–500, and 0.4–500 µg/L with coefficients of determination of 0.9999, 0.9991, and 0.9994 in water, serum and plasma samples, respectively. The inter‐ and intra‐assay precisions (n = 3) were respectively in the ranges of 1.8–4.6% and 1.9–4.0% at three concentration levels of 10, 50, and 100 µg/L. The limits of detection were also in the range of 0.02–0.1 µg/L.     

A novel LC‐MS/MS method for determination of tissue distribution and excretion of timosaponin B‐II in rat biological matrices

Timosaponin B‐II (TB‐II) is a natural bioactive steroid glycoside extracted from the Chinese medicinal herb Anemarrhena asphodeloides Bge. (Fam. Liliaceae). It has been demonstrated to have a good anti‐inflammatory effect and a low bioavailability (1.1%). Clinical research has focused on developing it into a completely new medicine. In this study, a rapid and sensitive analytical method based on LC‐MS/MS has been developed for the determination of TB‐II in rat biological matrices (tissues, bile, urine and feces samples). The analytes and internal standard were isolated from 100 μL samples by solid‐phase extraction and then separated using a DIKMA Inertsil ODS‐3 column (5 µm, 2.1 × 150 mm) with an isocratic mobile phase consisting of acetonitrile–0.05% formic acid (35:65) at a flow rate of 0.25 mL/min. Calibration curves (1/χ²‐weighted) offered satisfactory linearity (r² ≥ 0.990) within the test range. The accuracy, precision, recoveries and matrix effects were satisfactory in all the biological matrices examined. The assay was successfully applied to a tissue distribution and excretion study in rats. The preclinical data are useful for the design of clinical trials of TB‐II. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and simple HPLC method for the determination of curcumin in rat plasma: assay development,validation and application to a pharmacokinetic study of curcumin liposome

This paper describes a sensitive, specific and rapid high‐performance liquid chromatography (HPLC) method for the determination of curcumin in rat plasma. After a simple step of protein precipitation in 96‐well format using acetonitrile containing the internal standard (IS), emodin, plasma samples were analyzed by reverse‐phase HPLC. Curcumin and the IS emodin were separated on a Diamonsil C₁₈ analytical column (4.6 × 100 mm, 5 µm) using acetonitrile–5% acetic acid (75:25, v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was sensitive with a lower limit of quantitation of 1 ng/mL, with good linearity (r² ≥ 0.999) over the linear range 1–500 ng/mL. All the validation data, such as accuracy and precision, were within the required limits. A run time of 3.0 min for each sample made high‐throughput bioanalysis possible. The assay method was successfully applied to the study of the pharmacokinetics of curcumin liposome in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of homocysteine,methionine and cysteine in maternal plasma after delivery by HPLC‐fluorescence detection with DBD‐F as a label

An HPLC‐fluorescence (FL) method for determination of sulfur‐containing amino acids such as homocysteine (Hcy), methionine (Met) and cysteine (Cys) in human plasma was developed. The sulfur‐containing amino acids were labeled with 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F). Calibration curves in the range of 1–100 µm (Hcy and Met) and 5–500 µm (Cys) indicated good linearities (r ≥ 0.998). The limits of detection at a signal‐to‐noise ratio of 3 were 0.13 (Hcy), 0.02 (Met) and 0.11 µm (Cys), respectively. Acceptable results for accuracy and precision of intra‐ and inter‐day measurements were obtained. The results of Hcy and Cys obtained by the proposed method indicated good correlations with the conventional method (r > 0.911, n = 20). Furthermore, the method was applied to determination of the sulfur‐containing amino acids in maternal plasma (n = 200) after delivery. The concentrations of Hcy, Met and Cys as a median (inter quartile range, Q₁ and Q₃) were 5.37 (3.32–7.79) μm , 25.20 (20.10–31.06) μm and 147.25 (102.81–189.31) μm , respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Comprehensive profiling of CTP-binding proteins using a biotinylated CTP affinity probe

Recent studies have shown that CTP may act as a ligand to regulate the activity of its target proteins in many biological processes. However, proteome-wide identification of CTP-binding proteins remains challenging. Here, we employed a biotinylated CTP affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics approach to capture, identify and quantify CTP-binding proteins in human cells. By performing two types of competitive SILAC experiments with high vs. low concentrations of CTP probe (100 vs. 10 µmol/L) or with CTP probe in the presence of free CTP, we identified 90 potential CTP-binding proteins which are involved in a variety of biological processes, including protein folding, nucleotide binding and cell-cell adhesion. Together, we developed a chemical proteomic method for uncovering the CTP-binding proteins in human cells, which could be widely applicable for profiling CTP-binding proteins in other biological samples.     

Effervescent tablet‐assisted demulsified dispersive liquid–liquid microextraction based on solidification of floating organic droplet for determination of methadone in water and biological samples prior to GC‐flame ionization and GC‐MS

A novel effervescent tablet‐assisted demulsified dispersive liquid–liquid microextraction based on the solidification of floating organic droplet was developed to determine methadone prior to gas chromatography with flame ionization detection and gas chromatography with mass spectrometry. In this method, a tablet composed of citric acid, sodium carbonate, and 1‐undecanol was utilized. The resulting effervescent tablet generated carbon dioxide in situ to disperse 1‐undecanol in the sample. Thus, the dispersive and extraction processes were performed in one synchronous step. An aliquot of acetonitrile as the demulsifier solvent was used for the separation of two phases instead of centrifugation. Under optimal conditions, the developed method was linear up to 50 000 µg/L with correlation coefficients higher than 0.99. Moreover, limits of detection and limits of the quantification were in the range of 3‐10  and 7‐30 µg/L in water and biological samples, respectively. Intra‐ and interday precisions (n = 6) of the spiked methadone at a concentration level of 50 µg/L were over ranges of 5.1‐6.8% and 5.7‐7.1%, respectively. The preconcentration factors and recovery values were obtained in the range of 140‐145 and 98.1 to 101.6% in real samples, respectively.     

Development and validation of a UHPLC–MS/MS method for the simultaneous determination of five bioactive flavonoids in rat plasma and comparative pharmacokinetic study after oral administration of Xiaochaihu Tang and three compatibilities

An accurate, rapid, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of baicalin, wogonoside, baicalein, wogonin, and oroxylin A in rat plasma. Then, the stability of baicalin and baicalein in the preparation of plasma sample was systematically investigated. The Waters BEH C₁₈ column was used with a gradient mobile phase system of acetonitrile and water containing 0.1% formic acid. The analytes were detected in the multiple reaction monitoring mode with positive electrospray ionization. 100 μL fresh plasma was added with 50 μL antioxidant reagent (1 mol/L HCl containing 0.5% Vitamin C), and liquid–liquid extraction with ethyl acetate was used to extract the analytes from plasma. Lower limits of quantification of baicalin, wogonoside, baicalein, wogonin, and oroxylin A were 21.9, 4.80, 1.20, 0.848, and 0.800 ng/mL, respectively. The mean extract recoveries of five flavonoids were 69.1∼89.2%, and the precision and accuracy were within the acceptable limits. This method was further successfully applied to the comparative pharmacokinetic study of these five flavonoids in rats after oral administration of Xiaochaihutang and three compatibilities. The obtained results may be helpful to reveal the mechanism of Xiaochaihutang formula compatibility.     

A rapid and sensitive LC‐MS/MS method for the determination of linarin in small‐volume rat plasma and tissue samples and its application to pharmacokinetic and tissue distribution study

A rapid and sensitive LC‐MS/MS method was developed for the determination of linarin in small‐volume rat plasma and tissue sample. Sample preparation was employed by the combination of protein precipitation (PPT) and liquid–liquid extraction (LLE) to allow measurement over a 5‐order‐of‐magnitude concentration range. Fast chromatographic separation was achieved on a Hypersil Gold column (100 × 2.1 mm i.d., 5 µm). Mass spectrometric detection was achieved using a triple‐quadrupole mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. Quantification was performed using selected reaction monitoring of precursor‐product ion transitions at m/z 593 → 285 for linarin and m/z 447 → 271 for baicalin (internal standard). The total run time was only 2.8 min per sample. The calibration curves were linear over the concentration range of 0.4–200 µg/mL for PPT and 0.001–1.0 µg/mL for LLE. A lower limit of quantification of 1.0 ng/mL was achieved using only 20 μL of plasma or tissue homogenate. The intra‐ and inter‐day precisions in all samples were ≤14.7%, while the accuracy was within ±5.2% of nominal values. The validated method has been successfully applied to pharmacokinetic and tissue distribution study of linarin. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of carbapenems in water samples by UHPLC–MS/MS

A rapid and reliable method for the detection of five carbapenems (biapenem, imipenem, doripenem, meropenem, and faropenem) in water was developed and validated. After acidification of water samples with acetic acid, carbapenems were isolated using a Bond Elut PPL cartridge. The target compounds were separated using ultra high performance liquid chromatography with a chromatographic run time of 5 min and detected on a triple quadrupole mass spectrometer operated in positive electrospray ionization and multiple reaction monitoring mode. Mean recoveries were in the range of 76.6–106.5%, with satisfactory intraday and interday relative standard deviations lower than 10.0 and 10.8%, respectively. The limits of detection and quantification were in the ranges of 0.05–0.2 µg/L and 0.1–0.5 µg/L, respectively, depending on the analyte. The proposed method was applied to the analysis of river samples and wastewater samples from swine farms, and no carbapenems were detected in the collected samples.     

Polymer phase transition characteristics coupled with GC‐MS for the determination of phthalate esters

Phthalate esters are easily released from plastics materials and migrate into the soil and water environment, causing serious pollution and posing a great threat to the health of human beings. A novel temperature‐sensitive extractant combined with liquid–liquid microextraction was developed to preconcentrate three phthalates in the water environment. To optimize the extraction efficiency for the three phthalate esters, various parameters, including polymer molecular weight, salt type, salt addition, adsorption time, desorption solvent, desorption volume, and desorption time have been studied. Under optimal conditions, limits of detection and limits of quantification were in the range of 0.007–0.120 and 0.021–0.350 µg/L, respectively. Linearities varied in the range of 5–1000 µg/L, with the correlation coefficients of 0.9867–0.9997. The preconcentration factors were in the range of 25–75. The relative recoveries of the three phthalate esters were in the range of 82.2–105.6% at the spiked levels. The relative standard deviations were in the range of 0.7–9.2% based on triplicate measurements. The results indicate that the temperature‐sensitive material is a good extractant for phthalate esters in water samples.     

Use of recombinantly produced <Superscript>15</Superscript>N<Subscript>3</Subscript>-labelled nicotianamine for fast and sensitive stable isotope dilution ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry

Nicotianamine (NA) is an important metal chelator, implicated in the intra- and intercellular trafficking of several transition metal ions in plants. To decipher its roles in physiological processes such as micronutrient acquisition, distribution or storage, fast and sensitive analytical techniques for quantification of this non-proteinogenic amino acid will be required. The use of a recombinant Schizosaccharomyces pombe strain expressing a nicotianamine synthase (NAS) gene allowed for the production of [¹⁵N₃]-NA, which was enriched from cell extracts through cation exchange and used for stable isotope dilution analysis of NA. Such an approach should be widely applicable to important bioanalytes that are difficult to synthesize. The analytical procedure comprises mild aqueous extraction and rapid Fmoc derivatization, followed by fast separation using ultra-performance liquid chromatography (UPLC) and sensitive detection by positive ion electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a chromatographic cycle time of only 8 min. Derivatization was optimized with respect to incubation time and species suitable for quantification. The limit of detection was 0.14 to 0.23 pmol in biological matrices with the response being linear up to 42 pmol. Recovery rates were between 83% and 104% in various biological matrices including fission yeast cells, fungal mycelium, plant leaves and roots.     

Ion pair assisted micro matrix solid phase dispersion extraction of alkaloids from medical plant

A novel micro matrix solid phase dispersion method was successfully used for the extraction of quaternary alkaloids in Phellodendri chinensis cortex. The elution of target compounds was accomplished with sodium hexanesulfonate as the eluent solvent. A neutral ion pair was formed between ion-pairing reagent and positively charged alkaloids in this process, which was beneficial for selectively extraction of polar alkaloids. Several parameters were optimized and the optimal conditions were listed as follows: silica gel as the sorbent, silica to sample mass ratio of 1:1, the grinding time of 1 min. The exhaustive elution of targets was achieved by 200 µL methanol/water (9:1) containing 150 mM sodium hexane sulfonate at pH 4.5. The method validation covered linearity, recovery, precision of intraday and interday, limits of detection, limits of quantitation, and repeatability. This established method was rapid, simple, environmentally friendly, and highly sensitive.     

An ultra high performance liquid chromatography with tandem mass spectrometry method for simultaneous determination of thirteen components extracted from Radix Puerariae in rat plasma and tissues: Application to pharmacokinetic and tissue distribution study

A rapid and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for simultaneous determination of thirteen bioactive components (gallic acid, protocatechuic acid, puerarin, p‐hydroxycinnamic acid, daidzin, ononin, daidzein, naringenin, genistein, apigenin, formononetin, biochanin A, and β‐sitosterol) of Radix Puerariae extract in rat plasma and tissues. The plasma and tissues samples were pretreated by protein precipitation extraction, and umbelliferone and rutin were used as internal standards. Sample separation was performed on a ZORBAX RRHD Eclipse plus C₁₈ column (2.1 mm × 50 mm, 1.8 µm, Agilent) with a mobile phase consisting of methanol–water (containing 0.1% formic acid). The mass spectrometry analysis was conducted in positive and negative ionization modes with multiple reaction monitoring. The lower limit of quantitation range for the 13 analytes was 0.2?35 ng/mL. The intra‐ and inter‐day precision of all the analytes were less than 10.92%, with an accuracy ranging from ?13.10 to 11.96%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied to pharmacokinetic and tissue distribution study of the 13 bioactive components in rats after oral administration of R. Puerariae extract.     

A validated LC‐MS/MS method for rapid determination of brazilin in rat plasma and its application to a pharmacokinetic study

Brazilin is a major homoisoflavonoid component isolated from the dried heartwood of traditional Chinese medicine Caesalpinia sappan L., which is a natural red pigment used for histological staining. Herein a sensitive, specific and rapid analytical LC‐MS/MS method was established and validated for brazilin in rat plasma. After a simple step of protein precipitation using acetonitrile, plasma samples were analyzed using an LC‐MS/MS system. Brazilin and the IS (protosappanin B) were separated on a Diamonsil C₁₈ analytical column (150 × 4.6 mm, 5 µm) using a mixture of water and 10 mm ammonium acetate in methanol (20:80, v/v) as mobile phase at a flow rate of 0.6 mL/min. The method was sensitive with a lower limit of quantitation of 10.0 ng/mL, with good linearity (r² ≥ 0.99) over the linear range 10.0–5000 ng/mL. All the validation data, such as accuracy and precision, matrix effect, extraction recovery and stability tests were within the required limits. The assay method was successfully applied to evaluate the pharmacokinetics parameters of brazilin after an oral dose of 100 mg/kg brazilin in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

The bioanalysis of vinorelbine and 4‐O‐deacetylvinorelbine in human and mouse plasma using high‐performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4‐O‐deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 µL plasma aliquot was protein precipitated with acetonitrile–methanol (1:1, v/v) containing the internal standard vinorelbine‐d3 and 20 µL volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using isocratic elution with 1 mm ammonium acetate–ammonia buffer pH 10.5–acetonitrile–methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4‐O‐deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 µL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4‐O‐deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre‐)clinical pharmacologic studies with vinorelbine in humans and mice. Copyright © 2009 John Wiley & Sons, Ltd.