Highly sensitive HPLC method for the determination of galantamine in human plasma and urine through derivatization with dansyl chloride using fluorescence detector

A new method based on fluorescence derivatization with 5‐(dimethylamino) naphthalene‐1‐sulfonyl chloride (dansyl chloride) was developed for the quantitative determination of galantamine in human plasma and urine using high‐performance liquid chromatography. The reaction between galantamine and dansyl chloride was optimally realized in 30 min at room temperature and pH 10.5, with a reagent to galantamine molar ratio of 2.13. The derivative was extracted with dichloromethane, and the extract was dried under a nitrogen stream and dissolved in the mobile phase. Chromatographic analysis was performed with an Inertsil C₁₈ column and a mobile phase comprising 40% acetonitrile and 60% 10 mM o‐phosphoric acid, 1.2 ml/min. The injection volume was 20 μl. The derivatives were detected with a fluorescence detector (excitation 375 nm/emission 537 nm). The retention time for the dansyl derivative of galantamine was 16.8 min. Linearity was observed between 125 and 2000 ng/ml in water, urine and plasma. The limit of detection and limit of quantification for the developed method were 6.27–70.99 and 18.81–212.97 ng/ml, respectively. Per cent recovery was calculated as 95.15 for urine and 95.78 for plasma. Interday repeatability values for urine and plasma samples (n = 6) at three different concentrations were calculated as a per cent relative standard deviation of 0.24–0.59 and 0.35–0.56. The corresponding per cent relative standard deviation values for intraday repeatability were 0.13–0.51 and 0.04–0.15, respectively.     

A simple FRET system using two‐color CdTe quantum dots assisted by cetyltrimethylammonium bromide and its application to Hg(II) detection

In this study, we developed a novel simple fluorescence resonance‐energy transfer (FRET) system between two‐color CdTe quantum dots (QDs) assisted by cetyltrimethylammonium bromide (CTAB). Mercaptopropionic (MPA)‐capped CdTe QDs serving as both donors and acceptors were successfully synthesized by changing the refluxing time in aqueous solution. CTAB micelles formed in water and minimized the distance between the donors and acceptors significantly by electrostatic interactions, improving FRET efficiency. Several factors that affected the fluorescence spectra of the FRET system were investigated. The prepared FRET system was feasible as an effective fluorescent probe to detect Hg(II) in aqueous solution. At pH 7.0, a linear relationship between the quenched fluorescence intensity of orange‐emitting acceptors (QDs_(A)) and Hg(II) concentration was acquired in the range 5–250 nmol/L with a detection limit of 1.95 nmol/L. The developed method showed excellent analytical performance for Hg(II) with high sensitivity and acceptable selectivity, reproducibility and stability. This finding indicated that the method has a promising potential application for environmental monitoring. This study demonstrated the great promise of QDs for expedient, low‐cost and high‐sensitivity detection of Hg(II).     

Optimization of ASE and SPE conditions for the HPLC‐FLD detection of piperazine in chicken tissues and pork

Accelerated solvent extraction (ASE) and solid‐phase extraction (SPE) conditions were optimized by a high‐performance liquid chromatography‐fluorescence detector (HPLC‐FLD) method for the detection of piperazine in chicken tissues and pork. Piperazine residues were determined by precolumn derivatization with trimethylamine and dansyl chloride. Samples were extracted with 2% formic acid in acetonitrile using an ASE apparatus and purified using a Strata‐X‐C SPE column. The monosubstituted product of the reaction of piperazine with dansyl chloride was 1‐dansyl piperazine (1‐DNS‐piperazine). Chromatographic separations were performed on an Athena C₁₈ column (250 × 4.6 mm, id: 5 μm) with gradient elution using ultrapure water and acetonitrile (5:95, V/V) as the mobile phase. The calibration curves showed good linearity over a concentration range of LOQ‐200.0 μg/kg with a coefficient of determination (R²) ≥ .9992. The recoveries and relative standard deviations (RSD values) ranged from 78.49% to 97.56% and 1.19% to 5.32%, respectively, across the limit of quantification (LOQ) and 0.5, 1, and 2.0 times the maximum residue limit (MRL; μg/kg). The limits of detection (LODs) and LOQs were 0.96 to 1.85 μg/kg and 3.20 to 5.50 μg/kg, respectively. The method was successfully applied for the validation of animal products in the laboratory.     

High‐Throughput Chiral LC–MS/MS Method Using Overlapping Injection Mode for the Determination of Pantoprazole Enantiomers in Human Plasma with Application to Pharmacokinetic Study

A sensitive and high‐throughput chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of R‐pantoprazole and S‐pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid–liquid extraction in 96‐well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ‐RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 μL/min. The enantiomers were quantified on a triple‐quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole‐d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r² > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R‐pantoprazole and from 92.5% to 96.5% for S‐pantoprazole and the IS‐normalized matrix factor was 0.98 to 1.07 for R‐pantoprazole and S‐pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569–575, 2016. © 2016 Wiley Periodicals, Inc.     

Expression of genes encoding the luciferase from Photobacterium leiognathi in Escherichia coli Rosetta (DE3) and its application in NADH detection

Cloning of genes encoding the luciferase from Photobacterium leiognathi YL in Escherichia coli Rosetta (DE3) was performed successfully and the expressed forms of lux AB were purified to homogeneity. Experimental measurements revealed that luciferase from Photobacterium leiognathi YL has good thermal stability and a high residual activity at extreme pH values, which are extremely important for its various ecological, industrial and medical applications. Furthermore, we made a first attempt for quantitative detection of NADH by recombinant E. coli Rosetta (DE3) coupled enzyme system. A good linear relationship between luminescence intensity and NADH with low (1–12 nmol/L) and high (10–500 nmol/L) concentration was observed, whose standard curve was y = 772.97× + 4041.1, R² = 0.9884 and y = 1710× + 4.99 × 10⁵, R² = 0.9727, respectively. Our results demonstrate a high sensitivity of recombinant E. coli coupled enzyme system to NADH on the basis of high soluble expression of recombinant luciferase and continuous and stable expression of some NAD(P)H‐dependent flavin mononucleotide (FMN) reductases.     

Enhancement of chemiluminescence of the KIO4–luminol system by gallic acid,acetaldehyde and Mn2+: application for the determination of catecholamines

Chemiluminescence (CL) from the oxidation of luminol with potassium periodate in strong alkaline solutions was greatly enhanced by the combined effect of gallic acid, acetaldehyde and Mn²⁺. The CL spectra exhibited only one emission band at 425 nm, indicating 3‐aminophthalate as the emitting species. Various scavengers for superoxide anion, hydroxyl radical and singlet oxygen quenched the CL emission very efficiently (74–100%), suggesting the possible involvement of these reactive oxygen species (ROS) in the CL reactions. It is postulated that oxidation of gallic acid and acetaldehyde by periodate catalyzed by Mn²⁺ generates these ROS, which then react with luminol to enhance the CL emission. We also found that the enhanced CL emission was strongly inhibited by catecholamines, probably because of their effective scavenging of ROS. Based on this observation, a simple, rapid and sensitive new CL method was developed for the determination of catecholamines. The detection limits (3σ) for dopamine, l‐ dopa, norepinephrine and epinephrine were 0.63, 1.37, 0.56 and 14.3 nmol/L, respectively. The linear range was 1–10 nmol/L; relative standard deviations were 0.71–1.34% for 0.1 µmol/mL catecholamines. This CL method was applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Seasonal variation and correlation analysis of vitamin D and parathyroid hormone in Hangzhou,Southeast China

This study aimed to describe the 25‐hydroxyvitamin D (25(OH)D) and parathyroid hormone (PTH) status of Southeast Chinese individuals influenced by season. The secondary aim was to determine the cutoff for sufficient 25(OH)D in a four‐season region. From January 2011 to June 2014, a total of 17 646 individuals were evaluated in our study. The serum levels of PTH were detected simultaneously in 5579 cases. A total of 25(OH)D and intact PTH were measured by the electrochemiluminescent immunoassay. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D and PTH were studied. The mean 25(OH)D concentration in our study was 43.00(30.40) nmol/L. The prevalence of insufficiency (25(OH)D < 50 nmol/L) was 62.87% and that of deficiency (<30 nmol/L) was 28.54%. Mean serum 25(OH)D levels revealed a limited sinusoidal profile throughout the year and were significantly higher in Autumn. On the other hand, PTH levels showed an opposite response to seasonal effects relative to 25(OH)D. Age, BMI and daylight were not significantly correlated with 25(OH)D and serum PTH reached a plateau at higher values of serum 25(OH)D of 42.86 nmol/L. This study demonstrated that Vitamin D insufficiency is highly prevalent in Southeast China. The concentration of 25(OH)D in the male group was generally higher than that in the female group. Seasonal variation was an important aspect of 25(OH)D and PTH concentration. This study revealed that the optimal serum threshold of 25(OH)D for bone health should be between 40 and 50 nmol/L for Southeast Chinese individuals.     

Simultaneous determination of piroxicam and norfloxacin in biological fluids by high‐performance liquid chromatography with fluorescence detection at zero‐order emission mode

A new highly sensitive high‐performance liquid chromatographic method with fluorescence detection (HPLC–FLD) in zero‐order emission mode was developed for the first time for the simultaneous determination of piroxicam (PRX) and norfloxacin (NRF) in biological fluids. The fluorescence detector wavelengths were set at 278 nm for excitation and zero‐order mode for emission. The zero‐order emission mode produced greater sensitivity for the measurement of both drugs than a fixed emission wavelength (446 nm). The new developed method was validated according to International Conference of Harmonization (ICH) guidelines. Linearity was found to be over concentration ranges 0.001–20 μg/ml and 0.00003–0.035 μg/ml for PRX and NRF, respectively. The limits of detection were 4.87 × 10^?4 and 1.32 × 10^?5 μg/ml for PRX and NRF, and the limits of quantitation were 1.47 × 10^?3 and 4.01 × 10^?5 μg/ml, respectively. The current fluorescence method was found to be more sensitive than most commonly used analytical methods and was successfully applied for simultaneous determination of PRX and NRF in biological fluids (serum and urine) with recoveries ranging from 91.67% to 100.36% for PRX and from 96.00% to 101.43% for NRF.     

Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.     

Hg2+‐selective ratiometric and colorimetric probe based on dansyl–rhodamine and its staining function in cell imaging

A new ratiometric probe composed of a dansyl–rhodamine dyad for the detection of Hg²⁺ via fluorescence resonance energy transfer was designed and synthesized. Rhodamine, dansyl chloride, and hydrazide were selected as the acceptor, donor, and reaction site, respectively. It displayed high selectivity and sensitivity to Hg²⁺ with obvious colour change and fluorescence change due to Hg²⁺‐assisted hydrolysis of rhodamine hydrazide. A good linear relationship ranging from 0 to 16 μM and 0–28 μM for the Hg²⁺ concentration was found based on absorbance and fluorescence assay, respectively. Detection limits of absorbance and fluorescence for Hg²⁺ were calculated to be 1.22 μM and 9.10 μM, respectively.     

Eco‐friendly synthesis of gelatin‐capped bimetallic Au–Ag nanoparticles for chemiluminescence detection of anticancer raloxifene hydrochloride

This study described the utility of green analytical chemistry in the synthesis of gelatin‐capped silver, gold and bimetallic gold–silver nanoparticles (NPs). The preparation of nanoparticles was based on the reaction of silver nitrate or chlorauric acid with a 1.0 wt% aqueous gelatin solution at 50°C. The gelatin‐capped silver, gold and bimetallic NPs were characterized using transmission electron microscopy, UV–vis, X‐ray diffraction and Fourier transform infrared spectroscopy, and were used to enhance a sensitive sequential injection chemiluminescence luminol–potassium ferricyanide system for determination of the anticancer drug raloxifene hydrochloride. The developed method is eco‐friendly and sensitive for chemiluminescence detection of the selected drug in its bulk powder, pharmaceutical injections and biosamples. After optimizing the conditions, a linear relationship in the range of 1.0 × 10^–9 to 1.0 × 10^–1 mol/L was obtained with a limit of detection of 5.0 × 10^–10 mol/L and a limit of quantification of 1.0 × 10^‐9 mol/L. Statistical treatment and method validation were performed based on ICH guidelines. Copyright © 2016 John Wiley & Sons, Ltd.     

Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—Comparison with HPLC methodology

In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 μL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard? column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters^® Quattro Premier? XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9 > 145.6 and 193.9 > 147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97–113%) and the method showed good linearity to 10,000 nmol/L (r² = 0.999). Within-batch and between-batch imprecision was <10% and bias <15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC–MS/MS and HPLC showed good agreement between the two methods but this LC–MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.     

Immunogenic assessment of plant‐produced human papillomavirus type 16 L1/L2 chimaeras

Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus‐like particle (VLP) type–restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second‐generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV‐16 L1‐based chimaeras, containing cross‐protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV‐16 L2 amino acid 108–120, 56–81 or 17–36 substituted into the C‐terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium‐mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108–120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti‐L1 and anti‐L2 responses in mice, and antisera neutralized homologous HPV‐16 and heterologous HPV‐52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.     

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose‐dependent manner

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real‐time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α‐MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL‐2‐antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA‐6 or peroxisome proliferator‐activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α‐MHC, and GATA‐4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.     

Deep soil flipping increases carbon stocks of New Zealand grasslands

Sequestration of soil organic carbon (SOC) has been recognized as an opportunity to off‐set global carbon dioxide (CO₂) emissions. Flipping (full inversion to 1–3 m) is a practice used on New Zealand's South Island West Coast to eliminate water‐logging in highly podzolized sandy soils. Flipping results in burial of SOC formed in surface soil horizons into the subsoil and the transfer of subsoil material low in SOC to the “new” topsoil. The aims of this study were to quantify changes in the storage and stability of SOC over a 20‐year period following flipping of high‐productive pasture grassland. Topsoils (0–30 cm) from sites representing a chronosequence of flipping (3–20 years old) were sampled (2005/07) and re‐sampled (2017) to assess changes in topsoil carbon stocks. Deeper samples (30–150 cm) were also collected (2017) to evaluate the changes in stocks of SOC previously buried by flipping. Density fractionation was used to determine SOC stability in recent and buried topsoils. Total SOC stocks (0–150 cm) increased significantly by 69 ± 15% (179 ± 40 Mg SOC ha^‐1) over 20 years following flipping. Topsoil burial caused a one‐time sequestration of 160 ± 14 Mg SOC ha^‐1 (30–150 cm). The top 0–30 cm accumulated 3.6 Mg SOC ha^‐1 year^‐1. The chronosequence and re‐sampling revealed SOC accumulation rates of 1.2–1.8 Mg SOC ha^‐1 year^‐1 in the new surface soil (0–15 cm) and a SOC deficit of 36 ± 5% after 20 years. Flipped subsoils contained up to 32% labile SOC (compared to <1% in un‐flipped subsoils) thus buried SOC was preserved. This study confirms that burial of SOC and the exposure of SOC depleted subsoil results in an overall increase of SOC stocks of the whole soil profile and long‐term SOC preservation.     

Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds

A new trypsin inhibitor (EATI) was isolated from Entada acaciifolia (Benth.) seeds. EATI is a competitive inhibitor with a molecular mass of 20 kDa and an inhibition stoichiometry of 1:1 for bovine trypsin. The dissociation constant (K_i) calculated was 1.75 nmol/L, displaying a high affinity between enzyme and inhibitor. Both Native PAGE and RP-HPLC revealed that EATI is composed of four isoinhibitors that share the amino acid composition and the amino-terminal sequence homolog to Kunitz-type inhibitors. EATI is stable to denaturation by heat (up to 70 °C), pH (2–10), urea (8 mol/L) and its inhibitory activity was unaltered in different concentrations of DTT (up to 100 mmol/L). CD analysis revealed that EATI in reduced form underwent structural modifications associated with a decrease in thermal and pH stabilities, suggesting that their disulfide bonds are not involved in the structuring of its reactive site, but are important for maintenance of its conformational stability. This behavior makes EATI one of the few inhibitors described in the literature with high DTT resistance.     

Micelle‐enhanced direct spectrofluorimetric method for the determination of linifanib: Application to stability studies

A new simple stability‐indicating spectrofluorimetric method has been developed and validated for the determination of the tyrosine kinase inhibitor, linifanib (LNF). The proposed method makes use of the native fluorescence characteristics of LNF in a micellar system. Compared with aqueous solutions, the fluorescence intensity of LNF was greatly enhanced upon the addition of Tween‐80. The relative fluorescence intensity of LNF was measured in a diluting solvent composed of 2% Tween‐80: phosphate buffer pH 8.0 (20: 80, v/v) using excitation and emission wavelengths of 290 and 450 nm, respectively. The proposed method was fully validated as per the ICH guidelines. The recorded fluorescence intensity of LNF was rectilinear over a concentration range of 0.3–2 μg/ml with a high correlation coefficient (r = 0.9990) and low limits of detection (0.091 μg/ml) and quantitation (0.275 μg/ml). The applicability of the method was extended to study the inherent stability of LNF under different stress degradation conditions including, alkaline, acidic, oxidative, photolytic and thermal degradation. Moreover, the method was utilized to study the kinetics of the alkaline and oxidative degradation of LNF. The pseudo‐first order rate constants and half‐lives were calculated.     

Enhanced chemiluminescence for trazodone trace analysis based on acidic permanganate oxidation in concurrent presence of rhodamine 6G

A new sensitized chemiluminescence method by acidic permanganate oxidation was developed for the sensitive determination of trazodone. A fluorescent dye as used rhodamine 6G to increase a chemiluminescence intensity. Under optimum conditions, the liner range of the calibration curve was obtained for 1–5000 nmol/L. The limit of detection was calculated from 3σ of a blank was 0.23 nmol/L. The coexistent ions and substances had no interference with the chemiluminescence measurement. The chemiluminescence spectra were measured to elucidate a possible mechanism for the system. The present method was satisfactorily used in the determination of the drugs in pharmaceutical samples and animal serums.     

Analysis of flumequine enantiomers in rat plasma by UFLC‐ESI‐MS/MS

In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra‐fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid–liquid extraction using methyl tert‐butyl ether as the extraction solvent. Direct resolution of the R‐ and S‐isomers was performed on a CHIRALCEL OJ‐RH column (4.6 × 150 mm, 5 μm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5–500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43–8.68 and 94.24–106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies.     

Dual effects of [Tyr6]‐γ2‐MSH(6–12) on pain perception and in vivo hyperalgesic activity of its analogues

[Tyr⁶]‐γ2‐MSH(6–12) with a short effecting time of about 20 min is one of the most potent rMrgC receptor agonists. To possibly increase its potency and metabolic stability, a series of analogues were prepared by replacing the Tyr⁶ residue with the non‐canonical amino acids 3‐(1‐naphtyl)‐L ‐alanine, 4‐fluoro‐L ‐phenylalanine, 4‐methoxy‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine. Dose‐dependent nociceptive assays performed in conscious rats by intrathecal injection of the MSH peptides showed [Tyr⁶]‐γ2‐MSH(6–12) hyperalgesic effects at low doses (5–20 nmol) and analgesia at high doses (100–200 nmol). This analgesic activity is fully reversed by the kyotorphin receptor‐specific antagonist Leu–Arg. For the two analogues containing in position 6, 4‐fluoro‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine, a hyperalgesic activity was not observed, while the 3‐(1‐naphtyl)‐L ‐alanine analogue at 10 nmol dose was found to induce hyperalgesia at a potency very similar to γ2‐MSH(6–12), but with longer duration of the effect. Finally, the 4‐methoxy‐L ‐phenylalanine analogue (0.5 nmol) showed greatly improved hyperalgesic activity and prolonged effects compared to the parent [Tyr⁶]‐γ2‐MSH(6–12) compound. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.