Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [2H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Determination of valproic acid and its metabolites using gas chromatography with mass-selective detection: application to serum and urine samples from sheep

An improved method for the quantitative determination of valproic acid (VPA) and sixteen of its metabolites has been developed using gas chromatography-mass spectrometry with selected-ion monitoring. The method is applicable to serum or urine and all metabolites are measured in a single chromatographic run of 29.5 min. Ions selected for quantitative purposes were the characteristic [M-57]⁺ ions of the tert.-butyldimethylsilyl (tBDMS) derivatives. The method utilizes heptadeuterated VPA as well as six heptadeuterated metabolites as internal standards [i.e. 2-[²H₇]propyl-2-pentenoic acid (2-ene[²H₇]VPA), 2-[²H₇]propyl-4-pentenoic acid (4-ene[²H₇]VPA), 2-[²H₇]propyl-3-oxopentanoic acid (3-keto[²H₇]VPA), 2-[²H₇]propyl-4-oxopentanoic acid (4-keto[²H₇]VPA), 2-[²H₇]propyl-3-hydroxypentanoic acid (3-OH[²H₇]VPA), 2-[²H₇]propyl-5-hydroxypentanoic acid (5-OH[²H₇]VPA)]. The method demonstrates very good accuracy and precision over a large range of concentrations for VPA and all metabolites measured in both human and sheep biological fluids. The assay was applied to the analysis of VPA and metabolites in serum and urine samples collected from three non-pregnant ewes following intravenous bolus administration of a mixture of VPA and [¹³C₄]VPA. Sheep were observed to produce measurable quantities of the majority of metabolites found in humans, with the notable exception of the di-unsaturated compounds (i.e. 2,3′-diene VPA and 2,4-diene VPA). The pharmacokinetics and metabolism of VPA and [¹³C₄]VPA appear to be equivalent in the sheep model. The elimination half-life of VPA and [¹³C₄] VPA in the ewe were estimated to be approximately 3.5 ± 0.4 and 3.2 ± 0.4 h, respectively.     

Determination of N-acetylneuraminic acid by gas chromatography-mass spectrometry with a stable isotope as internal standard

N-Acetylneuraminic acid was determined by gas chromatography-mass spectrometry using selected ion-monitoring technique with N-[²H₃]acetylneuraminic acid as an internal standard. M-COOTMS fragments at $\text{m}\text{z}$ 624 of trimethylsilyl derivatives of N-acetylneuraminic acid and at $\text{m}\text{z}$ 627 of that of the internal standard were used as monitoring ions. The standard curve obtained was linear in the range of over 10³, and the lower limit for quantitation was estimated to be a few hundred picograms. This method was used to measure total N-acetylneuraminic acid in the plasma of healthy humans and patients with lung cancer. The total N-acetylneuraminic acid level in the plasma was two to three times higher in the patients than in controls. A few hundred nanoliters of plasma was sufficient for the analysis. The mass fragmentogram of plasma gave a good signal/noise ratio, and measurements were very specific, accurate, and reproducible.     

An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability,bioconversion, and vitamin A status in humans

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [¹³C₁₀]β-carotene and 1 mg [¹³C₁₀]retinyl acetate in human subjects over a 2 week period. A reverse phase C₁₈ column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [¹²C]β-carotene and [¹²C] retinoids; m/z 547→330 and m/z 274→98 for [¹³C₁₀]β-carotene and [¹³C₅] cleavage products; and m/z 279→100 for metabolites of [¹³C₁₀]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [¹³C₁₀]retinyl acetate with [¹³C₁₀]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual''s vitamin A status.     

Determination of glyceryl trinitrate in human plasma by gas chromatography—negative ion chemical ionization—selected ion monitoring

A specific, sensitive and accurate quantitation method for glyceryl trinitrate was developed using gas chromatography—negative ion chemical ionization—selected ion monitoring with dichloromethane as a reagent gas. [¹⁵N₃] and [²H₅, ¹⁵N₃] variants were synthesized from non-labelled or [²H₈]glycerol and [¹⁵N]nitric acid. The former variant was used for preventing adsorption of glyceryl trinitrate onto active sites on column materials and the latter was used as an internal standard for quantitation of glyceryl trinitrate in biological fluids by selected ion monitoring. The quantitation limit of this method is 0.1 ng/ml of human plasma. When glyceryl trinitrate was administered intravenously in the dose of 4 μg/kg to patients receiving hypotensive anesthesia for surgical operation, the plasma levels exhibited a biexponential decay. The mean and standard deviation of half-lives of the α and β phases were found to be about 0.41 ± 0.13 and 5.34 ± 1.60 min, respectively.     

Mono- and bis-cobalt complexes with carbonyl, nitrosyl, and monocarbollide ligands

Reaction of [Co(CO)₃(NO)] with [2-NMe₃-closo-2-CB₁₀H₁₀] in refluxing CH₂Cl₂ affords the mono- and di-cobalt complexes [1-NMe₃-2-CO-2-NO-closo-2,1-CoCB₁₀H₁₀] (3) and [2,7-{Co(CO)(NO)}-7-(μ-H)-1-NMe₃-2-CO-2-NO-closo-2,1-CoCB₁₀H₉] (4), respectively, of which 4 contains formally both Co(I) and Co(-I) centers. Compound 4 reacts with CO to give 3, or with donor ligands L in the presence of Me₃NO to afford simple substituted species, [1-NMe₃-2-L-2-NO-closo-2,1-CoCB₁₀H₁₀] (compounds 5; L = PEt₃, PPh₃, CNBu^t).     

Simultaneous determination, in calf urine, of twelve anabolic agents as heptafluorobutyryl derivatives by capillary gas chromatography-mass spectrometry

A method to determine twelve anabolic hormones (diethylstilbestrol, hexestrol, dienestrol, 17β-estradiol, 19-nortestosterone, testosterone, 1-dehydrotestosterone, 17α-methyltestosterone, progesterone, estrone, 17α-ethynilestradiol, and trenbolone) is presented. Urine samples were extracted with octadecylsilica columns and clean-up was performed in two steps with basic alumina and silica solid-phase extraction cartridges. The extracts obtained were derivatized with heptafluorobutyric anhydride and analyzed by GC-MS. Stability of derivatives was good and compounds having keto groups produced enol derivatives that were stable also. SIM mode was applied to increase the sensitivity and, when possible, the higher m/z ions were selected to improve identification. Repeatability of the chromatographic analysis was evaluated on the basis of area repeatability, and the coefficient of variation obtained was lower than 13%. Absolute recoveries were in the range 35–60% (dehydrotestosterone and estrone <20%) with coefficients of variation between 14 and 37% for the whole procedure. [²H₃]Testosterone and [²H₈]diethylstilbestrol were evaluated to improve quantitative data. The recovery of [²H₃]testosterone was found to be equal to or slightly higher than that of the other hormones, but the recovery of [²H₈]diethylstilbestrol was lower than any other. [²H₃]Testosterone was the most suitable for use as an internal standard, as its addition at the beginning of analytical procedure, corrected recovery results and greatly improved precision. Corrected recoveries from urine ranged from 72–110%, and coefficients of variation ranged from 6–15%, except for testosterone which yielded slightly higher values. The limit of detection was 0.5 ng/ml for all the compounds studied.     

Application of liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the analysis of stable isotope enrichments of phenylalanine and tyrosine

Whole-body protein synthesis and breakdown are measured by a combined tracer infusion protocol with the stable isotope amino acids l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine that enable the measurement of the phenylalanine to tyrosine conversion rate. We describe a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the measurement of very low tracer–tracee ratios (TTR) of the amino acids l-phenylalanine and l-tyrosine in human plasma. TTR calibration curves of the tracers l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine were linear (r² > 0.99) in the range between 0.01% and 5.0% TTR and lowest measurable TTR for the tracers was 0.01% at a physiological concentration of 60 μM. The method was applied successfully to plasma samples from a clinical study reaching a steady state enrichment plateau (mean ± SD) of 3.33 ± 0.19% for l-[ring-²H₅]-phenylalanine, 2.40 ± 0.43% for l-[ring-²H₂]-tyrosine and 0.29 ± 0.07% for l-[ring-²H₄]-tyrosine, respectively. The LC–MS/MS method can be applied for measurement of very low plasma enrichments of phenylalanine and tyrosine for the determination of whole-body protein synthesis and breakdown rates in humans.     

UV-Vis and fluorimetric Al, Zn, Cd and Pb complexation studies of two 3-hydroxyflavones and a 3-hydroxythioflavone

The complexation of Al³⁺, Zn²⁺, Cd²⁺ and Pb²⁺ by the 3-hydroxyflavones: 3-hydroxy-2-(2-methoxyphenyl)-4H-1-benzopyran-4-one (H1) and 3-hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one (H2), and by the 3-methoxythioflavone: 3-hydroxy-2-(2-methoxyphenyl)-4H-1-benzopyran-4-thione (H3) have been studied spectrophotometrically and fluorimetrically to determine the corresponding complexation constants, K_sp and K_fl, in 5:95 water:ethanol (v/v) solution for which [HClO₄] was either 10⁻² or 10⁻⁵ mol dm⁻³ and I = 0.10 mol dm⁻³ (NaClO₄) at 298.2 K. Complexation occurs dominantly through the deprotonated ligand for [Al(1)]²⁺ and [Al(2)]²⁺ for which log K_sp = 4.51 and 4.73, respectively, in 10⁻² mol dm⁻³ HClO₄ and 4.21 and 4.61 in 10⁻⁵ mol dm⁻³ HClO₄. For Pb²⁺ complexation by H1, H2 and H3 is characterized by log K_sp = 2.20, 2.57 and 3.22, respectively, in 10⁻² mol dm⁻³ HClO₄ and 4.70, 5.38 and 5.74 in 10⁻⁵ mol dm⁻³ HClO₄. Equilibrium mixtures of [Pb(H1)]²⁺ and [Pb1]⁺, [Pb(H2)]²⁺ and [Pb2]⁺, and [Pb(H3)]²⁺ and [Pb3]⁺ appear to be formed. Complexation of Zn²⁺ and Cd²⁺ by all three ligands was only detected in 10⁻⁵ mol dm⁻³ HClO₄. For Zn²⁺ complexation by H1, H2 and H3 log K_sp = 3.22, 3.74 and 4.46 and for Cd²⁺ the corresponding values are 2.39, 2.40 and 3.72 for Cd²⁺. Only [Al1]²⁺ and [Al2]²⁺ show significant fluorescence and are characterized by log K_fl = 6.30 and 7.49 in 10⁻² mol dm⁻³ HClO₄.     

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3α,11β,17α,21-tetrahydroxy-5β-pregnane-20-one), allo-tetrahydrocortisol (allo-THF, 3α,11β,17α,21-tetrahydroxy-5α-pregnane-20-one) and tetrahydrocortisone (THE, 3α,17α,21-trihydroxy-5β-pregnane-11,20-dione) in human plasma and urine is described. [1,2,3,4,5-²H₅]THF (THF-d₅), allo-[1,2,3,4,5-²H₅]THF (allo-THF-d₅) and [1,2,3,4,5-²H₅]THE (THE-d₅) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M−30]⁺) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.     

Determination of salbutamol enantiomers in human plasma and urine by chiral high-performance liquid chromatography

Enantiomers of salbutamol were directly separated (R_s=1.16) and quantitated at therapeutic concentrations after solid-phase extraction from human plasma and urine by normal-phase high-performance liquid chromatography on a chiral column with fluorescence detection. The assay was linear for each enantiomer between 1.25 and 500 ng ml⁻¹ and had a minimum limit of detection of 250 pg ml⁻¹. A 3-ml plasma or 1-ml urine sample was required for quantitation at therapeutic doses. Inter-day variation was 50% for S-(+)- and 6.5% for R-(−)-salbutamol. The assay was used to compare enantioselective disposition after single doses of racemate by the intravenous, oral and rectal routes.     

PLASMA CHOLINE: ITS TURNOVER AND EXCHANGE WITH BRAIN CHOLINE1

—The kinetics of plasma choline (Ch) and the uptake of plasma Ch into the brain were studied by means of intravenous infusion of [²H₄]Ch at various rates into anaesthetized and conscious rats. [²H₄]Ch levels in both arterial and venous plasma at steady state were linearly related to the infusion rate; however, unlabelled Ch levels were independent of infusion rate. [²H₄]Ch levels were higher in the arterial plasma than in the venous plasma, while unlabelled Ch levels were higher in the venous plasma than in the arterial plasma. It was concluded that Ch is being generated in the brain and is released into the venous effluent. The supply of Ch to the plasma is not decreased if the plasma Ch level is increased. The clearance and turnover of Ch in the compartment of its initial distribution are 75 ml kg^-1 min^-1 and 716 nmol kg^-1 min^-1, respectively. The uptake of plasma Ch into the brain is not saturated even at very high levels of plasma Ch.     

Transportation of de novo synthesized jasmonoyl isoleucine in tomato

In plants, jasmonic acid (JA) and its derivatives are thought to be involved in mobile forms of defense against biotic and abiotic stresses. In this study, the distal transport of JA-isoleucine (JA-Ile) that is synthesized de novo in response to leaf wounding in tomato (Solanum lycopersicum) plants was investigated. JA-[¹³C₆]Ile was recovered in distal untreated leaves after wounded leaves were treated with [¹³C₆]Ile. However, as [¹³C₆]Ile was also recovered in the distal untreated leaves, whether JA-Ile was synthesized in the wounded or in the untreated leaves was unclear. Hence, stem exudates were analyzed to obtain more detailed information. When [¹³C₆]Ile and [²H₆]JA were applied separately into the wounds on two different leaves, JA-[¹³C₆]Ile and [²H₆]JA-Ile were detected in the stem exudates but [²H₆]JA–[¹³C₆]Ile was not, indicating that JA was conjugated with Ile in the wounded leaf and that the resulting JA-Ile was then transported into systemic tissues. The [²H₃]JA-Ile that was applied exogenously to the wounded tissues reached distal untreated leaves within 10 min. Additionally, applying [²H₃]JA-Ile to the wounded leaves at concentrations of 10 and 60 nmol/two leaves induced the accumulation of PIN II, LAP A, and JAZ3 mRNA in the distal untreated leaves of the spr2 mutant S. lycopersicum plants. These results demonstrate the transportation of de novo synthesized JA-Ile and suggest that JA-Ile may be a mobile signal.     

GC–MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK_L 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC–MS method for the analysis of S-nitrosothiols and their ¹⁵N- or ²H–¹⁵N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S¹⁵NACET or d₃-S¹⁵NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO₂]^?) for SNACET and m/z 47 (i.e., [¹⁵NO₂]^?) for S¹⁵NACET/d₃-S¹⁵NACET for compound X, and m/z 157 for SNACET and m/z 160 for d₃-S¹⁵NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S¹⁵NACET/d₃-S¹⁵NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0–10 μM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200 nM in total. Oral administration of S¹⁵NACET to rabbits (40–63 μmol/kg body weight) resulted in formation of ALB-S¹⁵NO, [¹⁵N]nitrite and [¹⁵N]nitrate in plasma.     

Determination of free salsolinol concentrations in human urine using gas chromatography—mass spectrometry

The urine concentrations of free salsolinol were determined in six healthy volunteers, using a gas chromatographic—mass spectrometric method with electron-capture negative-ion chemical ionization after derivatization with pentafluoropropionyl anhydride. The sensitivity of this method allows the quantification of salsolinol concentrations of 0.55 pmol/ml. The synthesis of [²H₄]salsolinol from dopamine and [²H₄]acetaldehyde via a Pictet—Spengler condensation is described; [²H₄]salsolinol was used as the internal standard for salsolinol quantification. The urine concentrations of free salsolinol ranged from ca. 1 to 6 pmol/ml.     

Electrospray mass spectrometry of cis-[Ru(NO)Cl(bpy)2] (bpy=2,2-bipyridine): fragmentation from desolvated {[Ru(NO)Cl(bpy)2], Cl} ion pairs by electron transfer and internal Lewis base pathways

The electrospray mass spectrum (ESI-MS) of cis-[Ru(NO)Cl(bpy)₂]Cl₂ (bpy=2,2^′-bipyridine), obtained from 50% CH₃OH/50% H₂O as the mobile solvent, exhibited ruthenium-containing ions derived from a {[Ru^II(NO⁺)Cl(bpy)₂]²⁺, Cl⁻}⁺ ion pair (m/z=514) and [Ru^II(NO⁺)Cl(bpy)₂]²⁺ (m/z=239.5). [Ru^IIICl(bpy)₂]²⁺, from the loss of NO from the 239.5 ion, is detected at m/z=224.5. Only the m/z 514 ion pair is detected when 100% CH₃OH mobile solvent is used, but the presence of even small amounts of water prompted the additional detection of the m/z 239.5 and m/z 224.5 ions under tandem MS-MS conditions. Ruthenium-chloro-containing ions appear as a characteristic collection of eight main, and four lesser, intense ions created from combinations ¹⁰⁴Ru, ¹⁰²Ru, ¹⁰¹Ru, ⁹⁹Ru, ⁹⁸Ru, ⁹⁶Ru, ³⁵Cl and ³⁷Cl isotopes with minor contributions from ¹³C, etc. For convenience of discussion, only the most abundant m/z species are mentioned herein as representative of all the isotopically distributed ions.Four fragmentation channels are detectable from the m/z=514 chloride ion pair: (1) the loss of HCl (main channel; ca. 50% of fragmentation events), (2) the loss of NO (ca. 12% ), (3) the loss of bpy (minor pathway), and (4) the loss of Cl atom (ca. 38% ).Loss of NO from ion m/z 514 yields ion m/z 484, which is the precursor of ions m/z 448 (by loss of HCl), m/z 328 (by loss of bpy) and m/z 292 (by loss of HCl and bpy). Loss of HCl from ion m/z 514 generates ion m/z 478, [Ru^II(NO⁺)Cl(bpyH)(bpy-H)]⁺, deprotonated at the ortho C-H of one bpy ligand. In MS-MS experiments, the m/z 478 ion was established to undergo loss of NO, producing ion m/z 448, rejoining further fragmentation process for ion m/z 448 at this point. Loss of neutral bipyridine from m/z 514 in low yield produces ion m/z 358, which undergoes further loss of NO to form [RuCl₂(bpy)]⁺ ion (m/z=328). MS-MS “neutral loss of 30” spectra confirmed the NO loss events as part of the fragmentation sequence for all four pathways.A fourth species of m/z=479 from the “514” ion is obtained by an internal electron transfer from Cl⁻ of the ion pair, and loss of the resultant neutral Cl atom. The product [Ru^II(NO^·)Cl(bpy)₂]⁺ “479” fragment undergoes facile loss of NO to generate [Ru^IICl(bpy)₂]⁺ (m/z=449). Ion m/z 449 gives rise to ions m/z 413 (loss of HCl) and m/z 257(loss of HCl and bpy). MS-MS experiments confirm the neutral loss of Cl from the m/z 514 ion, and the formation of the m/z 449 ion via m/z 479 and m/z 514 parents. This pathway was not observed in a prior study for the related complex, [Ru(NO)Cl(dpaH)(dpa⁻)]⁺ (dpaH=2,2^′-dipyridylamine), which does not have an external Cl⁻ in an ion pair.     

Relationship between NaCl- and H2O2-Induced Cytosolic Ca2+ Increases in Response to Stress in Arabidopsis

Salinity is among the environmental factors that affect plant growth and development and constrain agricultural productivity. Salinity stress triggers increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Ca²⁺ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS). It is well established that ROS also triggers increases in [Ca²⁺]_i. However, the relationship and interaction between salinity stress-induced [Ca²⁺]_i increases and ROS-induced [Ca²⁺]_i increases remain poorly understood. Using an aequorin-based Ca²⁺ imaging assay we have analyzed [Ca²⁺]_i changes in response to NaCl and H₂O₂ treatments in Arabidopsis thaliana. We found that NaCl and H₂O₂ together induced larger increases in [Ca²⁺]_i in Arabidopsis seedlings than either NaCl or H₂O₂ alone, suggesting an additive effect on [Ca²⁺]_i increases. Following a pre-treatment with either NaCl or H₂O₂, the subsequent elevation of [Ca²⁺]_i in response to a second treatment with either NaCl or H₂O₂ was significantly reduced. Furthermore, the NaCl pre-treatment suppressed the elevation of [Ca²⁺]_i seen with a second NaCl treatment more than that seen with a second treatment of H₂O₂. A similar response was seen when the initial treatment was with H₂O₂; subsequent addition of H₂O₂ led to less of an increase in [Ca²⁺]_i than did addition of NaCl. These results imply that NaCl-gated Ca²⁺ channels and H₂O₂-gated Ca²⁺ channels may differ, and also suggest that NaCl- and H₂O₂-evoked [Ca²⁺]_i may reduce the potency of both NaCl and H₂O₂ in triggering [Ca²⁺]_i increases, highlighting a feedback mechanism. Alternatively, NaCl and H₂O₂ may activate the same Ca²⁺ permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways.     

Interactions of verapamil,D 600, flunarizine and nifedipine with cerebral histamine-receptors

Experiments conducted on membrane fractions of guinea-pig brain using ligand-binding techniques have shown that certain Ca²⁺-antagonists interact with histamine (H₁ or H₂) receptors. Flunarizine (inhibition constant, K_i ∼ 86 nM) was nearly as potent as diphenhydramine (K_i ∼ 44 nM) in inhibiting [³H]pyrilamine binding to cerebellar H₁-receptors, whereas verapamil, D 600 and nifedipine did not interact with this site. Regarding [³H]tiotidine binding to H₂-receptors of cerebral cortex, verapamil (K_i ∼ 1400 nM) and D 600 (K_i ∼ 1240 nM) were nearly as potent as cimetidine (K_i ∼ 910 nM) whereas flunarizine and nifedipine were inactive. The interaction of flunarizine with H₁-receptors might explain, in part, its sedative side-effect. The interaction of verapamil with H₂-receptors, demonstrated here for the first time, might be involved in the anti-arrhythmic action of this agent.     

A >22 Å long metal-complex anion and a 1D cationic coordination polymer, both based on 4,4′-bipyridine-N,N′-dioxide, yielding a 3D and 2-fold interpenetrated fsc net of the hydrogen-bonded metal-organic 1D polymer

The cationic one-dimensional (1D) coordination polymer chain ¹_∞{[Co(μ-bpdo)(H₂O)₄]²⁺} and the metal-complex anion trans-[Co(SO₄)₂(bpdo)₂(H₂O)₂]²⁻, both based on the 4,4′-bipyridine-N,N′-dioxide (bpdo) ligand, form a complementary supramolecular pair ¹_∞{[Co(μ-bpdo)(H₂O)₄]²⁺}_nn[Co(SO₄)₂(bpdo)₂(H₂O)₂]²⁻ (1) with respect to charge balance and hydrogen bonding. With a length of >22.14 ? along the bpdo-Co-bpdo axis the metal-complex trans-[Co(SO₄)₂(bpdo)₂(H₂O)₂]²⁻ is one of the longest and anisotropic counter anions (aspect ratio 22.14:8.11:4.17) observed so far in coordination polymers. Hydrogen-bonding of the anion links the cationic metal-organic 1D polymer into a 2-fold interpenetrated three-dimensional (3D) fsc (or sqc11) 4,6-c 2-nodal net of stoichiometry (4-c)(6-c) with square-planar, 4-connected (Co in anion) and octahedral, 6-connected (Co in cation) nodes in a 1:1 ratio. The 4-c point symbol is (4⁴.6²), the 6-c one (4⁴.6¹⁰.8) yielding a point symbol for the fsc net of (4⁴.6²)(4⁴.6¹⁰.8). The synthesis of 1 requires the presence of a Schiff base. Synthesis under the same conditions in the absence of the Schiff base yields the molecular complex and cocrystal [Co(bpdo)(H₂O)₅]SO₄·1/2bpdo (2) which is related (as pseudo-polymorph) to the known solvate [Co(bpdo)(H₂O)₅]SO₄·2H₂O (3) (CSD Refcodes RAXMUZ and RAXMUZ01).