Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, d-Trp7,9, mePhe8]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, d-Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

Effects of morphine-3-glucuronide on the antinociceptive activity of peptide and nonpeptide opioid receptor agonists in mice

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a μ-opioid receptor agonist, of U-50,488H, a κ-opioid receptor agonist, of [ -Pen², -Pen⁵]enkephalin (DPDPE), a δ₁-opioid receptor agonist, and of [ -Ala²,Glu⁴]deltorphin II (deltorphin II), a δ₂-opioid receptor agonist in mice. Morphine administered ICV (2.5 μg/mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 μg/mouse; ICV), and deltorphin II (15 μg/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of μ-, κ-, δ₁-, or δ₂-opioid receptor agonists.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Model experiments on squid axons and NG108-15 mouse neuroblastoma × rat glioma hybrid cells

Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na⁺ current, the M current and the low-voltage-activated Ca²⁺ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves (Proc R Soc Lond B (1993) 253, 61–68) studied the persistent or steady-state Na⁺ current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na⁺ current (PF_peak and PF_ss) as a function of voltage. Whereas PF_peak starts to rise at −50 mV and reaches a maximum at +40 to +50 mV, PF_ss only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na⁺ current turns on in the threshold region and saturates at around 0 mV. Schmitt and Meves (Pflügers Arch (1993) 425, 134–139) recorded M current, a non-inactivating K⁺ current, from NG108-15 neuroblastoma × glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased I_M, the effective concentrations being 0.1–1 μM and 5–25 μM, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19–31 peptide, an effective inhibitor of PKC, in a concentration of 1 μM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves (Pflügers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca²⁺ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > −20 mV, AA (25–100 μM) decreased l-v-a and h-v-a I_Ca. The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a I_Ca. The AA effect was not prevented by 50 μM eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19–31 peptide and not mimicked by 0.1–1 μM PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed.     

The effects of Ca and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied β-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5–8 nmol/min per ml ghosts are remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (K_a) for (±)-isoprenaline from 0.1 to 0.6 μM. The apparent dissociation constant for propranolol (0.01 μM) remained unchanged. The K_a values for isoprenaline in native reticulocytes and in resealed ghosts were identi The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal β-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca²⁺ concentrations ranging between 0.1 and 1 μM. GTP stimulated iosprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3–5 but did not change the K_a value for isoprenaline. K_a values for the guanylnucleotides in different experiments varied between 0.3 and 2 μM. Ca²⁺ concentrations up to 4.6 μM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their K_a values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native β-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

A Xanthine-Derivative K+-Channel Opener Protects against Serotonin-Induced Cardiomyocyte Hypertrophy via the Modulation of Protein Kinases

This study investigated whether KMUP-1, a xanthine-derivative K⁺ channel opener, could prevent serotonin-induced hypertrophy in H9c2 cardiomyocytes via L-type Ca²⁺ channels (LTCCs). Rat heart-derived H9c2 cells were incubated with serotonin (10 μM) for 4 days. The cell size increased by 155.5%, and this was reversed by KMUP-1 (≥1 μM), and attenuated by the LTCC blocker verapamil (1 μM) and the 5-HT_2A antagonist ketanserin (0.1 μM), but unaffected by the 5-HT_2B antagonist SB206553. A perforated whole-cell patch-clamp technique was used to investigate Ca²⁺ currents through LTCCs in serotonin-induced H9c2 hypertrophy, in which cell capacitance and current density were increased. The LTCC current (I_Ca,L) increased ~2.9-fold in serotonin-elicited H9c2 hypertrophy, which was attenuated by verapamil and ketanserin, but not affected by SB206553 (0.1 μM). Serotonin-increased I_Ca,L was reduced by KMUP-1, PKA and PKC inhibitors (H-89, 1 μM and chelerythrine, 1 μM) while the current was enhanced by the PKC activator PMA, (1 μM) but not the PKA activator 8-Br-cAMP (100 μM), and was abolished by KMUP-1. In contrast, serotonin-increased I_Ca,L was blunted by the PKG activator 8-Br-cGMP (100 μM), but unaffected by the PKG inhibitor KT5823 (1 μM). Notably, KMUP-1 blocked serotonin-increased I_Ca,L but this was partially reversed by KT5823. In conclusion, serotonin-increased I_Ca,L could be due to activated 5-HT_2A receptor-mediated PKA and PKC cascades, and/or indirect interaction with PKG. KMUP-1 prevents serotonin-induced H9c2 cardiomyocyte hypertrophy, which can be attributed to its PKA and PKC inhibition, and/or PKG stimulation.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Sequences of initiator and elongator methionine tRNAs in bean mitochondria

Summary Two bean mitochondria methionine transfer RNAs, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced usingin vitro post-labeling techniques.One of these tRNAs^Met has been identified by formylation using anE. coli enzyme as the mitochondrial tRNA_F ^Met. It displays strong structural homologies with prokaryotic and chloroplast tRNA_F ^Met sequences (70.1–83.1%) and with putative initiator tRNA_m ^Met genes described for wheat, maize andOenothera mitochondrial genomes (88.3–89.6%).The other tRNA^Met, which is the mitochondrial elongator tRNA_F ^Met, shows a high degree of sequence homology (93.3–96%& with chloroplast tRNA_m ^Met, but a weak homology (40.7%) with a sequenced maize mitochondrial putative elongator tRNA_m ^Met gene.Bean mitochondrial tRNA_F ^Met and tRNA_m ^Met were hybridized to Southern blots of the mitochondrial genomes of wheat and maize, whose maps have been recently published (15, 22), in order to locate the position of their genes.     

Ecophysiology of the marine cyanobacterium, Lyngbya majuscula (Oscillatoriaceae) in Moreton Bay, Australia

Large blooms of the marine cyanobacterium Lyngbya majuscula in Moreton Bay, Australia (27°05′S, 153°08′E) have been re-occurring for several years. A bloom was studied in Deception Bay (Northern Moreton Bay) in detail over the period January–March 2000. In situ data loggers and field sampling characterised various environmental parameters before and during the L. majuscula bloom. Various ecophysiological experiments were conducted on L. majuscula collected in the field and transported to the laboratory, including short-term (2 h) ¹⁴C incorporation rates and long-term (7 days) pulse amplitude modulated (PAM) fluorometry assessments of photosynthetic capacity. The effects of L. majuscula on various seagrasses in the bloom region were also assessed with repeated biomass sampling. The bloom commenced in January 2000 following usual December rainfall events, water temperatures in excess of 24 °C and high light conditions. This bloom expanded rapidly from 0 to a maximum extent of 8 km² over 55 days with an average biomass of 210 g_dw⁻¹ m⁻² in late February, followed by a rapid decline in early April. Seagrass biomass, especially Syringodium isoetifolium, was found to decline in areas of dense L. majuscula accumulation. Dissolved and total nutrient concentrations did not differ significantly (P > 0.05) preceding or during the bloom. However, water samples from creeks discharging into the study region indicated elevated concentrations of total iron (2.7–80.6 μM) and dissolved organic carbon (2.5–24.7 mg L⁻¹), associated with low pH values (3.8–6.7). ¹⁴C incorporation rates by L. majuscula were significantly (P < 0.05) elevated by additions of iron (5 μM Fe), an organic chelator, ethylenediaminetetra-acetic acid (5 μM EDTA) and phosphorus (5 μM PO₄⁻³). Photosynthetic capacity measured with PAM fluorometry was also stimulated by various nutrient additions, but not significantly (P > 0.05). These results suggest that the L. majuscula bloom may have been stimulated by bioavailable iron, perhaps complexed by dissolved organic carbon. The rapid bloom expansion observed may then have been sustained by additional inputs of nutrients (N and P) and iron through sediment efflux, stimulated by redox changes due to decomposing L. majuscula mats.     

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO₃⁻, nmol cm^− 2 s^− 1 − 1,76 ± 0,11_control × 1,29 ± 0,08_{REN 10 μM}; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO₃⁻, nmol cm^− 2 s⁻ 1 − 0.80 ± 0.07_control × 0.60 ± 0.06_{REN 1 μM}; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na⁺/H⁺exchanger and H⁺-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.     

Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

High-performance liquid chromatographic assay for melanotan-1 ([Nle-DPhe]α-melanocyte-stimulating hormone) in biological matrices

The overall objective of this research was to develop a sensitive, specific, and stability-indicating HPLC assay for the determination of the [Nle⁴-DPhe⁷]α-melanocyte-stimulating hormone analog known as Melanotan-1 (MT-1) in biological matrices, i.e., cell culture transport media and human plasma. Separation was accomplished isocratically within 8.0 min using a C₈ reversed-phase column. The mobile phase consisted of 0.1 M phosphate buffer-acetonitrile (80:20, v/v) with 18 μl/l triethylamine at pH 2.50. The flow-rate was 1 ml/min with detection at 214 nm. Standard curves (n = 5) were linear over the concentration range 100–1000 ng/ml. The precision, accuracy, intra- and inter-day variations were good with C.V.s typically within 8.7% for concentrations greater than 100 ng/ml. This method was applied to a study of the transport of MT-1 in the Caco-2 cell monolayer model.     

High-performance liquid chromatography coupled to ion spray mass spectrometry for the determination of colchicine at ppb levels in human biofluids

An original method based upon high-performance liquid chromatography coupled to ion spray mass spectrometry (HPLC-ISP-MS) has been developed for the identification and quantification of colchicine (COL) in human blood, plasma or urine. After single-step liquid-liquid extraction by dichloromethane at pH 8.0 using tofisopam (TOF) as an internal standard, solutes are separated on a 5-μm C₁₈ Microbore (Alltech) column (250×1.0 mm, I.D.), using acetonitrile-2 mM NH₄COOH, pH 3 buffer (75:25, v/v) as the mobile phase (flow-rate 50 μl/min). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with a ISP interface (nebulizing and curtain gas: N₂, quality U; main settings: ISP, +4.0 kV; OR, +50 V; Q0, −10 V; Q1, −13 V; electron multiplier, +2.2 kV); MS data are collected as either total ion current (TIC, m/z 100–500 or 380–405), or selected ion monitoring (SIM) at m/z 400 and 383 for COL and TOF, respectively. COL mass spectrum shows a prominent molecular ion [M+H]⁺ at m/z 400. Increasing OR potential fails to provide a significant fragmentation. Retention times are 2.70 and 4.53 min for COL and TOF, respectively. The quantification method shows a good linearity (r = 0.998) over a concentration range from 5 to 200 ng/ml. The lower limit of detection in SIM mode is 0.6 ng/ml COL, making the method convenient for both clinical and forensic purposes.     

Microbore high-performance liquid chromatographic determination of cisapride in rat serum samples using column switching

For the determination of cisapride from serum samples, an automated microbore high-performance liquid chromatographic method with column switching has been developed. After serum samples (100 μl) were directly injected onto a Capcell Pak MF Ph-1 pre-column (10×4 mm I.D.), the deproteinization and concentration were carried out by acetonitrile–phosphate buffer (20 mM, pH 7.0) (2:8, v/v) at valve position A. At 2.6 min, the valve was switched to position B and the concentrated analytes were transferred from MF Ph-1 pre-column to a C₁₈ intermediate column (35×2 mm I.D.) using washing solvent. By valve switching to position A at 4.3 min, the analytes were separated on a Capcell Pak C₁₈ UG 120 column (250×1.5 mm I.D.) with acetonitrile–phosphate buffer (20 mM, pH 7.0) (5:5, v/v) at a flow-rate of 0.1 ml/min. Total analysis time per sample was 18 min. The linearity of response was good (r=0.999) over the concentration range of 5–200 ng/ml. The within-day and day-to-day precision (CV) and inaccuracy were less than 3.7% and 3.8%, respectively. The mean recovery was 96.5±2.4% with the detection limit of 2 ng/ml.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05–100 mg/l for rat blood (r²>0.9995, n=6) and dog blood (r²>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-μl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1–104.1% and 1.5–6.8% for rat blood and 95.4–105.7% and 1.4–5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8–105.3% and 3.7–12.6% for rat blood and 87.5–107.1% and 2.9–15.3% for dog blood, respectively. The absolute recoveries were 82.4–93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.