High-performance liquid chromatographic determination of spectinomycin in swine,calf and chicken plasma using post-column derivatization

An HPLC method for the determination of spectinomycin in swine, calf and chicken plasma at 0.1 μg/ml or higher is described. The clean-up is based upon ion-pair solid-phase extraction on a High Hydrophobic C₁₈ column treated with sodium dioctyl suflosuccinate. After elution with methanol, spectinomycin is chromatographed on a Spherisorb SCX column using 0.1 M sodium sulphate solution (pH 2.6)-acetonitrile (80:20, v/v) as mobile phase. Fluorescence detection is at an excitation wavelength of 340 nm and an emission wavelength of 460 nm after post-column oxidation with sodium hypochlorite followed by derivatization with o-phthaldialdehyde. Mean recoveries were 99 ± 2% (n = 6), 99 ± 2% (n = 7) and 104 ± 2% (n = 6) for swine, calf and chicken plasma, respectively, at the 0.1 μg/ml level.     

Sensitive assay for measuring amoxicillin in human plasma and middle ear fluid using solid-phase extraction and reversed-phase high-performance liquid chromatography

We developed a sensitive assay to measure amoxicillin in human plasma and midle ear fluid (MEF) using solid-phase extraction and reversed-phase HPLC. Amoxicillin and cefadroxil, the internal standard, were extracted from 50–200 μl of sample with Bond Elut C₁₈ cartridges. The exact was analyzed on a 15 cm × 2 mm, 5μm Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer (pH = 6.5) and 5 mM tetrabutylammonium. The average absolute recovery of amoxicillin and cefadroxil were 91.2 ± 16.6% and 91.0 ± 6.8%, respectively. The limit of quantitation was 0.125 μg/ml with 200 μl sample size. The linear range was from 0.125 to 35.0 μg/ml with correlation coefficients greater than 0.999. These analytic conditions produced a highly sensitive amoxicillin assay in human body fluids without derivatization.     

Determination of phosphonoformate (foscarnet) in calf and human serum by automated solid-phase extraction and high-performance liquid chromatography with amperometric detection

An isocratic high-performance liquid chromatographic method with automated solid-phase extraction has been developed to determine foscarnet in calf and human serums. Extraction was performed with an anion exchanger, SAX, from which the analyte was eluted with a 50 mM potassium pyrophosphate buffer, pH 8.4. The mobile phase consisted of methanol–40 mM disodium hydrogenphosphate, pH 7.6 containing 0.25 mM tetrahexylammonium hydrogensulphate (25:75, v/v). The analyte was separated on a polyether ether ketone (PEEK) column 150×4.6 mm I.D. packed with Kromasil 100 C₁₈, 5 μm. Amperometric detection allowed a quantification limit of 15 μM. The assay was linear from 15 to 240 μM. The recovery of foscarnet from calf serum ranged from 60.65±1.89% for 15 μM to 67.45±1.24% for 200 μM. The coefficient of variation was ≤3.73% for intra-assay precision and ≤7.24% for inter-assay precision for calf serum concentrations ranged from 15 to 800 μM. For the same samples, the deviation from the nominal value ranged from −8.97% to +5.40% for same day accuracy and from −4.50% to +2.77% for day-to-day accuracy. Selectivity was satisfactory towards potential co-medications. Replacement of human serum by calf serum for calibration standards and quality control samples was validated. Automation brought more protection against biohazards and increase in productivity for routine monitoring and pharmacokinetic studies.     

Simultaneous determination of plasma phenytoin and its primary hydroxylated metabolites in carbon tetrachloride-intoxicated rats by high-performance liquid chromatography

We have developed a simple and sensitive method for the simultaneous determination of phenytoin (PHT), 5(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in rat plasma by high-performance liquid chromatography. The three substances were separated on a reversed-phase column (5 μm TSK gel ODS-80TM, 250 mm × 4.6 mm I.D.) using acetonitrile-0.008 M NaH₂PO₄ (pH 6) (35:65, v/v) as a mobile phase at a flow-rate of 0.8 ml/min. Absorbance was monitored at 215 nm. The quantification limit was 50 ng/ml for each of PHT, m-HPPH and p-HPPH. The mean recoveries for DPH, m-HPPH and p-HPPH from plasma were 95.6±3.6, 94.5±4.2 and 98.6±2.9%, respectively.     

1,3-β-d-Glucan synthase ofNeurospora crassa: Partial purification and characterization of solubilized enzyme activity

1,3-β-d-Glucan synthase activity ofNeurospora crassa was rendered soluble by treatment of crude protoplast lysates with 0.1% 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 600 mM glycerol. Approximately 50% of enzyme activity was solubilized; soluble enzyme activity was purified 5.6-fold with a net 38% recovery by sucrose gradient density sedimentation. Partially purified enzyme activity had a half-life of 60 ± 10 h at 4°C, aK_m,app of 0.75 ± 0.05 mM, and a V_max,app of 35 ± 1 enzyme units/mg protein.     

High-performance liquid chromatography with electrochemical detection for the determination of 7-monohydroxyethylrutoside in plasma

MonoHER (7-monohydroxyethyl rutoside) is a semisynthetic flavonoid, which can be used as a modulator for doxorubicin-induced cardiotoxicity. To study the pharmacokinetics of monoHER in mice and human an HPLC procedure was developed to measure the level of monoHER in plasma. After extraction of monoHER with methanol, the supernatant was equally diluted (v/v) with 25 mM phosphate buffer (pH 3.33). This solution was analysed by HPLC, using a reversed-phase ODS column, with a mobile phase consisting of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogen phosphate (pH 3.4), 10 mM acetic acid and 36μM EDTA. The retention time of monoHER was about 5.2 min. The lower limit of quantification of monoHER was set at 0.3 μM and the calibration line was linear up to 75 μM. The within-day accuracy and precision of the quality control samples (0.45, 1.0, 10 and 40 μM) were better than 15 and 13%, respectively. The between-day accuracy and precision were less than 3, 20%, respectively. The recovery of monoHER (using quality control concentrations) was concentration independent and ranged from 90.5 to 95.3% except for the lowest quality control, 0.45 μM, of which the recovery was 85%. The concentration of monoHER in plasma decreased with 10% when stored at −80°C for one month and with 20% when stored at −20°C for 3 weeks. The repeated injection of monoHER in aliquots of 10 μM, stored in the autosampler tray (4°C), showed a consistent decrease during a run: 15% over 24 h. To compensate for this decrease, sample duplicates were analysed in a mirror image sequence.     

Pharmacokinetic studies of amiloride and its analogs using reversed-phase high-performance liquid chromatography

We have studied the pharmacokinetics of amiloride and its analogs. A high-performance liquid chromatographic method has been adapted for the measurement of amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N,N-hexamethylene)amiloride (HMA) in mouse plasma, kidney, liver and tumor tissues. The method uses a C₈ preparative solid-phase column, followed by separation using a reversed-phase C₁₈ column (250×4 mm I.D., 5 μm particle size) with detection by ultraviolet absorption at 365 nm. Reversed-phase separations were performed at ambient temperature using a non-linear gradient method with two different mobile phases: mobile phase A was 100% acetonitrile while mobile phase B was 0.15 M perchloric acid at pH 2.20 (flow-rate was 1.2 ml/min). The retention times for amiloride, benzamil (used as an internal standard), EIPA and HMA are 13.4, 19.5, 21.8 and 23.5 min, respectively. The calibration curves are linear over the range of 0.1–50 μM in plasma and in tissues. The half-lives of amiloride, EIPA and HMA (and their confidence intervals) in plasma after intraperitoneal injection of drugs into mice were 68.8±0.2, 31.2±2.5 and 39.3±7.9 min, respectively. Amiloride was detected as a metabolite of EIPA but not of HMA. When EIPA was injected at a dose of 10 μg/g body weight, it was cleared rapidly from liver, but concentrations > 1 μM were sustained for at least 2 h in murine kidney and in a transplantable tumor.     

High-performance liquid chromatography separation and quantitation of ofloxacin enantiomers in rat microsomes

A sensitive, simple and accurate method for determination of enantiomers of ofloxacin in microsomal incubates was developed by chiral ligand-exchange RP-HPLC with fluorescence detection to examine stereoselective metabolism of ofloxacin in the glucuronidation process. The C₁₈ stationary phase was used as analytical column. The solution of chiral mobile phase additive was made up of 6 mM l-phenylalamine mixed with 3 mM CuSO₄ in water. Mobile phase consisted of the solution of chiral mobile phase additive–methanol (86:14).The fluorescence detector was operated at λ_ex 330 nm and λ_em 505 nm. The flow-rate of mobile phase was set at 1.0 ml/min. The achiral ODS column offers good separation of the two enantiomers in less than 25 min. The recovery of the assay was 97.9±6.1% (n=10) for S-ofloxacin and 99.6±6.0% (n=10) for R-ofloxacin. The method provides a high sensitivity and good precision (RSD<10%). The LOD was 0.6 μM for both enantiomers and the LOQ was 5.70±0.45 μM (n=8) for S-ofloxacin and 5.66±0.47 μM (n=8) for R-ofloxacin. The standard curves showed excellent linearity over the concentration range 5.5–2078 μM for S-(−)-ofloxacin and R-(+)-ofloxacin. The enantioselective method developed has been applied to determine the stereoselectivity of glucuronidation metabolism of ofloxacin optical isomers in rat liver microsomes.     

Normal or induced secretory patterns of luteinising hormone and follicle-stimulating hormone in anoestrous gonadotrophin-releasing hormone-immunised and cyclic control heifers

The objective was to determine the effect of gonadotrophin-releasing hormone (GnRH), GnRH analogue (GnRH-A) or oestradiol administration on luteinising hormone (LH) and follicle-stimulating hormone (FSH) release in GnRH-immunised anoestrous and control cyclic heifers. Thirty-two heifers (477 ± 7.1 kg) were immunised against either human serum albumin (HSA; controls; n = 8), or a HSAGnRH conjugate. On day 70 after primary immunisation, control heifers (n = 4 per treatment; day 3 of cycle) received either (a) 2.5 μg GnRH or (b) 2.5 μg of GnRH-A (Buserelin®) and GnRH-immunised heifers (blocked by GnRH antibody titre; n = 6 per treatment) received either (c) saline, (d) 2.5 μg GnRH, (e) 25 μg GnRH or (f) 2.5 μg GnRH-A, intravenously. On day 105, 1 mg oestradiol was injected (intramuscularly) into control (n = 6) and GnRH-immunised anoestrous heifers with either low (13.4 ± 1.9% binding at 1:640; n = 6) or high GnRH antibody titres (33.4 ± 4.8% binding; n = 6). Data were analysed by ANOVA. Mean plasma LH and FSH concentrations on day 69 were higher (P < 0.05) in control than in GnRH-immunised heifers (3.1 ± 0.16 vs. 2.5 ± 0.12 ng LH ml⁻¹ and 22.5 ± 0.73 vs. 17.1 ± 0.64 ng FSH ml⁻¹, respectively). The number of LH pulses was higher (P < 0.05) in control than in GnRH-immunised heifers on day 69 (3.4 ± 0.45 and 1.0 ± 0.26 pulses per 6 h, respectively). On day 70, 2.5 μg GnRH increased (P < 0.05) LH concentrations in control but not in GnRH-immunised heifers, while both 25 μg GnRH and 2.5 μg GnRH-A increased (P < 0.05) LH concentrations in GnRH-immunised heifers, and 2.5 μg GnRH-A increased LH in controls. FSH was increased (P < 0.05) in GnRH-immunised heifers following 25 μg GnRH and 2.5 μg GnRH-A. Oestradiol challenge increased (P < 0.05) LH concentrations during the 13–24 h period after challenge with a greater (P < 0.05) increase in control than in GnRH-immunised heifers. FSH concentrations were decreased (P < 0.05) for at least 30 h after oestradiol challenge. In conclusion, GnRH immunisation decreased LH pulsatility and mean LH and FSH concentrations. GnRH antibodies neutralised low doses of GnRH (2.5 μg), but not high doses of GnRH (25 μg) and GnRH-A (2.5 μg). GnRH immunisation decreased the rise in LH concentrations following oestradiol challenge.     

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C₁₈ column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 mM ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μM in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μM. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).     

Cortisol and cortisone levels in umbilical cord plasma and maternal plasma of normal pregnancies

A method for assaying cortisol and cortisone using chromatography on either paper or Sephadex LH-20 columns for isolation, followed by competitive protein binding, has been applied to umbilical cord and maternal plasma samples. In mixed cord plasma the mean cortisol concentration was 6.0 ± 0.8 μg/100 ml (n = 9) and the mean cortisone concentration was 13.5 ± 2.9 μg/100 ml (n = 9). In cord arterial plasma the mean cortisol concentration was 6.3 ± 2.9 μg/100 ml (n = 6) and the mean cortisone level was 10.1 ± 2.5 μg/100 ml (n = 6). For cord venous plasma, the mean level of cortisol was 5.6 ± 1.5 μg/100 ml (n = 6) and of cortisone was 13.5 ± 2.4 μg/100 ml (n = 6). Maternal plasma gave a mean value of cortisol of 42.3 ± 4.5 μg/100 ml (n = 6) and of cortisone of 6.2 ± 0.9 μg/100 ml. The results of this study suggest that the fetus at term-gestation produces cortisol. The significance of this production compared with placental transfer of maternal cortisol into the fetal circulation however is uncertain.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Formate and glucose stimulation of methane and hydrogen production in rumen liquor

Membrane-inlet mass spectrometry was used to investigate the effects of increasing the concentration of the rumen metabolites, formate and glucose, upon CH₄ and H₂ production during fermentation by unfractionated rumen liquor. Additions of formate up to 3.6 mM stimulated CH₄ and then excess H₂ production. Each addition caused a large accumulation of H₂ (>40 µM), which returned to in situ concentrations after periods of more than 1 h. Glucose additions up to 2.0 mM gave linear increases in CH₄ and H₂ production. The conversion of substrate carbon into CH₄ was found to decrease from 34% to 9% for formate, as concentrations were increased (1.6–3.6 mM); approximately 13.5% of the glucose carbon was converted to CH₄.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q₁₀ of 2.16. The K_m for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The K_m(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, K_m's were 340 μM for glycerol-3-P and 12 μM for NAD⁺. Effects of most inhibitors (of the forward reaction), glycerol-3-P (K_i = 2.4 mM), NAD⁺ (K_i = 0.2 mM), ATP, Mg·ATP, and P_i, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V_max activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.     

Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26

Variants of recombinant staphylokinase (Sak) were produced by site-specific mutagenesis of the unique Met-26 residue and purified to homogeneity from the cell extract of transformed E. coli. The desired mutations were confirmed by cDNA and amino-acid sequence analysis. Sak-M26L, Sak-M26C, Sak-M26R, Sak-M26V and Sak-M26A were selected for further analysis on the basis of their plasminogen activating activity. The specific fibrinolytic activities of Sak-M26L, Sak-M26C and Sak were comparable (76 000 ± 10 000, 75 000 ± 2400 and 78 000 ± 9700 HU/mg, respectively; mean ± S.E., n = 3 or 4). Active site exposure in equimolar (4.5 μM) mixtures with plasminogen at room temperature was more rapid with Sak-M26L than with Sak (quantitative exposure within 4 min and 8 min, respectively). Activation of 1 μM plasminogen by catalytic amounts (5 nM) of Sak-M26L initially appeared to be somewhat faster, but comparable 50 to 60% activation was obtained within 30 min. In contrast, Sak-M26R and Sak-M26V were virtually inactive, did not form active complexes with plasminogen and did not activate plasminogen. The catalytic efficiencies for plasminogen activation were comparable for plasmin-Sak-M26L, plasmin-Sak-M26C and plasmin-Sak (0.14 μM⁻¹ s⁻¹, 0.16 μM⁻¹ s⁻¹ or 0.12 μM⁻¹ s⁻¹, respectively). Comparable dose-dependent lysis of 0.06 ml ¹²⁵I-fibrin labeled human plasma clots submerged in 0.3 ml human plasma was obtained with Sak-M26L, Sak-M26C and Sak (concentration required for 50% lysis in 2 h, EC₅₀, of 17 ± 1.6 nM and 14 ± 2.5 nM, respectively), whereas Sak-M26R or Sak-M26V were inactive. Sak-M26A did not form a stable complex with plasminogen, as shown by gel filtration. These data establish that substitution of the unique Met residue in position 26 of the Sak sequence with Leu or Cys has little or no influence on its plasminogen activating or fibrinolytic potential. In contrast, substitution of Met-26 with either Arg or Val results in total loss of the functional activity. Thus, the amino acid in post 26 of Sak appears to be of crucial importance for the activation of plasminogen by staphylokinase.     

Determination of theophylline and its metabolites in human urine and plasma by high-performance liquid chromatography

A method for the quantitation of theophylline (13DMX) and the three metabolites, 1-methyluric acid (1MU), 3-methylxanthine (3MX) and 1,3-dimethyluric acid (13DMU) in human plasma and urine has been developed. The method is based on a simple one-step liquid-liquid extraction with ethylacetate-2 propanol followed by isocratic, reversed-phase high-performance liquid chromatography with UV detection (detection wavelength: 273 nm). The overall mean recoveries ranged from 86 to 95% for the four compounds. The detection limit was 1 μm for 1MU, 3MX and 13DMU and 2 μM for 13DMX in urine, and 0.1 μM for 1MU, 3MX and 13DMU and 0.2 μM for 13DMX in plasma. The intra-day and inter-day coefficient of variation was <6% and <9%, respectively, and the accuracy was within ±10% in both urine and plasma.The simple but sensitive method is highly suitable for the development of theophylline as a probe drug for assessing CYP1A2 activity in man.     

Ion-pair high-performance liquid chromatographic assay method for the assessment of clarithromycin stability in aqueous solution and in gastric juice

A simple and selective ion-pair HPLC method has been developed for the analysis of clarithromycin in aqueous solutions and in gastric juice. A Hypersil ODS 5-μm (150 × 4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile-aqueous 0.05 M phosphate buffer (pH 4.6) containing 5 mM 1-octanesulphonic acid (50:50, v/v). The column temperature was 50°C and detection was by UV absorption (210 nm). The limits of detection of 50-μl samples were 0.4 μg/ml (aqueous) and 0.78 μg/ml (0.5 ml gastric juice) or better. The assay was linear in the range of 1.56 to 100 μg/ml with r² values greater than 0.99. The recovery from the gastric juice samples was 98.5±2.9%. The method was applied successfully to determine the stability of clarithromycin in 0.01 M HCl and gastric juice.