Chiral resolution of the enantiomers of new selective CB(2) receptor agonists by liquid chromatography on amylose stationary phases

Analytical HPLC methods using derivatized amylose chiral stationary phases, Chiralpak AD-H and Chiralpak AS, were developed for the direct enantioseparation of eight substituted 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives with one stereogenic center. Baseline separation (R_s > 1.5) was always achieved on amylose based Chiralpak AD-H column to the difference with Chiralpak AS. Using UV detection, a linear response was observed within a 180–420 μmol L⁻¹ concentration range (r² > 0.991) for three racemic compounds 1, 3 and 4 with best pharmacological potentials; repeatability, limit of detection (LD) and quantification (LQ) were also determined: LD varied, for the solutes, from 0.36 to 2.56 μmol L⁻¹. Finally, the enantiopurity of these compounds was determined. Additionally, the effect of temperature variations upon isomer separations was investigated.     

A hand-held apparatus for “nose-only” exposure of mice to inhalable microparticles as a dry powder inhalation targeting lung and airway macrophages

Microparticles containing isoniazid and rifabutin were aerosolised using a simple apparatus fabricated from a 15-ml centrifuge tube. The dose available for inhalation by rodents was determined by collecting microparticles emitted at the delivery port. The dose available for inhalation was proportional to durations of exposure ranging from 10 to 90 s (10.5–13.5 CV%) and the weight of powder taken for fluidization (10–50 mg, r² = 0.982). The apparatus was then used to administer inhalations of microparticles to mice. Other groups of mice received free rifabutin orally, or by i.v. injection. Rifabutin was estimated in serum and tissues of dosed mice by HPLC. When 20 mg of microparticles were loaded in the apparatus, 2.5 mg were collected at the delivery port in 30 s of operation. Mice inhaled 300 μg of the 2.5 mg emitted at the delivery port. Airway and lung macrophages of mice receiving inhalations for 30 s accumulated 0.38 μg of rifabutin, while the amount in blood serum of these mice was 0.62 μg. In mice receiving 83 μg rifabutin i.v. or orally, the intracellular amounts were 0.06 and 0.07 μg respectively, while the amounts in serum were 1.02 and 0.80 μg. These observations confirmed that inhalation of microparticles targeted airway and lung macrophages.     

Drug permeability in 16HBE14o- airway cell layers correlates with absorption from the isolated perfused rat lung

The permeability of the lung is critical in determining the disposition of inhaled drugs and the respiratory epithelium provides the main physical barrier to drug absorption. The 16HBE14o- human bronchial epithelial cell line has been developed recently as a model of the airway epithelium. In this study, the transport of 10 low molecular weight compounds was measured in the 16HBE14o- cell layers, with apical to basolateral (absorptive) apparent permeability coefficients (P_app) ranging from 0.4 × 10⁻⁶ cm s⁻¹ for Tyr-D-Arg-Phe-Phe-NH₂ to 25.2 × 10⁻⁶ cm s⁻¹ for metoprolol. Permeability in 16HBE14o- cells was found to correlate with previously reported P_app in Caco-2 cells and absorption rates in the isolated perfused rat lung (k_a,lung) and the rat lung in vivo (k_{a,in vivo}) [Tronde, et al., 2003. J. Pharm. Sci. 92, 1216–1233; Tronde, et al., 2003. J. Drug Target. 11, 61–74]. Log linear relationships were established between P_app in 16HBE14o- cells and P_app in Caco-2 cells (r² = 0.82), k_a,lung (r² = 0.78) and k_{a,in vivo} (r² = 0.68). The findings suggest that permeability in 16HBE14o- cells may be useful to predict the permeability of compounds in the lung, although no advantage of using the organ-specific cell line 16HBE14o- compared to Caco-2 cells was found in this study.     

Method development and validation for isoflavones in soy germ pharmaceutical capsules using micellar electrokinetic chromatography

The separation of six soy isoflavones (Glycitein, Daidzein, Genistein, Daidzin, Glycitin and Genistin) was approached by a 3² factorial design studying MEKC electrolyte components at the following levels: methanol (MeOH; 0–10%) and sodium dodecylsulfate (SDS; 20–70 mmol L⁻¹); sodium tetraborate buffer (STB) concentration was kept constant at 10 mmol L⁻¹. Nine experiments were performed and the apparent mobility of each isoflavone was computed as a function of the electrolyte composition. A novel response function (RF) was formulated based on the productory of the mobility differences, mobility of the first and last eluting peaks and the electrolyte conductance. The inspection of the response surface indicated an optimum electrolyte composition as 10 mmol L⁻¹ STB (pH 9.3) containing 40 mmol L⁻¹ SDS and 1% MeOH promoting baseline separation of all isoflavones in less than 7.5 min.

The proposed method was applied to the determination of total isoflavones in soy germ capsules from four different pharmaceutical laboratories. A 2 h extraction procedure with 80% (v/v) MeOH under vortexing at room temperature was employed. Peak assignment of unknown isoflavones in certain samples was assisted by hydrolysis procedures, migration behavior and UV spectra comparison. Three malonyl isoflavone derivatives were tentatively assigned. A few figures of merit for the proposed method include: repeatability (n = 6) better than 0.30% CV (migration time) and 1.7% CV (peak area); intermediate precision (n = 18) better than 6.2% CV (concentration); recoveries at two concentration levels, 20 and 50 μg mL⁻¹, varied from 99.1 to 103.6%. Furthermore, the proposed method exhibited linearity in the concentration range of 1.6–50 μg mL⁻¹ (r² > 0.9999) with LOQ varying from 0.67 to 1.2 μg mL⁻¹. The capsules purity varied from 93.3 to 97.6%.     

Enantioselective LC analysis of synephrine in natural products on a protein-based chiral stationary phase

An enantioselective LC method with photodiode array detection (PAD) was developed for the enantioseparation of (±)-synephrine from C. aurantium L. var. amara fruits and phytotherapic derivatives by using a protein-based chiral stationary phase with cellobiohydrolase as the chiral selector (Chiral-CBH). Analyses were carried out on a Chiral-CBH column (100 × 4.0 mm i.d., 5 μm), with a mobile phase consisting of 2-propanol (5%, w/w) in sodium phosphate buffer (pH 6.0; 10 mM) and disodium EDTA (50 μM). The flow rate was 0.8 mL/min. Detection was set at 225 nm. To identify the order of elution, the racemate was resolved by the preparation of suitable diastereoisomeric salts with antipodes of appropriate organic acids.

Isolation of synephrine from C. aurantium fruits and phytoproducts was performed by solid-phase extraction (SPE) with a strong cation-exchange phase.

The method developed was validated and was found to be linear in the 0.40–40.14 μg/mL range (r² = 1.000, P < 0.0001) for both synephrine enantiomers. The limit of detection (LOD) for each enantiomer was 0.04 μg/mL. The limit of quantification (LOQ) for each enantiomer was 0.13 μg/mL. Intra-day precision (calculated as %R.S.D.) ranged from 0.03 to 0.24% for (−)-synephrine and from 0.03 to 0.35% for (+)-synephrine. Inter-day precision (calculated as %R.S.D.) ranged from 0.07 to 1.45% for (−)-synephrine and from 0.06 to 1.26% for (+)-synephrine. Intra- and inter-day accuracies (calculated as %recovery) were in the ranges of 97.4–100.6 and 98.0–101.6% for (−)-synephrine, and in the ranges 97.0–101.5 and 98.1–102.8% for (+)-synephrine.

The results of the application of the method to the analysis of C. aurantium samples showed that (−)-synephrine was the main component. (+)-Synephrine was not detected in C. aurantium fruits and was present in low concentration in the phytoproducts.     

Drug release mechanism of paclitaxel from a chitosan–lipid implant system: Effect of swelling, degradation and morphology

Localized and sustained delivery of anti-cancer agents to the tumor site has great potential for the treatment of solid tumors. A chitosan–egg phosphatidylcholine (chitosan–ePC) implant system containing PLA-b-PEG/PLA nanoparticles has been developed for the delivery of paclitaxel to treat ovarian cancer. Production of volumes of ascites fluid in the peritoneal cavity is a physical manifestation of ovarian cancer. In vitro release studies of paclitaxel from the implant were conducted in various fluids including human ascites fluid. A strong correlation (r² = 0.977) was found between the release of paclitaxel in ascites fluid and PBS containing lysozyme (pH 7.4) at 37 °C. The drug release mechanism for this system was proposed based on swelling, degradation and morphology data. In addition, in vitro release of paclitaxel was found to be a good indicator of the in vivo release profile (correlation between release rates: r² = 0.965). Release of paclitaxel was found to be sustained over a four-week period following implantation of the chitosan–ePC system into the peritoneal cavity of healthy Balb/C mice. Also, the concentrations of paclitaxel in both plasma and tissues (e.g. liver, kidney and small intestine) were found to be relatively constant.     

Validation of an isocratic LC method for determination of quercetin and methylquercetin in topical nanoemulsions

The aim of this study was to validate an isocratic LC method for the quantification of either quercetin (Q) or methylquercetin (MQ) incorporated in topical nanoemulsions. The analyses were performed at room temperature on a reversed-phase C₁₈ column using a mobile phase composed of methanol/water (70:30, v/v) and trifluoracetic acid 0.1% at 0.8 mL min⁻¹. The detection was carried out on a UV detector at 368 or 354 nm for Q and MQ, respectively. The linearity, in the range of 0.15–1.5 μg/mL, presented a determination coefficient (r²) higher than 0.99, calculated by the least square method for both flavonoids. No interferences from the excipients (egg-lecithin or octyldodecanol) were detected. The R.S.D. values for intra- and inter-day precision experiments were lower than 2% for both flavonoids. The recovery ranged from 98.9% to 103.46% for Q and from 98.9% to 102.92% for MQ.     

Further evidence of an association between adolescent bipolar disorder with smoking and substance use disorders: a controlled study

Although previous work suggests that juvenile onset bipolar disorder increases risk for substance use disorders and cigarette smoking, the literature on the subject is limited. We evaluated the association of risk for substance use disorders and cigarette smoking with bipolar disorder in adolescents in a case–control study of adolescents with bipolar disorder (n = 105, age 13.6 ± 2.5 years [mean]; 70% male) and without bipolar disorder (“controls”; n = 98, age 13.7 ± 2.1 years; 60% male). Rates of substance use and other disorders were assessed with structured interviews (KSADS-E for subjects younger than 18, SCID for 18-year-old subjects). Bipolar disorder was associated with a significant age-adjusted risk for any substance use disorder (hazard ratio[95% confidence interval] = 8.68[3.02 25.0], χ² = 16.06, p < 0.001, df = 1), alcohol abuse (7.66 [2.20 26.7], χ² = 10.2, p = 0.001, df = 1), drug abuse (18.5 [2.46 139.10], χ² = 8.03, p = 0.005, df = 1) and dependence (12.1 [1.54 95.50], χ² = 5.61, p = 0.02, df = 1), and cigarette smoking (12.3 [2.83 53.69], χ² = 11.2, p < 0.001, df = 1), independently of attention deficit/hyperactivity disorder, multiple anxiety, and conduct disorder (CD). The primary predictor of substance use disorders in bipolar youth was older age (BPD − SUD versus BPD + SUD, logistic regression: χ² = 89.37, p < 0.001). Adolescent bipolar disorder is a significant risk factor for substance use disorders and cigarette smoking, independent of psychiatric comorbidity. Clinicians should carefully screen adolescents with bipolar disorder for substance and cigarette use.     

Assay for nipecotic acid in small blood samples by gas chromatography-mass spectroscopy

An analytical method for nipecotic acid quantification in rat blood was developed utilizing a stable isotope internal standard and capillary gas chromatography–mass spectroscopy. The method involves a solid phase extraction step followed by a two-step derivatization. The analytes are separated by capillary gas chromatography and detected by selected ion monitoring of their base peaks at 180 and 185 m/z, respectively. The assay has a limit of detection (LOD) of 10 ng/ml and a limit of quantification of 26 ng/ml in 200 μl of rat whole blood. The linear range of the assay covers from 26 to 6500 ng/ml (r²=0.9996, n=9). The coefficient of variation was less than 10% at concentrations of 50, 1000 and 5000 ng/ml. The assay was used to characterize the pharmacokinetics of R-(-)-nipecotic acid in a rat. R-(-)-nipecotic acid clearance was 4.2 ml/min, its half-life was 1.5 h and its volume of distribution at steady state was 325 ml.     

Comparative evaluation between capillary electrophoresis and high-performance liquid chromatography for the analysis of florfenicol in plasma

A capillary electrophoresis (CE) and a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV detection have been developed for florfenicol analysis in plasma samples. The suitabilities of both methods for quantitative determination of florfenicol were approved through validation specification, such as linearity, precision, selectivity, accuracy, limit of detection and quantification. The capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assay were compared by analyzing a series of plasma samples containing florfenicol in different concentrations using the two methods. The extraction procedure is simple and no gradient elution or derivatization is required. Furthermore, the analysis time of the CE method is two times shorter than the respective parameter in HPLC and solvent consumptions is considerably lower. The calibration curve were linear to at least 0.05–10 μg/ml (r = 0.9998) and 0.1–10 μg/ml (r = 0.9998) for CE and HPLC, respectively. The separation efficiency are good for both methods. The detection limits for florfenicol were 0.015 μg/ml with CE and 0.03 μg/ml with HPLC and CE method gave lower value, even though UV detector was applied in the both cases. The both methods were selective, robust and reliable quantification of florfenicol and can be useful for clinical and biomedical investigations.     

Intrapulmonary pharmacokinetics and pharmacodynamics of meropenem

The objective of this study was to determine the plasma and intrapulmonary pharmacokinetic parameters of intravenously administered meropenem in healthy volunteers. Four doses of 0.5 g, 1.0 g or 2.0 g meropenem were administered intravenously to 20, 20 and 8 healthy adult subjects, respectively. Standardised bronchoscopy and timed bronchoalveolar lavage (BAL) were performed following administration of the last dose. Blood was obtained for drug assay prior to drug administration and at the time of BAL. Meropenem was measured in plasma, BAL fluid and alveolar cells (ACs) using a combined high pressure liquid chromatographic–mass spectrometric technique. Plasma, epithelial lining fluid (ELF) and AC pharmacokinetics were derived using non-compartmental methods. C_max/MIC₉₀ (where C_max is the maximum plasma concentration and MIC₉₀ is the minimum inhibitory concentration required to inhibit 90% of the pathogen), AUC/MIC₉₀ (where AUC is the area under the curve for the mean concentration–time data), intrapulmonary drug exposure ratios and percent time above MIC₉₀ during the dosing interval (%T > MIC₉₀) were calculated for common respiratory pathogens with MIC₉₀ values of 0.12–4 μg/mL. In the 0.5 g dose group, the C_max (mean ± S.D.), AUC_0–8 h and half-life for plasma were, respectively, 25.8 ± 5.8 μg/mL, 28.57 μg h/mL and 0.77 h; for ELF the values were 5.3 ± 2.5 μg/mL, 12.27 μg h/mL and 1.51 h; and for ACs the values were 1.0 ± 0.5 μg/mL, 4.30 μg h/mL and 2.61 h. In the 1.0 g dose group, the C_max, AUC_0–8 h and half-life for plasma were, respectively, 53.5 ± 19.7 μg/mL, 55.49 μg h/mL and 1.31 h; for ELF the values were 7.7 ± 3.1 μg/mL, 15.34 μg h/mL and 0.95 h; and for ACs the values were 5.0 ± 3.4 μg/mL, 14.07 μg h/mL and 2.17 h. In the 2.0 g dose group, the C_max, AUC_0–8 h and half-life for plasma were, respectively 131.7 ± 18.2 μg/mL, 156.7 μg h/mL and 0.89 h. The time above MIC in plasma ranged between 28% and 78% for the 0.5 g dose and between 45% and 100% for the 1.0 g and 2.0 g doses. In ELF, the time above MIC ranged from 18% to 100% for the 0.5 g dose and from 25% to 88% for the 1.0 g dose. In ACs, the time above MIC ranged from 0% to 100% for the 0.5 g dose and from 24% to 100% for the 1.0 g dose. Time above MIC in ELF and ACs for the 2.0 g dose was not calculated because of sample degradation. The prolonged T > MIC₉₀ and high intrapulmonary drug concentrations following every 8 h administration of 0.5–2.0 g doses of meropenem are favourable for the treatment of common respiratory pathogens.     

Improved liquid chromatographic method for the simultaneous determination of tramadol and its three main metabolites in human plasma, urine and saliva

Tramadol, an analgesic agent, and its main metabolites O-desmethyltramadol (M1), N-desmethyltramadol (M2) and O,N-didesmethyltramadol (M5) were determined simultaneously in human plasma, saliva and urine by a rapid and specific HPLC method. The sample preparation was a simple, one-step, extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith™ Performance RP-18e 100 mm × 4.6 mm column, using a mixture of methanol:water (19:81, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (λ_ex 200 nm/λ_em 301 nm) was used. The calibration curves were linear (r² > 0.996) in the concentration ranges in plasma, saliva and urine. The lower limit of quantification was 2.5 ng/ml for all compounds. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were acceptable in all analyzed body fluids The developed procedure was applied to assess the pharmacokinetics of tramadol and its main metabolites following administration of 100 mg single oral dose of tramadol to healthy volunteers.     

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C₁₈ column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l⁻¹ nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min⁻¹). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml⁻¹ (r > 0.9995) for amikacin and 8 to 105 μg ml⁻¹ (r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml⁻¹, respectively.

In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.3 min, LOD = 1.8 μg ml⁻¹, range 5–40 μg ml⁻¹, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).     

A bioassay for metals utilizing a human cell line

The purpose of this study was to assess the ability of the HepG2 cell line to function as a bioassay for metal contamination in sediments, using metallothionein (MT) as a biomarker of exposure. Sediments were collected from the eastern and western ends of Lake Erie, extracted using EPA method 200.7, and analyzed for cadmium (Cd), mercury (Hg) and lead (Pb) levels using ICP-AES. Sediment extracts were neutralized then used at a 2.5% final concentration in the exposure medium. MT levels were measured using the cadmium–hemoglobin affinity assay after a 48 h exposure. Fortified blanks from the ICP protocol served as positive controls. Also, HepG2 cells were exposed to Cd, Pb or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Additionally, cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005–0.1 mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5 mg/kg with lead the predominant metal, accounting for 100–88.9% of the total quantifiable metals in the sediments. The biomarker response (MT induction) was strongly correlated (r² = 0.9919, r² = 0.990) with total metal and lead levels in the sediments, respectively, which supports recent field studies indicating the biomarker can discern differences in the strength of the inducing agent. Statistically significant MT induction was associated with sediments which contained measurable Cd concentrations and no significant differences were observed when comparing Cd only and Cd + Pb exposed cells indicating no interactions between Cd and Pb were occurring and supporting our finding that Cd was the main inducing agent in sediment extracts. MT levels also increased significantly in a concentration-dependent manner when cells were exposed only to Cd. Results suggest this human bioassay and the MT biomarker of exposure may be useful for monitoring complex metal mixtures in aquatic sediments.     

HPLC assay with UV detection for determination of RBC purine nucleotide concentrations and application for biomarker study in vivo

ATP and other purine nucleotides are important biomarkers for ischemia and may have considerable potential as targets for management of ischemic heart disease and stroke. The main objective of the study is to develop a rapid HPLC assay, which has adequate sensitivity and specificity for measuring concentrations of ATP, ADP, AMP, GTP, GDP and GMP in erythrocytes (RBC). The assays used ion-pair chromatography coupled with ultraviolet detection at 254 nm to separate and detect the purine nucleotides. Using 50–100 μL of RBC lysate as blank biologic matrix, the assay was linear from 100 to 2000 μg/mL for ATP and ADP, and 20–400 μg/mL for AMP, GTP, and GDP with coefficients of determination (r²) >0.99. GDP and GMP were not measurable in the study because of low concentrations and interference from endogenous materials, respectively. The intra-assay and inter-assay variations over a period of 1 year were less than 10% and 20%, respectively for most of the nucleotides. The assay was successfully applied to two pilot biomarker studies to measure RBC concentrations of the purine nucleotides in rats under restraining and exercise conditions. Preliminary results showed that the RBC concentrations of ATP and GTP were higher in the spontaneously hypertensive rats (SHR) compared to the Sprague–Dawley (SD) rats, and that exercise increased RBC concentrations of ATP in rats treated with the calcium channel blocker diltiazem.     

Development and validation of two chromatographic methods for the quantification of E-6087 and one of its metabolites, E-6132, in rat plasma

E-6087 is a nonsteroidal anti-inflammatory compound under development that selectively inhibits cyclooxygenase-2. In vitro studies have shown that one of its metabolites, E-6132, also inhibits this enzyme. Due to chromatographic reasons, two reverse phase HPLC methods were developed and validated in order to elucidate which compound is responsible for the pharmacological activity in vivo. Chromatographic separation of E-6087 was achieved using acetonitrile–phosphate buffer (pH 2.5; 25 mM) (60:40, v/v) as mobile phase and two 4.6×150 mm×5 μm Inertsil ODS-2 columns. For E-6132, two Inertsil ODS-3 columns and 52% of acetonitrile were used instead. Internal standards and fluorescence detection differed between both methods. The same on-line solid-phase extraction method was used. Mean retention times for E-6087 and E-6132 were 15.2 (±1.3) and 36.1 (±0.6) min, respectively. The methods were selective and linear over the concentration range of 10–500 ng ml⁻¹ (r²>0.996) for E-6087 and 5–200 ng ml⁻¹ (r²>0.997) for E-6132. The limits of quantitation were 10 ng ml⁻¹ (E-6087) and 5 ng ml⁻¹ (E-6132) with a precision and accuracy <16% (E-6087) and <11% (E-6132). Mean recoveries from plasma were 43.2–61.9% (E-6087) and 60.4–65.2% (E-6132). For both compounds, both inter-assay and intra-assay precision and accuracy were within acceptable limits (<15%). As an example of the suitability of these methods, the results from a pharmacokinetic study are reported. After single oral administration of 5 mg kg⁻¹ of E-6087 to rats, plasma concentrations of E-6087 at peak time were higher than those of E-6132, suggesting that activity is mainly due to E-6087.     

Determination of 5-fluorouracil in human plasma by a simple and sensitive reversed-phase HPLC method

A simple and sensitive reversed-phase HPLC method with UV detection was developed and validated for the quantitation of 5-fluorouracil (5-FU) in human plasma. After acidification and salting out, 5-FU was extracted into ethyl acetate and back-extracted into a basic buffer. The extract was adjusted to neutral pH before being injected onto the HPLC column. 5-FU was separated from the matrix components on a YMC ODS-AQ column at 40°C using an aqueous mobile phase of 10 mM potassium phosphate at pH 5.5. A linear gradient of 0–25% methanol wash eluted late peaks, maintained column performance, and increased column stability. The run time was 20 min. The linear range was 25–300 ng ml⁻¹ (r²>0.999). The limit of quantitation was 25 ng ml⁻¹, with a signal-to-noise ratio of 23:1. Interday precision and accuracy of quality control samples were 6.2–8.4%, relative standard deviation and −0.1– + 1.9% relative error.     

Plasma and tonsillar tissue pharmacokinetics of teicoplanin following intramuscular administration to children

The population pharmacokinetics of teicoplanin in plasma and tonsillar tissue in children was determined following intramuscular administration. Thirty seven patients in all received either a single 5 mg/kg dose; 2 doses of 5 mg/kg, 12 h apart; 3 doses of 5 mg/kg, 12 h apart; or, a single 10 mg/kg dose. Limited data, comprising a maximum of 2 blood samples and 1 tonsillar sample were taken from each patient, with the maximum time being 48 h after the first dose of teicoplanin (in the 3×5 mg/kg dosing schedule). All plasma data were analyzed simultaneously by a maximum likelihood method employing a modified EM algorithm. A first-order absorption, one-compartment disposition model was fitted to the data. Mean parameter values (with lower and upper 95% confidence intervals) were: clearance/bioavailability, 0.024 L h⁻¹ kg⁻¹ (0.020–0.027); volume of distribution/bioavailability, 0.61 L kg⁻¹ (0.54–0.70); absorption rate constant, 0.43 h⁻¹ (0.31–0.61). A first-order transfer model for distribution of teicoplanin between plasma and tonsillar tissue was fitted to the tonsil data. The mean parameter values (95% confidence intervals) were: transfer rate constant between plasma and tonsils 0.49 h⁻¹ (0.35–0.67); transfer rate constant between tonsils and plasma 0.73 h⁻¹ (0.52–1.03). These rate constants correspond to a distribution half-life of 0.95 h and an equilibrium distribution concentration ratio between tonsillar tissue and plasma of 0.67. After normalising clearance and volume of distribution for body weight, there was no further influence of body weight on the pharmacokinetic parameters. Also, there was no effect of dose, and as two formulations were used, one for the 5 mg/kg dose and the other for the 10 mg/kg dose, no effect of formulation on the pharmacokinetics of teicoplanin after im (intramuscular) administration was found.     

Simultaneous determination of berberine and palmatine in rat plasma by HPLC-ESI-MS after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction and the pharmacokinetic application of the method

A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) method has been developed and validated for the identification and quantification of berberine and palmatine in rat plasma. After the addition of the internal standard (IS) and alkalization with 0.5 M sodium hydroxide solution, plasma samples were extracted by ethyl ether and separated by HPLC on a Shim-pack ODS (4.6 μm, 150 mm × 2.0 mm i.d.) column using a mobile phase composed of A (0.08% formic acid and 2 mmol/l ammonium acetate) and B (acetonitrile) with linear gradient elution. Analysis was performed on a Shimadzu LC/MS-2010A in selected ion monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. [M]⁺ = 336 for berberine; 352 for palmatine and [M + H]⁺ = 340 for IS were selected as detecting ions, respectively. The method was validated over the concentration range of 0.31–20 ng/ml for berberine and palmatine. Inter- and intra-CV precision (R.S.D.%) were all within 15% and accuracy (%bias) ranged from −5 to 5%. The lower limits of quantification were 0.31 ng/ml for both analytes. The extraction recovery was on average 68.6% for berberine, 64.2% for palmatine. The validated method was used to study the pharmacokinetic profile of berberine and palmatine in rat plasma after oral administration of Huang-Lian-Jie-Du decoction.     

Automated system for release studies of salicylic acid based on a SIA method

The aim of this work was to describe a fully automated system for the in vitro release testing of semisolid dosage forms based on SIA technique. The system was tested for monitoring release profiles of different ointments containing 3% of salicylic acid (Belosalic, Diprosalic, Triamcinolone S). The native fluorescence of salicylic acid was used for fluorimetric detection. Phosphate buffer pH 7.4 was the receptor medium; samples were taken at 10 min intervals during 6 h of the release test; and each test was followed by calibration with five standard solutions. The linear calibration range was 0.05–10 μg ml⁻¹ (r = 0.9996, six standards); the maximal SIA sample throughput for this system was 120 h⁻¹, sample volume being 50 μl and flow rate 50 μl s⁻¹. The detection limit for salicylic acid was 0.01 μg ml⁻¹.