Detection of modafinil in human urine by gas chromatography-mass spectrometry

The main purpose of this study was to detect and quantify modafinil in human urine by gas chromatography–mass spectrometry (GC–MS). Urinary samples were collected from three healthy male volunteers following oral administration of a clinical dose (100 mg) of modafinil (Provigil^®). Urine specimens were extracted with t-butylmethyl ether (TBME) prior to GC–MS analysis. The results demonstrate that the chromatographic characteristics and the mass spectrum of the unchanged parent drug extracted from urine samples were identical to that obtained from the authentic standard. The times for the unchanged modafinil to reach peak concentration in the urine of the three volunteers were at 2 h (6.14 μg/mL), 4 h (9.93 μg/mL) and 8 h (3.58 μg/mL), respectively. Total clearance occurred in approximately 48–72 h with 2–5% eliminated through urine as unchanged modafinil. The present study demonstrates that modafinil is detectable in the absence of hydrolysis and derivatization steps.     

Determination of lopinavir cerebral spinal fluid and plasma ultrafiltrate concentrations by liquid chromatography coupled to tandem mass spectrometry

A method for the determination of lopinavir (LPV) concentrations in cerebral spinal fluid (CSF) and plasma ultrafiltrate (UF) was developed and validated to analyze clinical specimens from patients receiving antiretroviral treatment with lopinavir/ritonavir. The CSF (400 μL sample volume) final calibration range for LPV was 0.313–25.0 ng/mL. The final calibration range for UF (50 μL sample volume) was 1.25–100 ng/mL. The samples were prepared using liquid–liquid extraction, concentrated, and analyzed using a reversed phase isocratic separation. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer. Isolation of LPV through chromatographic separation and proper selection of calibration matrix were important factors in achieving accurate results. Plasma UF was found to be an equivalent calibration matrix to CSF whereas plasma matrix produced a positive bias in samples with unknown concentrations. Artificial CSF media prepared chemically were biased and less superior than UF. Sources of plasma for the UF did not affect accuracy. Several CSF sources were tested for specificity of the method and LPV concentrations were accurately produced with atmospheric pressure chemical ionization source producing more accurate results than the electrospray source. The method successfully measured LPV concentrations in CSF that were previously undetectable by HPLC as well as UF from protein binding studies.     

Simultaneous determination of triamcinolone acetonide and oxymetazoline hydrochloride in nasal spray formulations by HPLC

A high-performance liquid chromatography (HPLC) method with UV detection at 232 nm was developed and validated for the simultaneous determination of triamcinolone acetonide (TAA) and oxymetazoline hydrochloride (OXY) in nasal spray formulations. The chromatographic system consisted of a μBondapak™ CN column (150 mm × 3.9 mm), 5 μm particle size with a mobile phase composition of acetonitrile:ammonium acetate (pH 5.0, 20 mM) (10:90, v/v) at a flow rate of 1.0 mL/min. Calibration curves were linear for both TAA and OXY in the concentration range of 2.5–25.0 μg/mL. The limit of detection and quantitation were 0.29 and 0.88 μg/mL for OXY and 0.24 and 0.73 μg/mL for TAA. The described method was further applied to the analysis and stability studies of two nasal spray formulations I and II prepared from TAA and OXY commercial nasal spray products. The stability of OXY and TAA in the commercial products and the nasal formulations I and II were analyzed after 30 days at room temperature and 30 days at 40 °C/60% relative humidity. The results of the stability study showed that OXY and TAA in the commercial nasal spray products and the nasal formulations I and II were stable at 20–25 °C (room temperature) but TAA was unstable at 40 °C/60% relative humidity. TAA exhibited more than 10% loss at 14 days in both the nasal formulations and in the commercial products. OXY showed increased degradation at 40 °C/60% relative humidity but <10%.     

Development and validation of a bioanalytical method using automated solid-phase extraction and LC-UV for the simultaneous determination of lumefantrine and its desbutyl metabolite in plasma

A bioanalytical method for the determination of lumefantrine (LF) and its metabolite desbutyl-lumefantrine (DLF) in plasma by solid-phase extraction (SPE) and liquid chromatography has been developed. Plasma proteins were precipitated with acetonitrile:acetic acid (99:1, v/v) containing a DLF analogue internal standard before being loaded onto a octylsilica (3 M Empore) SPE column. Two different DLF analogues were evaluated as internal standards. The compounds were analysed by liquid chromatography UV detection on a SB-CN (250 mm × 4.6 mm) column with a mobile phase containing acetonitrile–sodium phosphate buffer pH (2.0; 0.1 M) (55:45, v/v) and sodium perchlorate 0.05 M. Different SPE columns were evaluated during method development to optimise reproducibility and recovery for LF, DLF and the two different DLF analogues. The within-day precisions for LF were 6.6 and 2.1% at 0.042 and 8.02 μg/mL, respectively, and for DLF 4.5 and 1.5% at 0.039 and 0.777 μg/mL, respectively. The between-day precisions for LF were 12.0 and 2.9% at 0.042 and 8.02 μg/mL, respectively, while for DLF 0.7 and 1.2% at 0.039 and 0.777 μg/mL, respectively. The limit of quantification was 0.024 and 0.021 μg/mL for LF and DLF, respectively. Different amounts of lipids in plasma did not affect the absolute recovery of LF or DLF.     

Steady-state serum and intrapulmonary pharmacokinetics and pharmacodynamics of tigecycline

The steady-state serum and intrapulmonary pharmacokinetic and pharmacodynamic parameters of tigecycline were determined after intravenous administration in 30 subjects. Tigecycline was administered as a 100 mg loading dose followed by six 50 mg doses given every 12 h and was measured using HPLC/mass spectrometry. Ratios of tigecycline maximum serum concentration and area under the serum concentration–time curve to 90%—minimum inhibitory concentrations (C_max/MIC₉₀; AUC/MIC₉₀), and percentage time above MIC₉₀ were calculated for common respiratory pathogens (Streptococcus pneumoniae, Chlamydia pneumoniae, Mycoplasma pneumoniae, Moraxella catarrhalis and Haemophilus influenzae). The C_max (mean ± S.D.), AUC and half-life for serum were 0.72 ± 0.24 μg/mL, 1.73 ± 0.64 μg*h/mL and 15.0 ± 1.10 h; for lung epithelial lining fluid (ELF) the values were 0.37 μg/mL, 2.28 μg*h/mL and 39.1 h; and for alveolar cells (AC) were 15.2 μg/mL, 134 μg*h/mL and 23.7 h. Tigecycline was concentrated in AC: C_max/MIC₉₀ ratios ranged from 30.4 (H. influenzae) to 507 (S. pneumoniae); AUC/MIC₉₀ ratios ranged from 268 (H. influenzae) to 4467 (S. pneumoniae); and percentage dose interval above MIC₉₀ was 100% for the five respiratory pathogens. The C_max/MIC₉₀, AUC/MIC₉₀ ratios, T > MIC₉₀ and extended serum and intrapulmonary half-lives following the regimen used in this study are favourable for the treatment of tigecycline-susceptible pulmonary infections.     

Antibiotic resistance in Escherichia coli outpatient urinary isolates: final results from the North American Urinary Tract Infection Collaborative Alliance (NAUTICA)

The North American Urinary Tract Infection Collaborative Alliance (NAUTICA) study determined the antibiotic susceptibility to commonly used agents for urinary tract infections of outpatient Escherichia coli urinary isolates obtained from various geographic regions in the USA and Canada. NAUTICA involved 40 medical centres (30 from the USA and 10 from Canada). From April 2003 to June 2004 inclusive, each centre submitted up to 50 consecutive outpatient midstream urine isolates. All isolates were identified to species level by each laboratory's existing protocol. Susceptibility testing was determined using the Clinical and Laboratory Standards Institute (CLSI) microdilution method. Ampicillin (resistant ≥32 μg/mL), sulphamethoxazole/trimethoprim (SMX/TMP) (resistant ≥4 μg/mL), nitrofurantoin (resistant ≥128 μg/mL), ciprofloxacin (resistant ≥4 μg/mL) and levofloxacin (resistant ≥8 μg/mL) resistance breakpoints used were those published by the CLSI. Of the 1142 E. coli collected, 75.5% (862) were collected from the USA and 280 (24.5%) were from Canada. Patient demographics revealed a mean age of 48.1 years (range, 2 months to 99 years), with female patients representing 79.4% of patients and males representing 20.6%. Overall, resistance to ampicillin was 37.7%, followed by SMX/TMP (21.3%), nitrofurantoin (1.1%), ciprofloxacin (5.5%) and levofloxacin (5.1%). Resistance rates for all antimicrobials were higher in US medical centres compared with Canadian centres (P < 0.05). Fluoroquinolone resistance was highest in patients ≥65 years of age (P < 0.05). Resistance rates demonstrated considerable geographic variability both in the USA and Canada. This study reports higher rates of antibiotic resistance in US versus Canadian outpatient urinary isolates of E. coli and demonstrates the continuing evolution of resistance to antimicrobial agents.     

An ultra-rapid drug screening method for acetaminophen in blood serum based on probe electrospray ionization-tandem mass spectrometry

Poisoning incidents caused by drugs, accidental ingestion of poisons, attempted suicide, homicide, and exposure to toxic compounds occur frequently every year across the globe. This raises the need to rapidly identify toxic agents in poisoned patients in a clinical emergency setting. In addition, determining drug/poison concentration is undoubtedly necessary to arrive at a toxicological treatment plan. The purpose of this study was to develop an ultra-rapid drug screening method for the clinical treatment of poisoning. Probe electrospray ionization (PESI), one of the ambient ionization techniques, is able to detect compounds from various biological materials almost directly. We applied the PESI technique to the rapid detection of acetaminophen (APAP). Blood serum samples were diluted 100-fold with 10 mM ammonium formate/ethanol (1:1 v/v) solution including deuterium-labeled internal standards (IS; APAP-d4). Only 10 μL of the diluted sample was used for measurement. The tandem mass spectrometer (MS/MS) equipped with a PESI was used in selected reaction monitoring mode for the quantitation of APAP; the measurement time was only 18 s. Transitions were set at 152 > 110 for quantitation, 152 > 65 for qualifier, and 156 > 114 for IS (APAP-d4). All measurements were conducted in positive mode. The calibration curve (1/x²) was linear over the range of 1.56–200 μg/mL (r² = 0.998), and the limit of detection and quantitation were 0.37 μg/mL and 1.56 μg/mL, respectively. The accuracy (bias) and precision (RSD%) of the method were within an acceptable range (−0.15–2.8% and 2.3–6.1%, respectively) and matrix effect at 3 concentrations (95.1–104%) indicated that PESI-MS/MS is only slightly affected by matrices. In real forensic cases, quantitative values of APAP determined by the PESI-MS/MS were almost identical to those determined by the liquid chromatography-MS/MS method. Since PESI-MS/MS is a simple, reliable, and rapid determination method for toxic agents with virtually no need for blood serum pretreatment, it would be highly suitable for poisoning cases in clinical emergency settings. In the future, a method for simultaneous rapid determination of multiple toxic agents will be developed.     

A novel cationic cholesterol derivative,its formulation into liposomes,and the efficient transfection of the transformed human cell lines HepG2 and HeLa

A novel cationic cholesterol derivative, 3 β[N-(N',N',N'-trimethylaminopropane)-carbamoyl] cholesterol iodide (Chol-Q), has been formulated with equimolar amounts of dioleoyl phosphatidylethanolamine (DOPE) into stable unilamellar liposomes up to 100 nm in size for DNA delivery into mammalian cells. When compared with similarly constituted liposomes containing the tertiary analogue 3 β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) in a band shift assay, liposomes displayed similar DNA binding affinities and appeared to afford complete protection to plasmid DNA against serum nuclease catalysed degradation at liposome:DNA ratios (w/w) of 2.5:1, 5:1, and 10:1 in incubation mixtures containing 5% fetal bovine serum at 37 C for 90 min. Chol-Q liposomes were, however, markedly less toxic to cells in culture over a wide range of concentrations with cells numbering 76% of untreated controls at 37.5 μg/mL complete medium in the human hepatocellular carcinoma line HepG2 and 75% at 30 μg/mL in cervical carcinoma HeLa cells. At these levels of Chol-T liposomes, cell numbers were 37% and 15%, respectively. Gene transfer experiments with pSV2CAT and pRSVCAT plasmids in HepG2 cells showed maximum efficiency at a Chol-Q liposome:DNA ratio of 5:1 (w/w) and at a Chol-T liposome:DNA ratio of 10:1. In HeLa cells, both liposome preparations performed best at a ratio of 2.5:1. Differences in transfection efficiencies over the liposome range of 5-20 μg/ mL were rather less pronounced with Chol-Q lipoplexes suggesting a greater versatility of this system.     

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.     

Determination of ceftiofur in bovine plasma by HPLC-DAD

Ceftiofur sodium is a third generation broad-spectrum cephalosporin, formulated as an intramuscular injection, which is used to treat respiratory diseases in swine, ruminants and horses. The thioester bond on ceftiofur is rapidly cleaved to give desfuroylceftiofur which is further metabolized to a disulfide dimer and various desfuroylceftiofur-protein and amino acid conjugates. Methods of analysis of ceftiofur rely on cleavage by dithioerythritol to produce desfuroylceftiofur, which is further stabilized by derivatization to desfuroylceftiofur acetamide using iodoacetamide. Previous analytical methods have extracted derivatized analyte from plasma and tissue using solid-phase extraction clean-up steps followed by HPLC analysis with results reported as ceftiofur-free acid equivalents (CFAE). The simplified method presented here involves direct HPLC injection of a cleaved and derivatized sample following a protein precipitation step with calibration by external standardization and selectivity achieved based on chromatography and diode-array detection (DAD). The assay was linear over the calibration range 0.4–40 μg/ml in plasma. Intra-batch reproducibility R.S.D. was 10.3% and intra-batch sample repeatability R.S.D. was 2.1% at the 5 μg/ml level. The mean accuracy over the range of the calibration curve was −4.2% and the detection limit was 0.15 μg/ml. The assay was successfully applied to bovine plasma following intramuscular injection of ceftiofur sodium. This simplified method is suitable for pharmacokinetic applications involving ceftiofur at normal therapeutic levels.     

High-performance liquid chromatographic assay for morphine in small plasma samples: application to pharmacokinetic studies in rats

In order to perform a reliable pharmacokinetic study of morphine during subchronic treatment in rats, an easy, rapid, sensitive and selective analytical method was proposed and validated. The analyte and internal standard (naloxone) were extracted from plasma samples (100 μL) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography (HPLC) along with electrochemical detection (ED). Standard calibration graphs were linear within a range of 3.5–1000 ng/mL (r = 0.999). The intra-day coefficients of variation (CV) were in the range of 5.8–8.5% at eight concentration levels (7–1000 ng/mL) and the inter-day coefficient of variation ranged from 7.4 to 8.8%. The intra-day assay accuracy was in the range of −5–10% and the inter-day assay accuracy ranged from 3.0 to 9.3% of relative error (RE). The limit of quantification was 3.5 ng/mL using a plasma sample of 100 μL (15.8% of CV and 12.8% of RE). Plasma samples were stable for 2 months at −20 °C. This method was found to be suitable for pharmacokinetic studies in rats, after subcutaneous administration of morphine (5.6 mg/kg per day) in subchronic treatment for 6 and 12 days.     

Simultaneous determination of serum flecainide and its metabolites by using high performance liquid chromatography

Simultaneous determination of serum flecainide and its oxidative metabolites was carried out by using high performance liquid chromatography (HPLC) equipped with conventional octadecylsilyl silica (ODS) column and fluorescence detector. Flecainide and its metabolites, m-O-dealkylated flecainide (MODF) and m-O-dealkylated lactam of flecainide (MODLF) in serum were extracted with ethyl acetate. The recoveries of flecainide, MODF and MODLF were greater than 92, 93, and 60% with the coefficient of variations (CVs) less than 3.2, 5.8, and 5.3%, respectively. The calibration curves were linear at the concentration range of 50–1500 ng/mL for flecainide and 10–500 ng/mL for MODF and MODLF (r>0.999). The CVs for intra-day assay were 2.7–5.3% for flecainide, 3.0–4.2% for MODF, and 3.7–4.3% for MODLF, respectively. The CVs for inter-day assay were 7.0–8.4% for flecainide, 3.3–6.7% for MODF, and 4.4–7.7% for MODLF, respectively. This assay method can be used for assessing the metabolic ability of flecainide in the patients with tachyarrhythmia.     

Development and Validation of a Rapid RP-HPLC-DAD Analysis Method for the Simultaneous Quantitation of Paclitaxel and Lapatinib in a Polymeric Micelle Formulation

A robust and rapid analysis method was developed and validated for the simultaneous assay of paclitaxel (PTX) and lapatinib (LPT) in a polymeric micelle formulation as a novel drug delivery system using high-performance liquid chromatography (HPLC). The assay was performed using the C18 MZ-Analytical Column (5 μm, 150 × 4.6 mm, OSD-3) which was protected with the C18 pre-column (5 μm, 4.0 × 4.6 mm, OSD-3). The mobile phase was composed of acetonitrile and water (70/30; V/V) with a flow rate of 0.5 mL/min and detection wavelength of 227 nm. Accuracy was reported as the relative error and was found to be less than 6.8%. The interday assay was evaluated to be 3.22% and 5.76% RSD for PTX and LPT, respectively. The intraday precision was found to be at its maximum value of 5.83% RSD. The limit of detection for both PTX and LPT was found to be 1 µg/mL by means of the newly developed method. The limit of quantitation for PTX and LPT was found to be 5 µg/mL. The calibration curves for both drugs were linear in the concentration range of 5 to 80 μg/mL. In vitro release for both drugs from the polymeric micelle was evaluated using the newly developed analysis method.     

Simultaneous determination of tolperisone and lidocaine by high performance liquid chromatography

A reversed phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of tolperisone (TP) and lidocaine (LD) has been developed. The drugs were separated on a column (4.60×250 mm²) Spherisorb ODS (5 μm) using 5.5% triethylamine in 70/30 v/v acetonitrile/water as mobile phase 0.7 ml min⁻¹and UV detection at 254 nm. The detection limits for Tolperisone hydrochloride (TP·HCl) and lidocaine hydrochloride (LD·HCl) were 0.20 ng/20 μl and 100 ng/20 μl and the quantitation limits were 0.50 ng/20 μl and 250 ng/20 μl, respectively. Linear calibration curves over the ranges of 1–10, 10–100 and 150–500 μg ml⁻¹ for TP·HCl and 10–500 μg ml⁻¹ for LD·HCl were established. Different calibration slopes were found for TP probably owing to changes in refractive index due to increase in TP concentration. The average recoveries of the added TP in the samples (TP·HCl tablets and injection liquid). A solutions spiked with standard TP·HCl were 99.9 and 99.7% with the RSD (n=11) of 0.66 and 0.67%, respectively. The average recovery of the added LD in the sample (injection) spiked with standard LD·HCl was 98.9% with the RSD (n=11) of 0.59%. The proposed method has been applied to the determination of TP·HCl and LD·HCl in commercial products available in Thailand. Comparative determination of TP by UV spectrophotometry and LD by colorimetry were also carried out. The results obtained by both methods were in good agreement of those obtained by the proposed method verified by using t-test. The proposed RP-HPLC method is simple, accurate, reproducible and suitable for routine analysis.     

Cleavage and development in cultured preimplantation mouse embryos exposed to lidocaine

Two-cell preimplantation mouse embryos were exposed in vitro to lidocaine (0 to 1,000 μg/mL) for 72 h to determine the effects of this anesthetic on subsequent cleavage and development during prolonged exposures. Embryonic development was monitored each 24 h for 3 d. Lidocaine adversely affected the in vitro development of the mouse embryos, altering the distribution of the development stages at the evaluated culture tunes. The percentage of two-cell embryos that cleaved and developed to more advanced stages was decreased by the exposure to lidocaine. After 24 h of culture, two-cell embryos were arrested before completion of cellular division; this occured in 30% of the embryos at concentrations of 10 to 100 μg/mL and in 73.2% of the embryos with 1,000 μg/mL. After 48 h the blastomeres of the arrested embryos began to degenerate, showing lysis or fragmentation. At the lowest concentration, 14.9% of the arrested embryos exhibited the capacity to recover. These embryos continued their cleavage and normal development towards blastocyst formation. The cytotoxic effect and arrest at the two-cell stage were observed in a dose-dependent manner after 72 h of culture. We conclude that sensitivity to lidocaine embryotoxicity occurs during a window at the two-cell stage.     

高效液相色谱法测定小鼠肝脏中反式-白藜芦醇含量

建立一灵敏的HPLC法，用于测定小鼠肝脏中反式-白藜芦醇含量。取小鼠全部肝脏制成匀浆，用乙酸乙酯提取。分离有机层并挥干，残渣用0．2mL流动相溶解，离心，取上清液50此注入高效液相色谱仪。样品在岛津ODS柱（150mm×4．6mm,5μm）上分离，柱温35℃，检测波长305nm，流动相为甲醇-0．1mol／L乙酸溶液（4：6，v／v），流速为1mL／min。以信噪比3：1计，白藜芦醇在小鼠肝脏中的检测限为3．0ng／g；标准曲线的线性范围为5．0-120．0ng／g；浓度为6、10及80ng／g的平均回收率分别为102％、96．0％及91．2％；日内、日间精密度的RSD均小于5％。与已报道的其它白藜芦醇生物样品测定方法相比较，本法更为快速与灵敏，并具有良好的线性、选择性、准确度与精密度，可用于小鼠肝脏中白藜芦醇的药物动力学研究。     

Lactoferrin inhibits apoptosis through insulin-like growth factor I in primary rat osteoblasts

Aim： Excessive apoptosis of osteoblasts is the major cause of low bone mass, and bovine lactoferrin （bLF）, an iron-binding glycoprotein, might protect osteoblastic cells from apoptosis induced by serum withdrawal. The aim of this study was to elucidate the mechanisms underlying the anti-apoptotic action of bLF in rat osteoblasts in vitro. Methods： Primary rat osteoblasts were incubated in the presence of varying concentrations of bLF for 24 h. The expression of insulin-like growth factor I （IGF-I） and IGF-I receptor （IGF-IR） was measured uisng RT-PCR and Western blotting. Cell apoptosis was examined with flow cytometry. siRNAs targeting IGF-I was used in this study.

Results： Treatment of bLF （0.1–1000 μg/mL） dose-dependently increased the expression of IGF-I and IGF-IR in the osteoblasts. Treatment with bLF （10, 100 μg/mL） markedly inhibited the osteoblast apoptosis （with the rate of total apoptosis of 70% at 10 μg/mL）, but the high concentration of bLF （1000 μg/mL） significantly promoted the osteoblast apoptosis. Knockdown of the IGF-I gene in osteoblasts with siRNA markedly increased the osteoblast apoptosis.

Conclusion： Lactoferrin （10 and 100 μg/mL） effectively inhibits apoptosis of primary rat osteoblasts by upregulating IGF-I expression.     

Validated HPLC method for determination of LASSBio-581, a new heterocyclic N-phenylpiperazine derivative, in rat plasma

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C₁₈ column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25–8.0 μg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 μg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 μg/ml, 7.44 to 13.81% at 1.5 μg/ml and 6.10 to 13.94% at 6.0 μg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 μg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.     

HPLC quantification of two isomeric triterpenic acids isolated from Mitracarpus scaber and antimicrobial activity on Dermatophilus congolensis

Oleanolic (OA) and ursolic acids (UA) were isolated for the first time from the alcoholic extract of Mitracarpus scaber possessing antimicrobial effects on Dermatophilus congolensis. These two triterpenic acids were also active (MIC 15 μg/ml) on this causative agent of dermatophilosis in African animals.

To quantify OA and UA in M. scaber, a new, simple and rapid high-performance liquid chromatography (HPLC) method compatible with MS detection was developed and validated. The mobile phase acetonitrile:H₂O (85:15, v/v) was pumped through a C18 octadecylsilyl silica column at a flow rate of 0.6 ml/min and the eluate was monitored at 215 nm. The calibration curves constructed between 0.5 and 10 μg/ml showed linear relationships with good R² values. The developed method was precise and reproducible with relative standard deviations (RSD) for these two active constituents between 0.22–2.06% (intraday) and 1.61–3.72% (interday) for concentrations from 0.5 to 6 μg/ml. Limits of detection and quantification were, respectively, 0.2 and 0.5 μg/ml.     

Determination of cationic surfactants as the preservatives in an oral solution and a cosmetic product by capillary electrophoresis

In this study, a capillary electrophoresis method was developed for the determination of cationic surfactants, benzethonium and cetylpyridinium ions, which are commonly used as preservatives in various pharmaceutical and cosmetic products. Determination was performed in a fused-silica capillary using a mixed 75 mmol/L phosphoric acid and 50% acetonitrile electrolyte at pH 2.5. Analysis of benzethonium and cetylpyridinium ions was achieved in around 5 min. Repeatability in migration times (R.S.D.%) for benzethonium and cetylpyridinium ions were 0.3. The calibration curves were linear from 0.0125 to 0.400 mmol/L for benzethonium ions and from 0.025 to 0.400 mmol/L for cetylpyridinium ions. The minimum detection limits (signal-to-noise ratio = 3) are 1.47 and 4.30 μg/mL for benzethonium and cetylpyridinium ions, respectively. The method was applied to the analysis of benzethonium ion in a cosmetic product and cetylpyridinium ion in a mouthwash.