[The study on the fluorescence spectrometry for a novel photosensitizer of chlorine e6-C15-monomethyl ester and its application in biosamples analysis].

This paper studied the fluorescence spectral characteristic of chlorin e6-C15-monomethyl ester in different solvents to develop a fluorescence spectrometry for determining the concentration of chlorin e6-C15-monomethyl ester in plasma. By comparing the fluorescence spectral characteristics of chlorin e6-C15-monomethyl ester in six different solvents including methanol, ethanol, acetone, acetonitrile, phosphate buffered saline and water, the influence of different solvents on the fluorescence spectral characteristic of chlorin e6-C15-monomethyl ester has been examined. The results indicated that methanol and acetonitrile are the ideal solvent system, and the effect of phosphate buffered saline is better than water. The effect of organic solvent content and solution pH value on emission wavelength and fluorescence intensity was further evaluated. It was found that the fluorescence intensity of chlorin e6-C15-monomethyl ester was strong and stable in pH 7.2 phosphate buffered saline -acetonitrile (3 : 7) solution and can be detected at the excitation wavelength of 498.00 nm and the emission wavelength of 664.05 nm. Based on this, we developed the fluorescence spectrometry for determining chlorin e6-C15-monomethyl ester. It is specific and sensitive with good linearity over the range of 0.5-50 microg x mL(-1). The intra-batch and inter-batch precisions (RSD) were less than 10%, the extraction recoveries were all over 90%. The established method in this paper was simple, fast, effective and could be successfully applied to study the pharmacokinetics of chlorine e6-C15-monomethyl ester in SD rats.

Development and validation of a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry assay for the determination of neostigmine in small-volume beagle dog plasma and its application to a pharmacokinetic study.

A simple, rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of neostigmine in small-volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96-well filtration plate, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) with a mobile phase composed of methanol-water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 µL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor-to-product ion transitions m/z 223.0 → 72.0 and 306.0 → 140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1-100ng/mL for neostigmine (r ≥ 0.998). All the validation data, such as accuracy, intra-run and inter-run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs.

Development and validation of a rapid and sensitive assay for the determination of anisodamine in 50 µL of beagle dog plasma by LC-MS/MS.

A simple, rapid, high-throughput, and highly sensitive LC-MS/MS was developed to determine anisodamine in a small volume (50 µL) of beagle dog plasma using atropine sulfate as the internal standard. The analyte and internal standard were isolated from 50 µL plasma samples after a one-step protein precipitation using Sirocco 96-well protein precipitation filtration plates. The separation was accomplished on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) and the run time was 4 min. A Micromass Quatro Ultima mass spectrometer equipped with an ESI source was operated in the multiple reaction monitoring mode with the precursor-to-product ion transitions m/z 306.0→140.0 (anisodamine) and 290.0→123.9 (atropine) used for quantitation. The method was sensitive with a low LOQ of 0.05 ng/mL, and good linearity in the range 0.05-50 ng/mL for anisodamine (r(2) ≥ 0.995). All the validation data, such as accuracy, intra- and interrun precision, were within the required limits. The method was successfully applied to the pharmacokinetic study of anisodamine hydrochloride injection in beagle dogs.

Determination of an unstable pentapeptide, monocyte locomotion inhibitory factor, in dog blood by LC-MS/MS: application to a pharmacokinetic study.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and established for the quantitative determination of monocyte locomotion inhibitory factor, a pentapeptide (Met-Gln-Cys-Asn-Ser) produced by Entamoeba histolytica in axenic culture, in dog blood. The main challenge was the chemical and enzymatic instability of the peptide which was successfully overcome. After a simple protein precipitation, MLIF was separated from AS-5 (Met-Gln-Gly-Asn-Ser), acted as an internal standard, on a Gemini C18 column (5 µm, 50 mm × 4.6 mm i.d.) using a gradient elution of acetonitrile (0.2% formic acid) and water (0.2% formic acid) and detected by electrospray ionization tandem mass spectrometry. Excellent linearity was achieved (r>0.9943) over the linear range 5-1000 ng/ml using 0.2 ml blood sample. The validation results demonstrated that this method was specific, accurate and precise. It was successfully applied in measuring MLIF following intravenous infusion its administration at 0.2, 0.4, and 0.8 mg/kg in beagle dogs to support the pre-clinical pharmacokinetic study.

Determination of esculentoside A in dog plasma by LC-MS/MS method: application to pre-clinical pharmacokinetics.

A simple and rapid high performance liquid chromatography electrospray ionization ion-trap tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitative determination of esculentoside A (EsA) in dog plasma using ginsenoside Rg1 as the internal standard (IS). After liquid-liquid extraction (LLE) with n-butanol, the analyte and IS were separated on a Diamonsil C(18) (2.1 mm × 50 mm, 3 µm) column with the mobile phase of methanol-water containing 0.1% acetic acid (70:30, v/v) at a flow rate of 0.2 ml/min. An ion trap mass spectrometer equipped with an electrospray ionization source performed in selected reaction monitoring (SRM) mode was used as the detector. The precursor-product ion transitions were m/z 849.3 [M+Na](+)→m/z 805.3 for EsA and m/z 823.3 [M+Na](+)→m/z 643.3 for IS. The total chromatographic run time was 5 min. The method was sensitive enough with a lower limit of quantitation (LLOQ) of 5 ng/ml and had a good linearity (r(2)>0.997) over the linear range of 5-500 ng/ml. The mean extraction recovery of EsA from spiked plasma samples was over 75%. The intra- and inter-precisions were no more than 8.8% and accuracies were within the range of -4.6 to 8.7%. All the validated data were within the accepted criteria as stated in the FDA bioanalytical method validation guideline. The developed method was suitable for the quantification of EsA and successfully applied to the pharmacokinetic study of EsA after an oral administration to beagle dogs.

Simultaneous determination of amlodipine and bisoprolol in rat plasma by a liquid chromatography/tandem mass spectrometry method and its application in pharmacokinetic study.

A sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantitative determination of amlodipine and bisoprolol, using clenbuterol as the internal standard (IS). The analytes and IS were isolated from 100µL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase high performance liquid chromatography (RP-HPLC) separation was accomplished on a Diamonsil C(18) column (50mm×4.6mm, 5µm) with a mobile phase composed of methanol-water-formic acid (75:25:0.01, v/v/v) at a flow rate of 0.3mL/min. The method had a chromatographic total run time of 3min. Multiple reacting monitoring (MRM) transitions of m/z [M+H](+) 409.1→237.9 (amlodipine), m/z [M+H](+) 326.2→116.0 (bisoprolol) and m/z [M+H](+) 277.0→203.0 (clenbuterol, IS) were used to quantify amlodipine, bisoprolol and IS, respectively. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.2ng/mL for both amlodipine and bisoprolol, and the linear range was 0.2-50ng/mL for both amlodipine and bisoprolol (r(2)>0.9961). All the validation data, such as accuracy, precision and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic studies of amlodipine and bisoprolol in Sprague-Dawley (SD) rats.

ß-Cyclodextrin enhanced on-line organic solvent field-amplified sample stacking in capillary zone electrophoresis for analysis of ambroxol in human plasma, following liquid-liquid extraction in the 96-well format.

A field-amplified sample stacking (FASS) and capillary zone electrophoresis (CZE) method is described for the quantification of ambroxol hydrochloride in human plasma, following liquid-liquid extraction in the 96-well format. The separation was carried out at 25 °C in a 31.2 cm × 75 µm fused-silica capillary with an applied voltage of 15 kV. The background electrolyte (BGE) was composed of 6.25 mM borate-25 mM phosphate (pH 3.0) and 1mM ß-cyclodextrin. The detection wavelength was 210 nm. Clean-up and preconcentration of plasma biosamples were developed by 96-well format liquid-liquid extraction (LLE). In this study, FASS in combination with ß-cyclodextrin enhanced the sensitivity about 60-70 fold in total. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and robustness. The calibration graph was linear for ambroxol hydrochloride from 2 to 500 ng/ml. The lower limit of quantification was 2 ng/ml. The intra- and inter-day precisions of lowest limit of quantification (LLOQ) were 9.61 and 11.80%, respectively. The method developed was successfully applied to the evaluation of clinical pharmacokinetic study of ambroxol hydrochloride tablet after oral administration to 12 healthy volunteers.