Determination of free and encapsulated cytarabine and daunorubicin in rat plasma after intravenous administration of liposomal formulation using ultra-high performance liquid chromatography tandem mass spectrometry.

Liposome encapsulating cytarabine (CYT) and daunorubicin (DNR) is applied for treating Acute Myeloid Leukemia (AML) patients. To evaluate and compare relationship between the pharmacokinetics of free drug (drug which is not entrapped in liposomes) and liposome-encapsulated drug and the toxicity/efficacy, it is crucial to have trustworthy methods for separating the free and the encapsulated of the drug. In this study, methods were developed and validated to isolate and measure the free DNR/CYT (F-DNR/CYT), the encapsulated DNR/CYT (E-DNR/CYT) and the total DNR/CYT (T-DNR/CYT) in rat plasma. The methods involved solid-phase extraction (SPE) using reverse adsorbents for separating the F-DNR and E-DNR, SPE using cation exchange adsorbents for separating the E-CYT, ultrafiltration for isolating the F-CYT and protein precipitation (PPT) for releasing the T-DNR and T-CYT totally from the liposomal forms. The analytes were subsequently quantified on ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) individually with multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). The calibration curves showed good linear relationships over the concentration range of 0.22-44 µg/mL for E-DNR and T-DNR, 2-1000 ng/mL for F-DNR, 0.5-100 µg/mL for E-CYT and T-CYT, 4-2000 ng/mL for F-CYT respectively. For all the analytes, the within-and between-run precisions were less than13.6% and the accuracies (in terms of RE%) were within -12.5%. Besides, extraction recovery, matrix effect, dilution integrity and stability were also assessed. The methods were successfully applied to investigate the pharmacokinetics in Sprague-Dawley rats following i.v. administration liposomal formulation.

[Assay of Tetramethylpyrazine in Szechwan Lovage Rhizome and Cnidium Rhizome by HPLC-DAD-MS].

OBJECTIVE: To quantity the amount of tetramethylpyrazine in Szechwan Lovage Rhizome (Chuanxiong, the rhizome of Ligusticum chuanxiong Hort., CX) and Cnidium Rhizome(Japanese Chuanxiong, the rhizome of Cnidium officinale Makino, JCX) for quality assessment. METHODS: An HPLC-DAD-MS technique was employed to detect tetramethylpyrazine in 27 CX and 10 JCX samples. Tetramethylpyrazine was separated on a Waters Symmetry C,, column (250 mm x 4. 6 mm, 5 µm). The mobile phase was methanol-acetonitrile-water(27: 1: 72) at a flow rate of 1. 0 mL/min. The column temperature was 35 °C. DAD detection wavelength was 280 nm, while electrospray ionization detector was set at positive mode to collect MS spectrum. RESULTS: In the total of 37 herb samples, 11 samples were found to contain tetramethylpyrazine with the mean amount of 2. 19 µg/g(n = 11). 6 of 27 CX samples and 5 of 10 JCX sample were found the existence of tetramethylpyrazine with the amount of 0. 60 - 11. 75 µg and 0. 61 - 3. 05 µg/g,respectively. The correlation was not found between tetramethylpyrazine and the cultivation area, morphological character, processing or storage method for CX and JCX samples. It was possible that tetramethylpyrazine resulted from the microbes in soil. CONCLUSION: The developed method is accurate to quantify tetramethylpyrazine in CX and JCX herbs. Both the two herbs indeed contain tetramethylpyrazine, but it is not suitable to be a chemical marker to assess the quality of CX and JCX owing to low content.

[Chemical change of chuanxiong raw materials during storage].

OBJECTIVE: To evaluate the chemical changes in Chuanxiong raw material (CX), the rhizome of Ligusticum chuanxiong Hort., during CX storage and further assess its quality variety. METHODS: Four CX samples were sealed and stored at ambient temperature in room for two years. These samples were quantified on the amounts of characteristic chemical compounds by HPLC-DAD-(APCI) MS techniques. RESULTS: Eight characteristic peaks in HPLC fingerprint were found to be good separation and assigned as vanillin (1), ferulic acid (2), senkyunolide I (3), senkyunolide H (4), coniferyl ferulate (5), senkyunolide A (6), Z-ligustilide (7) and levistolide A(8), respectively based on their on-line APCI-MS data and UV spectra. After CX being stored, compounds 1 - 4, and 8 were decreased by 44.4%, 52.1%, 37.6%, 52.8% and 47.5% (n = 4), respectively, whilst compounds 5 - 7 were increased by 59.1%, 40.1% and 47.5% (n = 4), respectively. CONCLUSION: Multiple chemical compounds are found to be changed during CX storage, which results in the variety of quality and therapeutic effect because most of the tested compounds have been demonstrated to be bioactive by pharmacological study and clinical trials. It is suggested that CX should be stored under dark, cool and dry condition.

An ultra-high sensitive bioanalytical method for plasma melatonin by liquid chromatography-tandem mass spectrometry using water as calibration matrix.

For the endogenous substances with an ultra-low level in biological fluids, such as melatonin, the blank biological matrix is obviously not "blank". This problem leads to a serious issue of the bioanalytical methods development and validation by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This work developed and validated an ultra-high sensitive bioanalytical method for plasma melatonin by LC-MS/MS using water as calibration matrix. The lower limit of quantitation of the method was verified to be 1.0 pg/mL and the method exhibited a linear range of 1-5000 pg/mL. Potential matrix effects, accuracy and precision were fully monitored and validated by two complementary quality control approaches respectively using water and the pooled plasma as matrix. The intra-run and inter-run precisions were less than 11.5% and 12.2%, respectively, and the relative error was below ± 13.8% for all of 5 quality control levels. The method was successfully applied to investigate the daytime (8:00 AM-8:00 PM) baseline level of endogenous plasma melatonin, as well as the pharmacokinetic profiles of exogenous melatonin after oral administration in beagle dogs.

Meiothermus cateniformans sp. nov., a slightly thermophilic species from north-eastern China.

Two aerobic, non-motile and non-sporulating bacteria, strains LY1( T) and L462, were recovered from a hot spring located in the Qianshan area of north-eastern China. The novel strains had Gram-negative cell walls and grew at 30-66 degrees C (optimum 55-60 degrees C) and pH 5.5-10.0 (optimum pH 8.0-8.5). 16S rRNA gene sequence similarity analysis revealed that strains LY1( T) and L462 were most closely related to Meiothermus ruber ATCC 35948(T), M. taiwanensis WR-30(T) and M. cerbereus GY-1(T), with 97.1-98.4 % sequence similarity. Phylogenetic analysis indicated that the new isolates represented a novel species by forming a distinctive lineage within genus Meiothermus, which was confirmed by DNA-DNA reassociation values. Strain LY1( T) was obviously different from its closest relatives in a number of phenotypic characteristics, such as the inability to hydrolyse casein or to assimilate melibiose, trehalose, sucrose and lactose. Strain L462 was distinct in its ability to reduce nitrate. In addition, the novel strains were distinct in having larger amounts of anteiso-C(17 : 0) than their closest phylogenetic neighbours. On the basis of this polyphasic taxonomic characterization, the name Meiothermus cateniformans sp. nov. is proposed for the novel species, currently represented by the type strain LY1( T) (=CGMCC 1.6990(T) =JCM 15151(T)) and strain L462 (=CGMCC 1.6989 =JCM 15150).