Bruceines D and E are quassinoids from seeds of Brucea javanica (L.) Merr. exhibiting hypoglycemia effect. The crude drug is used as a traditional medicine by diabetes patients. The aim of this study is to understand the bioavailability and pharmacokinetics of both the bruceines D & E. A rapid and sensitive HPLC-MS/MS method was developed and validated for the quantification of both quassinoids, bruceines D & E in rat plasma. Both the bruceines D & E were separated with the Zorbax SBC-18 column with gradient elution and mobile phase system of acetonitrile and deionized water with 0.1% formic acid at a flow rate of 0.5mL/min. Analytes were detected in multiple reaction monitoring (MRM) mode with electrospray positive ionization. The quassinoids, namely bruceines D & E were detected with transitions of m/z 411.2→393.2 and m/z 395.2→377.2, respectively. Another quassinoid, eurycomanone was used as the internal standard with transition of m/z 409.2→391.2. The method was validated and conformed to the regulatory requirements. The validated method was applied to pharmacokinetic and bioavailability studies in rats. The pharmacokinetic study indicated both bruceine D and E were rapidly absorbed into the circulation system and reached its peak concentration at 0.54±0.34h and 0.66±0.30h, respectively. Bruceine E was eliminated slower than Bruceine D with t1/2 value almost increased two-fold compared to Bruceine D. In conclusion, a rapid, selective and sensitive HPLC-MS/MS method was developed for the simultaneous determination of both the bruceines D and E in rat plasma. Both bruceines D and E displayed poor oral bioavailability.
Enantioselective analysis is critically important in the pharmaceutical and agricultural industries. However, most of the methods reported were developed for the analysis of pure racemates acquired from chemical synthesis or purification. Direct analysis of chiral enantiomers in complex matrices has rarely been reported. This work demonstrated capillary electrophoresis-mass spectrometry (CE-MS) for the enantioselective analysis of botanical drugs for the first time, using a widely used botanical drug, Corydalis Rhizoma, as an example. The method was used for the simultaneous enantioselective analysis of dl-tetrahydropalmatine and (RS)-tetrahydroberberine (canadine) in Corydalis Rhizoma extract. Using (2-hydroxypropyl)-β-cyclodextrin as the chiral selector, a partial filling technique was used to avoid signal suppression and contamination of the MS detector. Post column organic modifier was used to assist with ionization in the flow through microvial CE-MS interface, therefore, organic solvents was not used in the background electrolyte. The completely aqueous background electrolyte contributed to better chiral separations. The CE-MS method established here can directly determine the analytes in their complex matrix without any pre-purification steps, while also offering high sensitivity and low operational costs (including sample, chiral selector and solvent). In the method validation process, good linearity (r > 0.993), sensitivity and accuracy (recoveries within 89.1-110.0%) were demonstrated. The CE-MS technique was shown to be able to provide good selectivity for the simultaneous chiral separation of multiple pairs of enantiomers in complex matrices.
Dye-ligand affinity chromatography in a stirred fluidized bed has been developed for the rapid recovery of malate dehydrogenase (MDH) from highly turbid baker's yeast cell homogenate in a single step. The most suitable dye, namely Reactive Orange 4, in its optimal immobilized concentration of 8.78 mg/mL was immobilized onto high-density STREAMLINE matrix. To further examine optimal adsorption and elution conditions, the enzyme recovery operation was carried out using unclarified cell homogenates in stirred fluidized bed system. Aiming to develop a non-specific eluent, namely NaCl, to effectively elute the MDH adsorbed, direct recovery of MDH from highly turbid cell homogenate (50% w/v) in a stirred fluidized bed adsorption system was performed. The proposed system successfully achieved a recovery yield of 73.6% and a purification factor of 73.5 in a single step by using 0.6 M NaCl as an eluent at a high liquid velocity of 200 cm/h.
A simple analytical method using HPLC with fluorescence detection was developed for the simultaneous determination of four lignans, phyllanthin (1), hypophyllanthin (2), phyltetralin (3) and niranthin (4) from Phyllanthus niruri L. in plasma. The method recorded limits of detection for 1, 2, 3 and 4 as 1.22, 6.02, 0.61 and 1.22 ng/ml, respectively, at a signal-to-noise ratio of 5:1 whereas their limits of quantification were 4.88, 24.41, 4.88 and 9.76 ng/ml, respectively, at a signal-to-noise ratio of 12:1. These values were comparable to those of other sensitive methods such as gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-MS (HPLC-MS) and HPLC-electrochemical detection (HPLC-ECD) for the analysis of plasma lignans. A further advantage over known methods was its simple protocol for sample preparation. The within-day and between-day accuracies for the analysis of the four lignans were between 87.69 and 110.07% with precision values below 10.51%. Their mean recoveries from extraction were between 91.39 and 114.67%. The method was successfully applied in the pharmacokinetic study of lignans in rats. Following intravenous administration, the lignans were eliminated slowly from the body with a mean clearance of 0.04, 0.01, 0.03 and 0.02 l/kg h and a mean half-life of 3.56, 3.87, 3.35 and 4.40 h for 1, 2, 3 and 4, respectively. Their peak plasma concentration upon oral administration was 0.18, 0.56, 0.12 and 0.62 microg/ml, respectively, after 1h. However, their absorption was incomplete with a calculated absolute oral bioavailability of 0.62, 1.52, 4.01 and 2.66% for 1, 2, 3 and 4, respectively.
A high-performance liquid chromatographic method with ultraviolet (UV) detection was developed for measuring cefotaxime in rat and human plasma. The method used direct injection of the plasma supernatant after deproteinization with 70% perchloric acid. Degradation of cefotaxime in acidic medium was retarded by adding phosphate buffer before centrifuging the sample. The mobile phase was 0.05 M aqueous ammonium acetate-acetonitrile-tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5. Analysis was run at a flow-rate of 1.0 ml/min, and a detection wavelength of 254 nm was used. The method has a quantification limit of 0.20 microgram/ml. The within- and between-day coefficients of variation and accuracy values were less than 8% and +/-3%, respectively, while the recovery values were greater than 87% over the concentration range tested (0.20-50 microgram/ml). The speed, sensitivity, specificity and reproducibility of this method make it particularly suitable for the routine determination of cefotaxime in human plasma. Moreover, only a relatively small sample plasma volume (100 microliter) is required, allowing this method to be applied to samples taken from neonates.
A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100-1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35°C and 100°C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35°C and 100°C, respectively. From the targeted metabolites identification, it was found that 3,4ɛ-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100°C.
Nicotine is a major addictive compound in cigarette. Its smoke is rapidly and extensively metabolized to several metabolites in human. Cotinine as a major metabolite of nicotine is commonly used as a biomarker to determine active and passive smokers. Cotinine has a longer half-life ( approximately 20 h) compared to nicotine ( approximately 2h). A simple, sensitive, rapid and high throughput GC-MS method was developed for simultaneous quantification of urinary nicotine and cotinine in passive and active smokers. In the sample preparation method, the analytes and internal standard were first basified and followed by liquid-liquid extraction. Upon completion, anhydrous sodium sulphate was added to the solvent mixture to trap moistures. The clear extract obtained was directly injected into GC-MS, operating under selective ion monitoring (SIM) mode. Calibration curves in the range of 0.5-5000 ng/mL of the analytes in urine matrix were established with linear correlation coefficients (r(2)) greater than 0.997. The limit of detection for both nicotine and cotinine were 0.20 ng/mL. The mean recoveries for nicotine and cotinine were 93.0 and 100.4%, respectively. The within- and between-assay accuracies were between 2.1 and 7.9% for nicotine and between 0.7 and 11.1% for cotinine. Within- and between-assay precisions of 3.3-9.5% for nicotine and 3.4-9.8% for cotinine were also achieved. The method can be used in routine assessment and monitoring of active smoking and exposure to environmental tobacco smoke. The applicability of the assay was demonstrated in a small-scale comparison study between smokers and non-smokers.
Hair nicotine is a known biomarker for monitoring long-term environmental tobacco smoke (ETS) exposure and smoking status. In general, hair nicotine assay involves alkaline digestion, extraction and instrumental analysis. The gas chromatography-mass spectrometry (GC-MS) assay currently developed has shown to be of high throughput with average approximately 100 hair samples being extracted and analyzed per day. This was achieved through simplified extraction procedure and shortened GC analysis time. The extraction was improved by using small volume (0.4 mL) of organic solvent that does not require further evaporation and salting steps prior to GC-MS analysis. Furthermore, the amount of hair utilized in the extraction was very little (5 mg) while the sensitivity and selectivity of the assay is equal, if not better than other established methods. The linearity of the assay (r(2)>0.995), limit of quantitation (0.04 ng/mg hair), within- and between-assays accuracies and precisions (<11.4%) and mean recovery (92.6%) were within the acceptable range.
PURPOSE: This study aimed to develop a sensitive, quantitative assay for the antimalarial piperaquine (PQ) and its metabolites M1 and M2 in human plasma.
RESULTS: Analytes were gradiently separated on a C18 column and detected with a Sciex API 4000 MS/MS with an ESI source operated in the positive ion mode with deuterated PQ as internal standard. The response was linear in the range 3.9-2508nM with a runtime of 7.0min per sample. The method was applied to clinical samples from healthy volunteers.
CONCLUSION: This LC-MS/MS method for the simultaneous quantitation of PQ and two of its metabolites in plasma may prove helpful for assessment of metabolite safety issues in vivo.
An aqueous two-phase flotation (ATPF) system based on polyethylene glycol (PEG) and sodium citrate (NaNO3C6H5O7·2H2O) was considered for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10. The effects of ATPF parameters namely phase composition, tie-line length (TLL), volume ratio between the two phases (VR), amount of crude load (CL), pH, nitrogen gas flow rate (FR) and flotation time (FT) on the performance of recovery were evaluated. BLIS was mainly concentrated into the upper PEG-rich phase in all systems tested so far. The optimum conditions for BLIS purification, which composed of PEG 8000/sodium citrate, were: TLL of 42.6, VR of 0.4, CL of 22% (w/w), pH 7, average FT of 30min and FR of 20mL/min. BLIS was partially purified up to 5.9-fold with a separation efficiency of 99% under this optimal conditions. A maximum yield of BLIS activity of about 70.3% was recovered in the PEG phase. The BLIS from the top phase was successfully recovered with a single band in SDS-gel with molecular weight of about 10-15kDa. ATPF was found to be an effective technique for the recovery of BLIS from the fermentation broth of P. acidilactici Kp10.