Simultaneous determination of four sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. extracts by high-performance liquid chromatography

Wilfortrine, wilfordine, wilforgine and wilforine are four major bioactive sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. The first analytical determination of the four major bioactive alkaloids is described. The four alkaloids are well-resolved within 15 min using the developed HPLC method. The identity of the analytes was confirmed by an HPLC-MS experiment, with all compounds being clearly assignable by atmospheric pressure chemical ionization (APCI) positive mode analysis. The method was validated for limit of qualification, linearity and inter-day variation of precision and accuracy. Seven T. wilfordii samples (extracts and commercial product) were successfully analysed.     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Determination of andrographolide in human plasma by high-performance liquid chromatography/mass spectrometry

In this paper, a rapid method based on high-performance liquid chromatography/electrospray-mass spectrometry (HPLC/ESI-MS) method for the quantitative determination of andrographolide (AND) in human plasma has been developed and validated. A liquid-liquid extraction (LLE) procedure was selected to isolate AND from biological matrixes. Isosorbide-5-mononitrate (IS-5-MN) was selected as the internal standard (IS). The correlation coefficient of the calibration curve was 0.998, in the range of 9.9-320.0 ng/mL. The validated method may be used to assess the bioavailability and pharmacokinetics of the drug.     

雷公藤植物内生Micromonospora sp. M66生产的一组吲哚生物碱

【目的】研究稀有放线菌——雷公藤内生小单孢菌(Micromonospora sp.M66)的次级代谢产物,为微生物药物或农用生物制剂开发提供结构多样的化合物资源。【方法】利用薄层层析、正(反)相硅胶柱层析、凝胶层析、液相色谱等技术对M66菌株中次级代谢产物进行分离纯化,利用波谱技术对化合物进行结构鉴定。【结果】最终分离纯化了7个单体化合物,结合质谱与核磁技术对这7个化合物进行了结构解析和鉴定,它们属于一组吲哚生物碱。化合物2是重要的植物生长调节剂,化合物3对淋巴细胞性白血病细胞P388、枯草芽孢杆菌和酿酒酵母的增殖有抑制作用,化合物6对金黄色葡萄球菌有很好的抑制作用。【结论】化合物3-7首次从小单胞菌中鉴定出来,表明该小单孢菌具有较强的利用吲哚或色氨酸合成次级代谢产物的能力和挖掘生物碱类药物的潜力。     

Determination of misoprostol acid in human plasma by liquid chromatography coupled to tandem mass spectrometry

A rapid and sensitive liquid chromatographic/tandem mass spectrometric method for determination of misoprostol acid, the active metabolite of misoprostol, was developed and validated. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a C(18) column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 367-249 and 296-269 was performed to quantify misoprostol acid and the internal standard hydrochlorothiazide, respectively. The method was linear in the concentration range of 10.0-3000 pg mL(-1) using 200 microL plasma. The lower limit of quantification was 10.0 pg mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 8.3%. Accuracy determined at three concentrations (25.0, 200 and 2700 pg mL(-1) for misoprostol acid) ranged from -0.5 to 1.2% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method was successfully used in a pharmacokinetic study of misoprostol in human plasma after an oral administration of 0.6 mg misoprostol.     

Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80 C(18) column with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. 5AC elutes at 5.0 +/- 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9-->113.0 for 5AC and m/z+ 242.0-->126.0 for 5-methyl-2'-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.     

Studies on Neotriptetraolide Isolated from Tripterygium wilfordii Hook. f.

A new diterpenoid bisepoxide, neotriptetraolide, was isolated from the leaves and root of Tripterygium wilfordii Hook. f. Neotriptetraolide was crystallized from CH3OH as colorless needle-like crystal, with amp of 237～238 ℃. Its molecular formula was C20H2608. Its structure was elucidated by means of spectral analysis (UV, IR, MS, 1H-NMR, 13C- NMR, 2D-COSY, 2D-NOESY, 13C-NOE, and selective long-range DEPT spectroscopy) and the computation of molecular mechanics and molecular graph.     

Determination of ketoconazole in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

Determination of ecabet in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a simple, robust and cost-effective assay for the determination of ecabet in human plasma. After a simple step of protein precipitation using methanol, plasma samples were analyzed by reverse phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) with valsartan as the internal standard (I.S.). Ecabet and the I.S. valsartan were separated on a Venusil MP C18 analytical column using methanol-10mM ammonium acetate (75:25, v/v, pH 3.0) as mobile phase at a flow rate of 1.0 mL/min. Ecabet and I.S. were eluted at 0.91 and 0.92 min, respectively, ionized in negative mode, and then detected by multiple reaction monitoring (MRM) essay. The MRM transitions of m/z 379.1-->m/z 277.1 and m/z 434.3-->m/z 350.1 were used to quantify ecabet and I.S., respectively. The assay was linear over the concentration range of 10-6000 ng/mL and was successfully applied to a pharmacokinetic study in healthy volunteers.     

Determination of clindamycin in human plasma by high-performance liquid chromatography using coupled columns

A rapid automated method has been developed for the determination of clindamycin, a lincosamide antibiotic, in human plasma. Coupled column HPLC was used after precipitation of plasma proteins with a saturated ammonium sulfate solution. As a first step, the drug and internal standard were trapped on a precolumn of LiChrospher 60RP-select B. A reversed-phase Nucleosil 100 C18 HD column then separated drug and internal standard from each other and from remaining plasma components. The assay was validated in the range 0.2–10.0 μg ml⁻¹ plasma. The results obtained for accuracy, intra- and inter-day precision complied very well with the generally accepted criteria for bioanalytical assays.     

Determination of soyasaponins Ba and Bb in human serum by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS-MS) has been developed and validated for the determination of soyasaponins Ba and Bb in human serum using glycyrrhizin as internal standard (I.S.). Soyasaponins Ba and Bb were extracted from human serum by liquid-liquid extraction and cleaned up by C(18) solid-phase extraction (SPE), followed by separation on a C(18) reversed-phase column using acetonitrile/water containing 0.025% acetic acid as a mobile phase for gradient elution. Soyasaponins Ba and Bb, and I.S. were ionized by negative ion pneumatically assisted electrospray and detected by HPLC-MS-MS in the multiple-reaction monitoring (MRM) mode using precursor-->product ion combinations at m/z 958-->940, 942-->924 and 822-->351, respectively. The calibration curves were linear (r(2)>0.991) in the concentration range of 0.5-100.0 ng/mL, with lower limits of quantification of 0.5 and 0.3 ng/mL for soyasaponins Ba and Bb, respectively, in human serum. Intra-day and inter-day relative standard deviations (R.S.D.) were less than 7.9 and 11.3%, respectively. The mean recoveries of soyasaponins Ba and Bb ranged from 92 to 101% and from 85 to 94%, respectively.     

Determination of beraprost in human plasma by a high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of beraprost, a stable, orally active prostacyclin analogue with vasodilatory, antiplatelet and cytoprotective effects. The analyte and internal standard, indomethacin, were extracted by solid-phase extraction using OASIS HLB cartridge. The chromatographic separation was performed on a C18 column with a mobile of 0.1% formic acid-methanol (30:70, v/v). The highest daughter ion of deprotonated analyte was quantitated in negative ionization by multiple reactions monitoring with a mass spectrometer. The mass transitions m/z 397>269 and m/z 356>312 were used to measure beraprost and internal standard, respectively. The assay exhibited a linear range from 0.02 to 2 ng/mL for beraprost in human plasma. The lower limit of quantitation was 20 pg/mL with a relative standard deviation of less than 20%. The method was validated with respect to linearity, sensitivity, specificity, recovery, accuracy and precision. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic study.     

Chiral evaluation of fluvastatin in human plasma by high-performance liquid chromatography electrospray mass spectrometry

The report describes for the first time the enantioselective analysis of fluvastatin in plasma using LC-MS-MS. The enantiomers of fluvastatin (FV) were extracted from plasma with diisopropyl ether at pH 5.0. The enantiomers were separated on a ChiralCel OD-R column with a mobile phase consisting of a mixture of acetonitrile, methanol and water (24:36:40) containing 0.1% formic acid. The protonated ions and their respective product ions were monitored in two functions, 410.6>348.2 for FV enantiomers and 307.1>161.6 for the internal standard (warfarin). Recoveries were higher than 90% and the quantitation limit was 1.5 ng mL(-1) plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of the intra- and interassay precision and accuracy were less than 10%. The method was applied to the investigation of the enantioselective pharmacokinetics of FV administered in a single dose of 40 mg (Lescol, Novartis, S?o Paulo, SP, Brazil) to a patient with primary hypertension and hypercholesterolemia and genotyped as CYP2C9*1/*1. The data showed higher plasma concentrations of the (-)-3S,5R-fluvastatin enantiomer, with an AUC (-)/(+) of 1.84. Oral clearance values (CL/F) were 29.27 and 49.58 L/h, respectively, for the (-)-3S,5R- and (+)-3R,5S-fluvastatin enantiomers.     

Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.     

Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies

A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 microl of human plasma by liquid-liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 microl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05-10 ng ml(-1). The limit of quantification (LOQ) was 0.05 ng ml(-1). This HPLC-MS-MS procedure was used to assess pharmacokinetic studies.     

Simultaneous determination of five toxic alkaloids in body fluids by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel analytical method was developed and validated for the rapid and simultaneous analysis of five toxic alkaloids: Brucine, Strychnine, Ephedrine, Aconitine and Colchicine, in blood and urine using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (HPLC-ESI-MRM) mode. The linear range was 0.05-50.0 ng mL(-1) for Brucine, 0.1-50.0 ng mL(-1) for Strychnine and Ephedrine, 0.01-10.0 ng mL(-1) for Aconitine and Colchicine. The limits of quantification for Brucine, Strychnine, Ephedrine, Aconitine and Colchicine were found to be 0.03, 0.05, 0.20, 0.05, 0.01 ng mL(-1), respectively. The average extraction recoveries in urine ranged from 96.0 to 114.0% and in whole blood were 94.0 to 113.0%. The intra-day and inter-day RSDs were less than 8.3 and 10.6%, respectively. The five alkaloids could be well separated within 7 min in a single run. The established method should be suitable for the determination of trace alkaloids in body fluids.     

Determination of tripdiolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reversed-phase high-performance liquid chromatography

Extracts of Tripterygium wilfordii Hook F. have been widely used in China to treat a variety of autoimmune and inflammatory diseases. The diterpenoids triptolide and tripdiolide are two major active components in the T. wilfordii ethyl acetate extract. An efficient solid-phase extraction and high-performance liquid chromatography (SPE-HPLC) method to measure triptolide content in the extract has been previously reported. However, a suitable means of tripdiolide quantification is not available because of interfering compounds in the extract that co-elute with tripdiolide. Therefore, this paper describes a method wherein tripdiolide content can be measured from a small amount of the extract. The extract solution (600 microL) was applied into an aminopropyl SPE tube. Triptolide was eluted with dichloromethane:methanol (1 mL, 49:1 v/v), followed by tripdiolide elution with dichloromethane:methanol (3 mL, 17:3 v/v). The tripdiolide eluate was analysed by HPLC using an isocratic solvent system and was quantified by measuring the peak area at 219 nm. The contents of triptolide and tripdiolide in the extract were determined to be 807.32 +/- 51.94 and 366.13 +/- 17.21 microg/g of extract, respectively. Since tripdiolide is biologically active and makes up a considerable portion of the extract, for extract quality control and standardisation purposes, it should be measured along with triptolide using the proposed SPE-HPLC method.     

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.