Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

Parallel extraction columns and parallel analytical columns coupled with liquid chromatography/tandem mass spectrometry for on-line simultaneous quantification of a drug candidate and its six metabolites in dog plasma.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%.     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.     

Determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxymethamphetamine in urine by online solid-phase extraction and ion-pairing liquid chromatography with detection by electrospray tandem mass spectrometry

A method using an online solid-phase extraction (SPE) and ion-pairing liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MS/MS) was developed for determination of amphetamine (Amp), methamphetamine (mAmp), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. A SPE cartridge column with both hydrophilic and lipophilic functions was utilized for online extraction. A reversed-phase C18 LC column was employed for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. This method was fully automated and the extraction and analysis procedures were controlled by a six-port switch valve. Recoveries ranging from 85-101% were measured. Good linear ranges (10-500 ng/mL) for Amp and mAmp were determined. For MDA, MDMA and MDEA, dual linear ranges were obtained from 5-100 and 100-500 ng/mL, respectively. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, ranged from 1-3 ng/mL. The applicability of this newly developed method was examined by analyzing several urine samples from drug users. Good agreement was obtained between the results from this method and a literature GC/MS method.     

适用于质谱分析的在线固相萃取除盐方法

建立了一个简单、快速、有效的适用于质谱或液相色谱-质谱联用的在线固相萃取(SPE)高通量除盐方法。方法分为单柱和双柱模式,借助于包含双梯度泵(上样泵/分析泵)、自动进样器和配有十通切换阀的柱温箱的高效液相色谱系统,完成样品的自动化在线除盐。单柱模式通过上样泵实现在SPE柱上进样和除盐,被分析物则保留在SPE柱上;除盐完成后,通过阀切换利用分析泵洗脱富集在SPE柱上的被分析物。双柱模式则在单柱模式基础上增加了1根SPE柱,在色谱管理软件控制下2根SPE柱轮流工作,高效率完成样品的在线除盐。该方法在结合质谱分析蛋白质、多肽等领域具有较好的应用前景。     

Direct-injection high performance liquid chromatography ion trap mass spectrometry for the quantitative determination of olanzapine,clozapine and N-desmethylclozapine in human plasma

A specific and sensitive direct-injection high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the rapid identification and quantitative determination of olanzapine, clozapine, and N-desmethylclozapine in human plasma. After the addition of the internal standard dibenzepin and dilution with 0.1% formic acid, plasma samples were injected into the LC/MS/MS system. Proteins and other large biomolecules were removed during an online sample cleanup using an extraction column (1 x 50 mm i.d., 30 microm) with a 100% aqueous mobile phase at a flow rate of 4 mL/min. The extraction column was subsequently brought inline with the analytical column by automatic valve switching. Analytes were separated on a 5 microm Symmetry C18 (Waters) analytical column (3.0 x 150 mm) with a mobile phase of acetonitrile/0.1% formic acid (20:80, v/v) at a flow rate of 0.5 mL/min. The total analysis time was 6 min per sample. The inter- and intra-assay coefficients of variation for all compounds were <11%. By eliminating the need for extensive sample preparation, the proposed method offers very large savings in total analysis time.     

A novel and sensitive UPLC–MS/MS method to determine mequitazine in rat plasma and urine: Validation and its application to pharmacokinetic studies

The aim of this study was to develop an analytical method to determine mequitazine in rat plasma and urine. Mequitazine was separated by UPLC–MS/MS equipped with a Kinetex core–shell C₁₈ column (50 × 2.1 mm, 1.7 μm) using 0.1% (v/v) aqueous formic acid and acetonitrile containing 0.1% (v/v) formic acid as a mobile phase by gradient elution at a flow rate of 0.3 mL/min. Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operating in multiple reaction monitoring positive ion mode. Mass transitions were m/z 323.3 → 83.1 for mequitazine and 281.3 → 86.3 for imipramine as internal standard. Liquid–liquid extraction with ethyl acetate and protein precipitation with methanol were used for sample extraction. Chromatograms showed that the method had high resolution, sensitivity and selectivity without interference from plasma constituents. Calibration curves for mequitazine in rat plasma and urine were 0.02–200 ng/mL, showing excellent linearity with correlation coefficients (r²) >0.99. Both intra‐ and inter‐day precisions (CV%) were within 4.08% for rat plasma and urine. The accuracies were 99.58–102.03%. The developed analytical method satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of mequitazine after oral and intravenous administration to rats.     

A direct injection high-throughput liquid chromatography tandem mass spectrometry method for the determination of a new orally active alphavbeta3 antagonist in human urine and dialysate

A generic high-throughput liquid chromatography (HTLC) tandem mass spectrometry (MS/MS) assay for the determination of compound I in human urine and dialysate (hemodialysis) was developed and validated. By using the HTLC on-line extraction technique, sample pretreatment was not necessary. The sample was directly injected onto a narrow bore large particle size extraction column (50 x 1.0 mm, 60 microm) where the sample matrix was rapidly washed away using a high flow rate (5 mL/min) aqueous mobile phase while analytes were retained. The analytes were subsequently eluted from the extraction column onto an analytical column using an organic-enriched mobile phase prior to mass spectrometric detection. The analytes were then eluted from the analytical column to the mass spectrometer for the determination. The linear dynamic range was 2.0-6000 ng/mL for the urine assay and 0.1-300 ng/mL for the dialysate assay. Intraday accuracy and precision were evaluated by analyzing five replicates of calibration standards at all concentrations used to construct the standard curve. For the urine assay, the precision (RSD%, n=5) ranged from 1.9 to 8.0% and the accuracy ranged from 87.8 to 105.2% of nominal value. For the dialysate assay, the precision (RSD%, n=5) ranged from 1.1 to 10.0% and the accuracy from 94.5 to 105.2% of nominal value. In-source fragmentation of the acyl glucuronide metabolite (compound III) did not interfere with the determination of parent compound I. The developed HTLC/MS/MS methodology was specific for compound I in the presence of compound III. Column life-time is increased and sample analysis time is decreased over traditional reversed-phase methods when direct injection assays for urine and dialysate are coupled with the technology of HTLC.     

Determination of basic drugs of abuse in human serum by online extraction and LC–MS/MS

A new quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to liquid chromatography (LC)–mass spectrometry (MS)/MS has been developed. The online extraction is carried out using two extraction columns simultaneously and one analytical column. One extraction column is loaded, while the other one is eluted by a gradient. The elution gradient also separates the analytes in the analytical column. For the sample preparation, serum is spiked with a mixture of deuterated analogues of the drugs. After protein precipitation with methanol/zinc sulphate, centrifugation, evaporation and reconstitution, the sample is injected into the LC system. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the protein precipitation step is over 80% for all analytes. Intra- and interday precision, as relative standard deviation, is lower than 6%, and in the case of accuracy, RE is lower than 15%. Only the most polar analytes showed matrix effects. The limits of quantitation for the analysed compounds vary between 0.5 and 2.8 ng/mL. The developed method was used to quantify basic drugs in samples “from driving under the influence of drugs” cases. The results were compared with those obtained by using solid-phase extraction–GC–MS.     

Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.     

HPLC determination of cis-diamminedichloroplatinum(II) in plasma and urine with UV detection and column-switching

A method for determining cis-diamminedichloroplatinum(II) (CDDP), an anticancer drug, in plasma and urine by HPLC with UV detection and column-switching has been developed. Typical conditions were as follows. An apparatus was composed of two columns, two pumps, a UV detector, a sample injector with a 100 microL loop, a switching valve, a column oven and a recorder. A Rheodyne model 7125 sample injector was used as the switching valve. A precolumn (4.6 mm ID x 25 cm) was packed with MCI GEL CK10S (a strong cation exchanger), and an analytical column (4.6 mm ID x 5 cm) was packed with MCI GEL CDR10 (a strong anion exchanger). Both columns were connected in series via the switching valve. The CDDP-containing fraction of the effluent from the precolumn was loaded to the analytical column by column-switching and the effluent from the analytical column was monitored at 210 nm. An eluent of 0.3 M sodium dihydrogen phosphate was pumped at a flow rate of 1 mL/min and the columns were maintained at 40 degrees C. CDDP was eluted at about 11 min and the identity of the peak of CDDP on the chromatogram was confirmed by its 3-dimensional chromatogram and analysis of platinum in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and concentration of CDDP up to 100 microM. Correlation efficients were 0.998 for plasma and 0.999 for urine. The detection limit was 0.1 microM for CDDP in both plasma and urine (S/N = 3,0.005 AUFS). The reproducibility was within 3% for 10 determinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of chemical derivatization to enhance liquid chromatography/tandem mass spectrometric determination of 1-hydroxypyrene, a biomarker for polycyclic aromatic hydrocarbons in human urine

This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 microL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.     

High turbulence liquid chromatography online extraction and tandem mass spectrometry for the simultaneous determination of suberoylanilide hydroxamic acid and its two metabolites in human serum

A reliable and sensitive method incorporating high turbulence liquid chromatography (HTLC) online extraction with tandem mass spectrometry (MS/MS), for simultaneous determination of suberoylanilide hydroxamic acid (SAHA) and its two metabolites, SAHA-glucuronide (M1) and 4-anilino-4-oxobutanoic acid (M2), in human serum, has been developed to support clinical studies. The HTLC technology significantly reduces the time required for sample clean-up since sample extraction and analysis are performed online. Clinical samples, internal standards (IS) and buffer are transferred into 96-well plates using a robotic liquid handling system. A 20 microL aliquot of prepared sample is directly injected into the HTLC/LC-MS/MS system where the matrix is rapidly washed away to waste and the analytes are retained on the narrow-bore extraction column (0.5 x 50 mm), using an aqueous mobile phase at 1.5 mL/min. Analytes are then eluted from the extraction column and transferred to the analytical column using a gradient mobile phase prior to detection by MS/MS. Interference with determination of SAHA from in-source dissociation of M1 is eliminated by the chromatographic separation. The resolution of SAHA and M1 did not change for more than 1500 serum sample injections by applying an acid wash (15% acetic acid) on the extraction column. The linear calibration ranges for SAHA, M1, and M2 are 2-500, 5-2000, and 10-2000 ng/mL, respectively. Assay intraday validation was conducted using five calibration curves prepared in five lots of human control serum. The precision expressed as relative standard deviation (RSD) is less than 6.8% and accuracy is 94.6-102.9% of nominal values for all three analytes. Assay specificity, freeze/thaw stability, storage stability, and matrix effects were also assessed.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Increasing throughput of parallel on-line extraction liquid chromatography/electrospray ionization tandem mass spectrometry system for GLP quantitative bioanalysis in drug development

An approach is described with turbulent flow on-line extraction liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for GLP quantitative bioanalysis of a drug candidate. Two systems were built in-house with standard laboratory parts and equipments. One system consisted of one gradient HPLC pump, one isocratic pump, one ten-port valve, two turbulent flow columns, one analytical column, one autosampler and one mass spectrometer. Using this system, an injection-to-injection cycle time of 0.8 min was achieved. By adding an additional valve, another analytical column and an isocratic pump, the injection-to-injection cycle time decreased to 0.4 min. Validation results from the two systems showed that precision and accuracy were acceptable for GLP quantitative analyses. The system was utilized to support sample bioanalysis of a drug candidate in a first-time in-human clinical trial.     

Quantitative determination of 8-isoprostaglandin F(2α) in human urine using microfluidic chip-based nano-liquid chromatography with on-chip sample enrichment and tandem mass spectrometry

Urinary 8-isoprostaglandin F(2α) (8-isoPGF(2α)) has been reported as an important biomarker to indicate the oxidative stress status in vivo. In order to quantitatively determine the low contents of 8-isoPGF(2α) (in sub- to low ng mL(-1) range) in physiological fluids, a sensitive detection method has become an important issue. In this study, we employed a microfluidic chip-based nano liquid chromatography (chip-nanoLC) with on-chip sample enrichment coupled to triple quadrupole mass spectrometer (QqQ-MS) for the quantitative determination of 8-isoPGF(2α) in human urine. This chip-nanoLC unit integrates a microfluidic switch, a chip column design having a pre-column (enrichment column) for sample enrichment prior to an analytical column for separation, as well as a nanospray emitter on a single polyimide chip. The introduction of enrichment column offers the advantages of online sample pre-concentration and reducing matrix influence on MS detection to improve sensitivity. In this study, the chip-nanoLC consisting of Zorbax 300A SB-C18 columns and Agilent QqQ Mass spectrometer were used for determining 8-isoPGF(2α) in human urine. Gradient elution was employed for effective LC separation and multiple reaction monitoring (MRM) was utilized for the quantitative determination of 8-isoPGF(2α) (m/z 353→193). We employed liquid-liquid extraction (LLE)/solid-phase extraction (SPE) for extracting analyte and reducing matrix effect from urine sample prior to chip-nanoLC/QqQ-MS analysis for determining urinary 8-isoPGF(2α). Good recoveries were found to be in the range of 83.0-85.3%. The linear range was 0.01-2 ng mL(-1) for urinary 8-isoPGF(2α). In addition, the proposed method showed good precision and accuracy for 8-isoPGF(2α) spiked synthetic urine samples. Intra-day and inter-day precisions were 1.8-5.0% and 4.3-5.8%, respectively. The method accuracy for intra-day and inter-day assays ranged from 99.3 to 99.9% and 99.4 to 99.7%, respectively. Due to its rapidity, enhanced sensitivity, and high recovery, this chip-nanoLC/QqQ-MS system was successfully utilized to determine the physiological biomarkers such as 8-isoPGF(2α) in human urine for clinical diagnosis.     

苯硼酸双柱固相微萃取-超高效液相色谱-串联质谱分析单胺类神经递质

本文构建了苯硼酸双柱固相微萃取-超高效液相色谱-串联质谱分析3种单胺类神经递质的方法。苯硼酸固相微萃取柱以4-乙烯基苯硼酸和甲叉双丙烯酰胺原位聚合法制备,该介质对含有顺式二羟基分子的化合物能够进行特异性捕获,并采取扫描电镜,红外光谱对其进行表征,研究了影响富集的各种条件。在优化条件下,线性度好,相关系数大于0.9971,检出限为23.6~152 pmol·L^-1,相对标准偏差(RSD)不大于5.9%。采用该方法对人体尿液和大鼠血清样品中3种单胺类神经递质进行分析,加标回收率为82.9%~113%,RSD为2.6%~7.7%。本文方法分析效率高,灵敏度高,准确度好,适用于尿液和大鼠血清样品中痕量单胺类神经递质的测定。     

Application of LC-MS/MS method for the in vivo metabolite determination of oleuropein after intravenous administration to rat

A highly sensitive, specific and simple LC‐MS/MS method was developed to investigate in vivo bio‐transformation of oleuropein in rat. Rat urine samples collected after the intravenous administrations were determined using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.80 mL/min on an RP‐C₁₈ column with a total run time of 30 min. This method has been successfully applied to simultaneous determination of oleuropein and its metabolite in rat urine. Oxygenation was found to be the major metabolic pathway of the oleuropein in rat after intravenous administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Moving from fast to ballistic gradient in liquid chromatography/tandem mass spectrometry pharmaceutical bioanalysis: Matrix effect and chromatographic evaluations

The paper describes the steps taken by the authors to move from a fast to a ballistic gradient in routine liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of plasma samples from pharmacokinetic (PK) profiling of new chemical entities. The reduction of column dimensions from 50 x 4.6 mm to 30 x 2.1 mm followed by optimization of chromatographic separation led to a decrease in the typical runtime from 5 (fast) to 2 min (ballistic) using an API4000 tandem mass spectrometer in Turbo Ionspray mode for detection. Three analytical standards representing typical molecular structures from our sample repository were used to spike plasma from four different species (rat, dog, human and mouse). Two different approaches were used to evaluate matrix effect: post-column infusion and comparison of the peak areas of neat standards and standards spiked after extraction into different pools of plasma; the influence of PEG400 as a typical dosing vehicle was also considered. Two different protein precipitation procedures were taken into account for sample extraction prior to injection. Peak shape, width and height, selectivity and sensitivity of the method were taken into account for chromatographic evaluation. The ballistic method was successfully cross-validated with the conventional fast gradient chromatographic assay.     

Simultaneous determination of tanshinone IIA and its three hydroxylated metabolites by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of tanshinone IIA and its three hydroxylated metabolites, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, in a rat liver microsome was developed and fully validated. A single step of liquid-liquid extraction with ethyl acetate was utilized in this method. Chromatographic separation of the sample matrix from the analytes and the internal standard diazepam was performed using a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1-500 ng/mL for all analytes. The intra- and inter-day precisions (RSD %) were within 15% and deviations of the assay accuracies were within 15.0% for all analytes. The analytes proved to be stable during sample storage, preparation and analyses. This validated method was successfully applied to the enzyme kinetic study of tanshinone IIA in liver microsome. The elimination of tanshinone IIA and formation of tanshinone IIB and hydroxytanshinone IIA in the liver microsome all exhibited a sigmoidal kinetics profile. The formation of przewaquinone A shows a typical hyperbolic profile. In addition, this method has now been applied in the analysis of other bio-samples including plasma, urine, bile and feces.