A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.     

Direct injection versus liquid-liquid extraction for plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry.

Direct injection versus liquid-liquid extraction for post-dose human plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) have been studied using a drug candidate compound. For the direct-injection method, an Oasis(R) HLB column (1 x 50 mm, 30 micrometer) was used as the on-line extraction column and a conventional Waters symmetry C18 column (3.9 x 50 mm, 5 micrometer) was used as the analytical column. Each plasma sample (100 microL) was mixed with 100 microL of a working solution of the internal standard in aqueous 0.05 M ammonium acetate (pH 6.9), and portions (10 microL) of these samples were then injected into the LC/MS/MS system. For the liquid-liquid extraction method, a YMC Basic C18 column (2.0 x 50 mm, 5 micrometer) was used as the analytical column. Each sample (0.5 mL) was extracted with methyl tert-butyl ether and the extract was reconstituted and injected into the LC/MS/MS system. The total analysis time for both methods was 2.0 min per sample. The accuracy, inter-day precision and intra-day precision obtained from the quality control samples were within 8% for both methods. The analysis results of post-dose human plasma samples showed that the deviations of 91% of the concentrations obtained using the direct-injection method were within +/-20% from the concentrations obtained using the liquid-liquid extraction method, and the overall average percentage deviation was -1.5%. The results showed that the two methods were equivalent in terms of total chromatographic run time, accuracy and precision. However, for a batch of 100 samples, the sample preparation time for the direct-injection method was only about 25% of the time required for liquid-liquid extraction. This decrease in sample preparation time resulted in the doubling of the overall sample analysis throughput.     

High-speed gradient parallel liquid chromatography/tandem mass spectrometry with fully automated sample preparation for bioanalysis: 30 seconds per sample from plasma

In this work, a high-throughput and high-performance bioanalytical system is described that is capable of extracting and analyzing 1152 plasma samples within 10 hours. A Zymark track robot system interfaced with a Tecan Genesis liquid handler was used for simultaneous solid-phase extraction of four 96-well plates in a fully automated fashion. The extracted plasma samples were injected onto four parallel monolithic columns for separation via a four-injector autosampler. The use of monolithic columns allowed for fast and well-resolved separations at a considerably higher flow rate without generating significant column backpressure. This resulted in a total chromatographic run cycle time of 2 min on each 4.6 x 100 mm column using gradient elution. The effluent from the four columns was directed to a triple quadrupole mass spectrometer equipped with an indexed four-probe electrospray ionization source (Micromass MUX interface). Hence, sample extraction, separation, and detection were all performed in a four-channel parallel format that resulted in an overall throughput of about 30 s per sample from plasma. The performance of this system was evaluated by extracting and by analyzing twelve 96-well plates (1152) of human plasma samples spiked with oxazepam at different concentrations. The relative standard deviation (RSD) of analyte sensitivity (slope of calibration curve) across the four channels and across the 12 plates was 5.2 and 6.8%, respectively. An average extraction recovery of 77.6% with a RSD of 7.7% and an average matrix effect of 0.95 with a RSD of 5.2% were achieved using these generic extraction and separation conditions. The good separation efficiency provided by this system allowed for rapid method development of an assay quantifying the drug candidate and its close structural analog metabolite. The method was cross-validated with a conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay.     

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.     

Effects of flow rate on high-throughput quantitative analysis of protein-precipitated plasma using liquid chromatography/tandem mass spectrometry

The effects of flow rate and column length on analyte response (peak area and height), total cycle time, column backpressure, and elution volume are presented. Rapid chromatographic separations and tandem mass spectrometric (MS/MS) detection are applied to the supernatant of protein-precipitated plasma standards containing four compounds from a drug discovery screen. The plasma samples were injected onto three C-18 columns (2 x 10,2.1 x 30 and 2.1 x 50 mm) at flow rates of 0.25, 0.50, 1.00 and 1.50 mL/min. The plasma samples were detected using a Sciex API 3000 tandem mass spectrometer operated in the Turbo Ionspray mode. A post-column split was used to maintain a flow rate of 0.25 mL/min into the mass spectrometer source to avoid differences in nebulization efficiency. The data show that diluted protein-precipitated plasma supernatants show average matrix effects (i.e. suppression) of 60.0% (2 x 10 mm), 89.3% (2 x 30 mm), and 76.7% (2 x 50 mm) of expected response at 10 ng/mL. Average matrix effects of 70.2% (2 x 10 mm), 88.9% (2 x 30 mm), and 81.2% (2 x 50 mm) of expected response at 1000 ng/mL plasma. The data also show if peak widths remain relatively constant, analytes are less sensitive as flow rates are increased. These data are consistent with the concentration-dependent relationship of ionspray in the range of flow rates studied. The data show that, while analyte response decreased proportionately to increases in flow rate, the analysis cycle times did not decrease proportionately.     

Post-column infusion study of the 'dosing vehicle effect' in the liquid chromatography/tandem mass spectrometric analysis of discovery pharmacokinetic samples

It has become increasingly popular in drug development to conduct discovery pharmacokinetic (PK) studies in order to evaluate important PK parameters of new chemical entities (NCEs) early in the discovery process. In these studies, dosing vehicles are typically employed in high concentrations to dissolve the test compounds in dose formulations. This can pose significant problems for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of incurred samples due to potential signal suppression of the analytes caused by the vehicles. In this paper, model test compounds in rat plasma were analyzed using a generic fast gradient LC/MS/MS method. Commonly used dosing vehicles, including poly(ethylene glycol) 400 (PEG 400), polysorbate 80 (Tween 80), hydroxypropyl beta-cyclodextrin, and N,N-dimethylacetamide, were fortified into rat plasma at 5 mg/mL before extraction. Their effects on the sample analysis results were evaluated by the method of post-column infusion. Results thus obtained indicated that polymeric vehicles such as PEG 400 and Tween 80 caused significant suppression (> 50%, compared with results obtained from plasma samples free from vehicles) to certain analytes, when minimum sample cleanup was used and the analytes happened to co-elute with the vehicles. Effective means to minimize this 'dosing vehicle effect' included better chromatographic separations, better sample cleanup, and alternative ionization methods. Finally, a real-world example is given to illustrate the suppression problem posed by high levels of PEG 400 in sample analysis, and to discuss steps taken in overcoming the problem. A simple but effective means of identifying a 'dosing vehicle effect' is also proposed.     

Optimizing an ultrafast generic high-performance liquid chromatography/tandem mass spectrometry method for faster discovery pharmacokinetic sample throughput

For higher throughput screening, where the number of new chemical entities (NCEs) to test is rapidly increasing, fast sample turnaround time is essential. In order to increase efficiency a generic high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method, with a cycle time of 85 s (42 injections/h), was created. This was accomplished through the use of a 1-min ballistic gradient and the optimization of the autosampler. The gradient was optimized by varying the organic mobile phase concentration and examining its ballistic characteristics with respect to matrix ion suppression and compound retention time. The autosampler time could be reduced by optimizing several parameters and by determining the source of most of the carryover in order to reduce the number of syringe and injector washes. Finally, the reliability of the new generic method is demonstrated by comparison of sample data with a standard 2-min linear gradient method that showed that the data sets were well correlated. For plasma AUC (ng.h/mL) of 28 NCEs, the regression line had a slope of 0.92 and the R2 was 0.929. The described method was found to be useful for both rat plasma and tissue samples.     

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.     

Determination of drug concentrations in plasma by a highly automated, generic and flexible protein precipitation and liquid chromatography/tandem mass spectrometry method applicable to the drug discovery environment

This report presents a highly automated procedure for the determination of drug concentrations in plasma samples. The method is generic, in that it has been applied without adaptation to many different drug candidate molecules, but is also flexible, in that variations in the nature and number of samples to be analyzed can be readily accommodated. The method includes preparation of dilutions of analyte stock solutions, spiking these into control plasma to generate analytical standards, and preparation of samples suitable for analysis by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) by precipitation of plasma proteins with acetonitrile, centrifugation, and dilution of the supernatants with HPLC buffer. All of these steps, apart from centrifugation, are performed without manual intervention on an automated liquid-handling workstation using 96-well plates. Analysis is by HPLC/MS/MS, using a generic HPLC gradient. Commercially available software was used for optimization of parameters for analysis by HPLC/MS/MS, integration of chromatographic peaks, and quantification of drug concentrations. The use of this methodology in our laboratory has greatly facilitated the analysis of small sample sets for a large number of analytes, a situation regularly encountered in an early drug discovery environment.     

Evaluation of several solid phase extraction liquid chromatography/tandem mass spectrometry on-line configurations for high-throughput analysis of acidic and basic drugs in rat plasma

Several configurations using 6- and 10-port switching valves were studied for high flow, on-line extraction of rat plasma coupled to an electrospray triple quadrupole mass spectrometer. Each plasma sample was diluted 1:1 with an aqueous internal standard solution. The sample was injected into a 2.1 x 20 mm cartridge column packed with 25 microm divinylbenzene/N-vinylpyrrolidone packing using 100% aqueous mobile phase at 4 mL/min. After sample loading and sample cleanup, the analytes were eluted from the extraction column with a 1.0-min gradient at 0.4 mL/min. The samples were either analyzed directly after elution from the extraction column or after additional separation using a short high performance liquid chromatography (HPLC) column. The different configurations were tested using an acidic drug (diflunisal) and a basic drug (clemastine) in rat plasma. On-line analysis was performed by injecting 200 microL of diluted plasma. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. All calibration standards gave relative standard deviations (RSDs) below 5%. The total time per sample was 3 min.     

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.     

Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high-performance liquid chromatography with tandem mass spectrometric detection

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50-microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide.     

Validated assay for the simultaneous determination of the anti-cancer agent gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma by high-performance liquid chromatography with tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the quantitative determination of gemcitabine (dFdC) and its metabolite 2',2'-difluorodeoxyuridine (dFdU) is presented. A 200-microL aliquot of human plasma was spiked with a mixture of internal standards, didanosine, lamivudine and fludarabine, and extracted using solid-phase extraction. Dried extracts were reconstituted in 1 mM ammonium acetate/acetonitrile (97:3, v/v) and 10-microL volumes were injected onto the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 bonded phase endcapped with polar groups (Synergi Hydro-RP column) using an eluent composed of 1 mM ammonium acetate (pH 6.8)/acetonitrile (94:6, v/v). Detection was performed by positive ion electrospray ionization followed by MS/MS. The assay quantifies a range from 0.5 to 1000 ng/mL for gemcitabine and from 5 to 10,000 ng/mL for dFdU using 200 microL of human plasma sample. Validation results demonstrate that gemcitabine and dFdU concentrations can be accurately and precisely quantified in human plasma. This assay is used to support clinical pharmacologic studies with gemcitabine.     

Confirmatory assay for the simultaneous detection of penicillins and cephalosporins in milk using liquid chromatography/tandem mass spectrometry

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection of residues of penicillins and cephalosporins in milk has been developed. After a simple extraction with acetonitrile, the extract was directly injected into the LC/MS/MS system on a C(18) column. A gradient consisting of acetonitrile and water, each containing 0.1% formic acid, was applied. The abundant parent ions [M + H](+) produced by positive electrospray ionisation were selected for fragmentation with argon. For each compound at least one fragment was recorded with multiple reaction monitoring. The limits of detection ranged from 1.5 to 25 microg/kg and the limits of quantification ranged from 4 to 50 microg/kg. Recoveries were examined at three levels (MRL, 0.5 x MRL, 2 x MRL) and ranged from 57 to 88%. The coefficients of variation obtained for the repeatability experiments were in agreement with those specified by the Horwitz equation. Linearity was checked by injecting extracts of samples spiked with increasing amounts of the different standards ranging from 0 to 150 microg/kg. The advantage of this method over existing methods is the very simple sample pre-treatment which makes the method very suitable for routine analysis.     

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.     

A highly automated 96-well solid phase extraction and liquid chromatography/tandem mass spectrometry method for the determination of fentanyl in human plasma

A high-throughput bioanalytical method based on automated sample transfer, automated solid phase extraction, and fast liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, has been developed for the determination of the analgesic fentanyl in human plasma. Samples were transferred into 96-well plates using an automated sample handling system. Automated solid phase extraction (SPE) was carried out using a 96-channel programmable liquid-handling workstation using a mixed-mode sorbent. The extracted samples were then dried down, reconstituted and injected onto a silica column using an aqueous/organic mobile phase with tandem mass spectrometric detection. The method has been validated over the concentration range 0.05-100 ng/mL fentanyl in human plasma, based on a 0.25-mL sample size. The assay is sensitive, specific and robust. More than 2000 samples have been analyzed using this method. The automation of the sample preparation steps not only increased the analysis throughput, but also facilitated the transfer of the method between different bioanalytical laboratories of the same organization.     

Determination of MK-0431 in human plasma using high turbulence liquid chromatography online extraction and tandem mass spectrometry

A robust and sensitive method using high turbulence liquid chromatography (HTLC) online extraction with tandem mass spectrometry (MS/MS) for the determination of MK-0431 in human plasma was developed and validated to support the clinical studies. This HTLC online extraction method eliminated the time-consuming offline sample extraction procedures and significantly increased productivity. A narrow bore large particle size reversed-phase column (Cyclone, 50 x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. The linear dynamic range of the calibration curve was 0.5 to 1000 ng/mL. Intraday validation was conducted using five calibration curves prepared in five lots of human control plasma, and the intraday precision (RSD%) was from 2.4 to 9.0% and the accuracy was from 98.0 to 103% of the nominal value. The intraday precision (RSD%, n = 5) for plasma quality control (QC) samples varied from 2.0 to 5.3% and accuracy from 103 to 105% of the nominal value. The interday precision (RSD%) for 100 sets of plasma QC samples in 29 analytical runs varied from 6.3 to 9.0% and the accuracy from 98.8 to 104% of the nominal value. No significant difference was observed between the interday and intraday precision and accuracy of the QC samples.     

Novel UHPLC‐MS/MS method for the determination of rotigotine in the plasma of patients with Parkinson's disease

A novel ultra‐high‐pressure liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of the dopamine receptor agonist rotigotine in human plasma. Following liquid–liquid extraction with tert‐ butyl methyl ether from 500 μL plasma, the chromatographic analysis was performed on a Gemini NX3 column using 5 mm pH 5.0 ammonium acetate–5 mm ammonium acetate in methanol as binary gradient mobile phase, at a flow rate of 0.3 mL/min. The MS/MS ion transitions were 316.00 → 147.00 for rotigotine and 256.10 → 211.00 for the internal standard (lamotrigine). The lower limit of quantitation was 50 pg/mL and the linearity was determined from 50 to 2500 pg/mL. The mean recovery was 96.9%. Both intra‐ and interassay imprecision and inaccuracy were ≤15% at all quality control concentrations. The method was successfully applied to measure morning trough plasma rotigotine concentrations in a series of patients with Parkinson's disease on chronic treatment. The present study describes the first fully validated method for rotigotine determination in human plasma.     

A UFLC‐MS/MS method with a switching ionization mode for simultaneous quantitation of polygalaxanthone III,four ginsenosides and tumulosic acid in rat plasma: application to a comparative pharmacokinetic study in normal and Alzheimer's disease rats

A fast, sensitive and reliable ultra fast liquid chromatography‐tandem mass spectrometry (UFLC‐MS/MS) method has been developed and validated for simultaneous quantitation of polygalaxanthone III (POL), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Re (GRe), ginsenoside Rg1 (GRg1) and tumulosic acid (TUM) in rat plasma after oral administration of Kai‐Xin‐San, which plays an important role for the treatment of Alzheimer's disease (AD). The plasma samples were extracted by liquid–liquid extraction using ethyl acetate–isopropanol (1:1, v/v) with salidrdoside as internal standard (IS). Good chromatographic separation was achieved using gradient elution with the mobile phase consisting of methanol and 0.01% acetic acid in water. The tandem mass spectrometric detection was performed in multiple reaction monitoring mode on 4000Q UFLC‐MS/MS system with turbo ion spray source in a negative and positive switching ionization mode. The lower limits of quantification were 0.2–1.5 ng/ml for all the analytes. Both intra‐day and inter‐day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 60.0%. The validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in normal and AD rat plasma. The results indicated that no significant differences in pharmacokinetic parameters of GRe, GRg1 and TUM were observed between the two groups, while the absorption of POL and GRd in AD group were significantly higher than those in normal group; moreover, the GRb1 absorbed more rapidly in model group. The different characters of pharmacokinetics might be caused by pharmacological effects of the analytes. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites

An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8 min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation.