Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

Narrow-bore sample trapping and chromatography combined with quadrupole/time-of-flight mass spectrometry for ultra-sensitive identification of in vivo and in vitro metabolites

The identification of in vitro and in vivo metabolites is vital to the discovery and development of new pharmaceutical therapies. Analytical strategies to identify metabolites at different stages of this process vary, but all involve the use of liquid chromatography separations combined with detection via mass spectrometry (HPLC/MS). Reported here is the use of narrow-bore column (0.5-1.0 mm i.d.) trapping of metabolites, followed by back-flushing onto a matching analytical column. Separated metabolites were then identified using quadrupole time-of-flight mass spectrometry (MS) and tandem MS. Metabolites in human plasma and from low-level in vitro incubations, that were not identified using standard HPLC/MS approaches, were characterized using the instrumental configuration described here.     

Increased throughput and reduced carryover of mass spectrometry-based proteomics using a high-efficiency nonsplit nanoflow parallel dual-column capillary HPLC system

We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 microm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet > or = 0.9, and peptides matched > or = 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events.     

Liquid chromatography/negative ion electrospray tandem mass spectrometry method for the quantification of rosuvastatin in human plasma: application to a pharmacokinetic study

A sensitive liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of rosuvastatin in human plasma. The plasma samples were prepared using liquid-liquid extraction with ethyl ether. Chromatographic separation was accomplished on a Zorbax XDB-C18 (150 mm x 4.6 mm i.d., 5 microm) column. The mobile phase consisted of methanol-water (75:25, v/v, adjusted to pH 6 by aqueous ammonia). Detection of rosuvastatin and the internal standard (IS) hydrochlorothiazide was achieved by ESI MS/MS in the negative ion mode. The lower limit of quantification was 0.020 ng/ml by using 200 microl aliquots of plasma. The linear range of the method was from 0.020 to 60.0 ng/ml. The intra- and inter-day precisions were lower than 8.5% in terms of relative standard deviation (RSD), and the accuracy was within -0.3 to 1.9% in terms of relative error (RE). Compared with the existing methods, the validated method offered increased sensitivity. The method was successfully applied for the evaluation of pharmacokinetics of rosuvastatin after single oral doses of 5, 10 and 20 mg rosuvastatin to 10 healthy volunteers.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system

The Eksigent Express 800 8-channel microfluidic HPLC system was investigated for carrying out multiparallel screening and development of fast normal phase chiral separations. In contrast to the familiar automated sequential chiral method development approaches that often afford a next day result, the multiparallel approach offers the exciting possibility of near "real time" method development, often affording an optimized method in less than 1 h. In this study, four column types (300 microm i.d.) with two different mobile phases are screened using a universal standard gradient approach. Interestingly, parallel method optimization following initial screening was shown to sometimes lead to surprising and unanticipated outcomes, emphasizing the value of the multiparallel screening approach. A variety of standard test racemates were analyzed, with optimized separation methods for most in the 1- to 2-min range. These results compare favorably with results obtained on a single channel conventional HPLC system using 4.6-mm i.d. columns. In addition, isocratic methods developed on the microbore columns are readily translated to the larger column format.     

Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography-mass spectrometry

A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35x0.3 mm I.D.; d(p) 30 micrometer) at high flow-rate conditions (450 microliter/min). The separation was performed on a microbore analytical column (55x2 mm I.D.; d(p) 3 micrometer) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.     

Monolithic silica liquid chromatography columns for the determination of cyclooxygenase II inhibitors in human plasma

Methods employing monolithic HPLC columns for the determination of the cyclooxygenase II inhibitors rofecoxib (I) and 3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one (DFP, III) in human plasma are described. Each analyte, together with an internal standard was extracted from the plasma matrix using solid-phase extraction in the 96-well format. The analytes were chromatographed on a Chromolith Speed Rod monolithic HPLC column (4.6 x 50 mm). Analyte detection for rofecoxib was via fluorescence following post-column photochemical derivatization. Detection for III was based on the native fluorescence of the compound. The precision, accuracy, and linearity of the methods were found to be comparable to those obtained using methods employing conventional packed HPLC columns. Use of the monolithic column permitted mobile phase flow-rates of up to 6.5 ml/min to be employed in the assays. The use of elevated flow-rates enabled the per sample analysis time to be reduced by up to a factor of 5 compared with assays based on packed HPLC columns. The results of experiments aimed at evaluating the ruggedness and reproducibility of monolithic columns employed in bioanalytical methods are presented.     

Highly sensitive and rapid determination of theophylline, theobromine and caffeine in human plasma and urine by gradient capillary high-performance liquid chromatography-frit-fast atom bombardment mass spectrometry

A sensitive and reliable analytical procedure has been established for the detection of theophylline (TH), theobromine (TB) and caffeine (CA) in human plasma and urine by gradient capillary high-performance liquid chromatography (HPLC)-frit-fast atom bombardment mass spectrometry (FAB-MS) (LC-frit-FAB-MS). Two capillary columns and a column-switching valve were used in this LC system to allow all of the sample injected to be introduced into the MS system. 7-Ethyltheophylline was used as the internal standard (I.S.). The xanthines in the specimen were extracted with an Extrelut column. The lowest detected amount was ca. 5 ng/ml using this method.     

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: application to pharmacokinetic studies

A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC-MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water-acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C(18) (100 mm x 2.0 mm i.d., 3 microm) column connected to a guard column MS-pursuit (0.20 mm x 0.20 mm i.d., 5 microm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r(2) values of 0.995 (CV=0.24%, n=10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C(max) (1667.25 ng/mL) and T(max) (3.89 h).     

The use of high-performance liquid chromatography for sample clean-up in mass fragmentographic assays

A novel high-performance liquid chromatography (HPLC) sample clean-up procedure for use in mass fragmentographic assays of (sub)-nanogram amounts of drugs in human plasma is described and compared with a conventional extraction sequence for sample purification. With the assay of the new antidepressant drug mianserin hydrochloride (Org GB 94) as an example, the HPLC procedure is discussed with respect to retention time, recovery, purification, column deterioration and convenience. It is demonstrated that HPLC sample clean-up is a useful and time-saving procedure for routine clinical analyses.     

Simultaneous determination of rosuvastatin and atorvastatin in human serum using RP-HPLC/UV detection: method development, validation and optimization of various experimental parameters

A novel, precise, accurate and rapid isocratic reversed-phase high performance liquid chromatographic/ultraviolet (RP-HPLC/UV) method was developed, optimized and validated for simultaneous determination of rosuvastatin and atorvastatin in human serum using naproxen sodium as an internal standard. Effect of different experimental parameters and various particulate columns on the analysis of these analytes was evaluated. The method showed adequate separation for rosuvastatin and atorvastatin and best resolution was achieved with Brownlee analytical C18 column (150×4.6 mm, 5 μm) using methanol-water (68:32, v/v; pH adjusted to 3.0 with trifluoroacetic acid) as a mobile phase at a flow rate of 1.5 ml/min and wavelength of 241 nm. The calibration curves were linear over the concentration ranges of 2.0-256 ng/ml for rosuvastatin and 3.0-384 ng/ml for atorvastatin. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for rosuvastatin were 0.6 and 2.0 ng/ml while for atorvastatin were 1.0 and 3.0ng/ml, respectively. All the analytes were separated in less than 7.0 min. The proposed method could be applied for routine laboratory analysis of rosuvastatin and atorvastatin in human serum samples, pharmaceutical formulations, drug-drug interaction studies and pharmacokinetics studies.     

High-performance liquid chromatographic analysis of abscisic acid in plant extracts.

A HPLC assay for ABA and bound ABA is described. The method uses conventional acid-base ether extractions for partial cleanup of the plant extracts followed by Sephadex G-25 column fractionation. The ABA is separated and detected by HPLC on a 3 m × 2.1 mm i.d. column of SCX-Zipax with a uv detector. The method is sensitive to less than 10 ng of ABA. HPLC analyses of ABA have been performed on extracts of leaves, stems, and roots of soybeans, pinto beans, cotton, apple seedlings, and the albedo and flavedo sections of orange peels.     

Disposable microbore high-pressure liquid chromatography columns for protein and peptide separations.

A range of high-performance liquid chromatography (HPLC) columns with internal diameters of 0.25 to 1.8 mm have been constructed by securing glass or plastic tubing into standard HPLC fittings. These were packed with chromatographic materials chosen for operation at moderate pressures with high flow rates. These columns were shown to be effective in a conventional HPLC instrument for peptide and protein separations in reverse-phase mode and for proteins in ion-exchange and size-exclusion modes. The simple construction and low cost of these microbore columns allow them to be considered as disposable. Using only small amounts of any type of packing material, they have the flexibility to be adapted to a wide range of analytical and micropreparative separations.     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution 150 × 4.6 mm I.D. column and transferred to a 150 × 1 mmI.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.     

Evaluation of four capillary columns for the analysis of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers in human serum for epidemiologic studies

The separation of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether (PBDE) congeners was evaluated on four capillary columns: 60 m x 0.25 mm i.d., 0.25 microm film thickness RTX-5MS and DB-XLB capillary columns, and 60 m x 0.18 mm i.d., 0.25 microm film thickness DB-XLB and DB-5MS capillary columns. Based on performance, capacity, and cost, the RTX-5MS (60 m x 0.25 mm i.d., 0.25 microm thickness) and the DB-XLB (60 m x 0.25 mm i.d., 0.25 microm film thickness) were selected for the analysis of human serum extracts by using gas chromatography/electron-capture detection. In contrast to previous studies, the oven temperature program affords the separation of congeners that are not separated by using other combinations of capillary columns, most notably PBDE-47 and PCB 180. In addition, the method enables determination of OCPs, PCBs, and PBDEs prevalent in a single extract of serum, which can lead to considerable time savings in the analysis of large number of samples collected for epidemiologic studies.     

Separation and determination of dexamethasone sodium phosphate in cochlear perilymph fluid by liquid chromatography with ultraviolet monitoring and electrospray ionization mass spectrometry characterization

The method for separation and determination of dexamethasone sodium phosphate (DexP) in cochlear perilymph fluid (CPF) of cavy was developed using HPLC with ultraviolet (UV) monitoring and electrospray ionization/mass spectrometry (ESI/MS) identification. The quantitative determination of DexP in CPF was achieved by HPLC with UV detection at 245 nm. The separation was carried out on a Phenomenex ODS(3) column ( 250 mm x 4.6 mm i.d., 5 microm) with the mobile phase of acetonitrile-5mmol/l ammonium acetate (23:77 (v/v)) at a flow rate of 1.0 ml/min. DexP was baseline separated from the matrices of CPF blanks within 15 min. The linearity ranged from 0.5 to 50 microg/ml. The limit of detection was 0.10 microg/ml. The recovery ranged from 98.5 to 100.8%. The relative standard deviations (R.S.D.s) of intra- and inter-day peak area were between 0.7-1.3 and 1.2-3.5%, respectively. Both full scan MS and MS2 of DexP with positive and negative polarity were obtained and elucidated. The specific ions were chosen to characterize DexP in the CPF sample. Using the proposed HPLC-UV-ESI/MS method, the concentration of DexP in CPF samples after both vein and middle ear injections were determined, and the relationships between concentration and time were obtained. This method offered reference data for clinical investigation of DexP to cure ear diseases.     

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.     

A sensitive LC-MS/MS method for the quantitative analysis of the Echinacea purpurea constituent undeca-2-ene-8,10-diynoic acid isobutylamide in human plasma

Echinacea purpurea is one of the most popular herbal medicines and is known for its immunostimulatory effects. Alkylamides are the main lipophilic components of E. purpurea that contribute to its pharmacological actions. For quantification in human plasma of one of these alkylamides, undeca-2-ene-8,10-diynoic acid isobutylamide, a sensitive LC-MS/MS assay has been developed and validated. Plasma samples were pretreated using liquid-liquid extraction with a mixture of diethyl ether and n-hexane (50:50, v/v). Dried extracts were reconstituted in 50 μL of acetonitrile-water (50:50, v/v) after which 15 μL of sample was injected into the HPLC system. HPLC was performed using a Polaris 3 C18-A column (50 mm×2 mm ID) and isocratic elution with acetonitrile-water (50:50, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. Subsequently, electrospray ionization in the positive ion mode followed by tandem mass spectrometry was performed for detection. The total run time was 3 min. The assay was validated over a concentration range from 0.05 to 50 ng/mL for undeca-2-ene-8,10-diynoic acid isobutylamide, with 0.05 ng/mL being the lower limit of quantification using 1.0 mL plasma samples. Inter-assay inaccuracy (±12.7%), within-day and between-day precisions (CV≤8.23%) were acceptable. Further, undeca-2-ene-8,10-diynoic acid isobutylamide was found to be chemically stable under relevant conditions. Finally, the applicability of this assay has been successfully demonstrated in a pharmacokinetic experiment in which a human volunteer ingested a commercial extract of E. purpurea.     

Use of online-dual-column extraction in conjunction with chiral liquid chromatography tandem mass spectrometry for determination of terbutaline enantiomers in human plasma

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a beta2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0-200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94-106%.