Using supported liquid extraction together with cellobiohydrolase chiral stationary phases-based liquid chromatography with tandem mass spectrometry for enantioselective determination of acebutolol and its active metabolite diacetolol in spiked human plasma.

A high through-put, sensitive, and enantioselective LC-MS/MS-based bioanalytical method was developed and validated for the simultaneous determination of individual acebutolol (AC) and its active metabolite-diacetolol (DC) enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP). Systematic optimization of chromatographic conditions including organic content, buffer concentration, and pH of mobile phases was conducted to improve the through-put for the direct separation of both AC and DC on CBH column during method development. Complete baseline separation of enantiomeric AC and DC was achieved within 1.5 min with a LC flow rate of 0.9 ml/min under method validation conditions. To further improve the assay through-put, supported liquid extraction (SLE) in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.0500-50.0 ng/ml for each AC and DC enantiomer using 0.100 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed

Determination of molindone enantiomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry using macrocyclic antibiotic chiral stationary phases.

A sensitive and selective bioanalytical assay was developed and validated for the determination of enantiomeric molindone in human plasma using high-performance liquid chromatography-tandem mass spectrometry along with supported liquid extraction procedures. The chiral separation was evaluated and optimized on macrocyclic antibiotic type chiral stationary phases (CSPs) based on teicoplanin aglycone (Chirobiotic TAG) in polar organic, polar ionic, and reversed-phase mode chromatography, respectively. Complete baseline separation was achieved on a Chirobiotic TAG column under isocratic condition in reversed-phase chromatography. The method validation was conducted using a Chirobiotic TAG column (100 mm x 2.1 mm) over the curve range 0.100-100 ng/ml for each molindone enantiomer using 0.0500 ml of plasma sample. The flow rate was 0.8 ml/min and the total run time was 9 min. Supported liquid extraction in a 96-well plate format was used for sample preparation. Parameters including recovery, matrix effect, linearity, sensitivity, specificity, carryover, precision, accuracy, dilution integrity, and stability were evaluated. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels were RSD

Determination of S-phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography-tandem mass spectrometric method.

S-phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S-phenylmercapturic acid in human urine. Isotope-labeled S-phenylmercapturic acid-d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S-phenylmercapturic acid (m/z 238 --> 109) and S-phenylmercapturic acid -d5 (m/z 243 --> 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400-200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved.

Automated 96-well solid phase extraction and hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the analysis of cetirizine (ZYRTEC) in human plasma--with emphasis on method ruggedness.

A high-throughput bioanalytical method based on automated sample transfer, automated solid phase extraction, and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) analysis, has been developed for the determination of cetirizine, a selective H(1)-receptor antagonist. Deuterated cetirizine (cetirizine-d(8)) was synthesized as described and was used as the internal standard. Samples were transferred into 96-well plates using an automated sample handling system. Automated solid phase extraction was carried out using a 96-channel programmable liquid-handling workstation. Solid phase extraction 96-well plate on polymer sorbent (Strata X) was used to extract the analyte. The extracted samples were injected onto a Betasil silica column (50 x 3, 5 microm) using a mobile phase of acetonitrile-water-acetic acid-trifluroacetic acid (93:7:1:0.025, v/v/v/v) at a flow rate of 0.5 ml/min. The chromatographic run time is 2.0 min per injection, with retention time of cetirizine and cetirizine-d(8) both at 1.1 min. The system consisted of a Shimadzu HPLC system and a PE Sciex API 3000 or API 4000 tandem mass spectrometer with (+) ESI. The method has been validated over the concentration range of 1.00-1000 ng/ml cetirizine in human plasma, based on a 0.10-ml sample size. The inter-day precision and accuracy of the quality control (QC) samples demonstrated <3.0% relative standard deviation (R.S.D.) and <6.0% relative error (RE). Stability of cetirizine in stock solution, in plasma, and in reconstitution solution was established. The absolute extraction recovery was 85.8%, 84.5%, and 88.0% at 3, 40, and 800 ng/ml, respectively. The recovery for the internal standard was 84.1%. No adverse matrix effects were noticed for this assay. The automation of the sample preparation steps not only increased the analysis throughput, but also increased method ruggedness. The use of a stable isotope-labeled internal standard further improved the method ruggedness. Practical issues of analyzing incurred samples were discussed. This HILIC-MS/MS method for analysis of citirizine in human plasma was successfully used to support clinical studies.

Development, validation and transfer of a hydrophilic interaction liquid chromatography/tandem mass spectrometric method for the analysis of the tobacco-specific nitrosamine metabolite NNAL in human plasma at low picogram per milliliter concentrations.

A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite. Deuterated NNAL (NNAL-d(4)) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL-gluc) in K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-glucuronidase to deconjugate the NNAL-gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL-d(4) of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00-1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL-gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay.

Direct injection of solid-phase extraction eluents onto silica columns for the analysis of polar compounds isoniazid and cetirizine in plasma using hydrophilic interaction chromatography with tandem mass spectrometry.

Isoniazid and cetirizine do not retain well on reversed-phase columns due to their high polarity. Silica columns, when operated under hydrophilic interaction conditions, do provide excellent retention of these compounds. We have developed simple and proof of concept analytical methods for the analysis of isoniazid and cetirizine in animal and human plasma, respectively. Both methods employed the approach of direct injection of solid-phase extraction (SPE) organic eluents onto silica columns for analysis, thus eliminating evaporation and reconstitution steps that are typically needed for reversed-phase liquid chromatographic analysis. Isoniazid was extracted from animal plasma samples using a Waters Oasis HLB 96-well plate and then eluted with acetonitrile, while cetirizine was extracted from human plasma with a Waters MCX mu-Elute plate and then eluted with acetonitrile containing 5% concentrated ammonium hydroxide. The direct injection of the SPE eluent onto the analytical column was necessary since significant loss of isoniazid was found during the evaporation and reconstitution steps. The method for isoniazid also enabled ultra-fast analysis due to the relatively low back-pressure exhibited by silica columns even under high flow conditions. Both methods show good linearity, accuracy and precision covering the range of 10-2000 ng/mL of isoniazid, and 1-1000 ng/mL of cetirizine in plasma. Substantial time savings were realized as a result of both the elimination of the evaporation and reconstitution steps and the fast chromatographic analysis.

A Novel Phenylene Topology: Total Syntheses of Zigzag [4]- and [5]Phenylene.

In agreement with theory, the title compounds 1 and 2, which were prepared by CpCo-catalyzed cycloisomerizations of appropriate oligoalkynylbenzenes, display physical properties that are in contrast with those of the corresponding linear isomers, but that are very similar to those of the angular topomers.