Journal of Chromatography B (v.877, #3)

We report here the development and validation of an LC/MS/MS method for the rapid and accurate quantitation of brimonidine in ocular tissues and fluids using brimonidine-d 4 as an internal standard (IS). Brimonidine was extracted from retina, iris/ciliary body, and vitreous humor samples with an acetonitrile:water (1:1) solution followed by sonication and vortexing. Aliquots of aqueous humor, iris/ciliary body, retina, and vitreous humor samples were diluted with acetonitrile containing IS and were separated on a reverse-phase HPLC column under isocratic conditions. Brimonidine (m/z transition: 292 → 212) and the internal standard (m/z transition: 296 → 216) were analyzed via multiple-reaction monitoring (MRM) in the positive electrospray mode on a 4000 Q TRAP® instrument. The total analysis time for each sample was less than 2.0 min. The calibration curves for brimonidine (1–1000 ng/mL) were constructed using a linear regression with 1/x 2 weighing. The lower limit of quantitation for brimonidine was 1.0 ng/mL for aqueous humor, 10 ng/g for iris/ciliary body, 12.5 ng/g for retina, and 1.6 ng/g for vitreous humor. Intra-day and inter-day estimates of accuracy and precision were within 15% of their nominal values indicating that the method is reliable for quantitation of brimonidine in ocular tissues and fluids.
Keywords: Brimonidine; Internal standard; Ocular tissues; LC/MS/MS; Bioanalysis; Pharmacokinetics;

The goal of this work was to determine the optimal conditions for separating trypsin (TRP) from alpha-chymotrypsin (ChTRP) and to apply them for trypsin purification from bovine pancreas by liquid–liquid extraction with polyethyleneglycol/sodium citrate (PEG/NaCit) aqueous two-phase systems. Partitioning behaviours of TRP and ChTRP are demonstrated to be very sensitive to variables such as PEG molecular weight, pH and tie line length. Aqueous two-phase systems (ATPSs) formed by PEG of MW 3350 and NaCit pH 5.20 showed the best separation capability. The addition of NaCl up to a final concentration of 7% (w/w) and the decrease of top/bottom volume ratio to 0.1 led to the recovery of 60% of pancreatic TRP in a concentrated form in the top phase with a 3-fold purification. Biomass presence up to 25% (w/w) of the total system mass did not affect significantly yield and purification parameters.
Keywords: Trypsin; Chymotrypsin; Partitioning; Purification; Aqueous two-phase systems; Pancreatic proteases;

Determination of a potent urokinase-type plasminogen activator, UK-356,202, in plasma at pg/mL levels using column-switching HPLC and fluorescence detection by Mark A.J. Bayliss; Richard F. Venn; Alan M. Edgington; Robert Webster; Donald K. Walker (121-126).
A rapid, sensitive and selective method using column-switching HPLC with fluorescence detection has been developed for the determination of UK-356,202, a potent urokinase-type plasminogen activator, in human plasma. A structural isomer of UK-356,202 is used as an internal standard. The lower limit of quantification is 20 pg/mL and the method is linear over a 100-fold concentration range. UK-356,202 is extracted from plasma simply through the removal of proteins by precipitation with acetonitrile. The HPLC system comprises three columns and the cycle time is 9.5 min per sample. The eluate from the extraction column is heart-cut onto a trace enrichment cartridge which is then back-flushed onto a narrow-bore Supelco ABZ+ Plus™ analytical column. The method has been used to analyze many thousands of samples from clinical and toxicological studies support. Its ruggedness is demonstrated by the use of a single extraction column for the analysis of over 1200 clinical samples.
Keywords: Urokinase-type plasminogen activator; Analysis; Column-switching; HPLC; Fluorescence detection; Plasma;

Fabrication of mono-sized magnetic anion exchange beads for plasmid DNA purification by Hai-Ping Zhang; Shu Bai; Liang Xu; Yan Sun (127-133).
Mono-sized magnetic polyglycidyl methacrylate (PGMA) microspheres cross-linked by divinylbenzene were fabricated by the dispersion polymerization and penetration–deposition method. The magnetic beads were nonporous, spherical, and mono-sized (1.0 μm). Moreover, it was superparamagnetic with a saturation magnetization of 16.5 emu/g. The prepared magnetic beads were then functionalized with high-density amino groups and used as anion exchangers to capture plasmid DNA from concentrated bacterial lysates of Escherichia coli without treatment with ribonuclease A. The adsorption capacity of plasmid DNA reached 90 μg/mg resin in the buffer solution of pH 6.5 and 0.5 mol/l NaCl content. The product was of high recovery yield (91.7% DNA) and of high purity with almost 100% protein and 62.5% RNA removal. Recycled use of the magnetic material for plasmid purification exhibited stability of the magnetic microspheres. The magnetic beads were also effective in the purification of DNA from original crude bacterial lysates. For example, 19.4 μg plasmid DNA (A 260/A 280  = 1.90) was isolated from 1.5 ml cell culture. The results presented in this article suggest that the prepared mono-sized magnetic adsorbent is suitable for high efficient isolation and purification of plasmid DNA from crude feedstock with high recovery yield and purity.
Keywords: Mono-sized bead; Magnetic microsphere; Anion exchange; Plasmid DNA; Adsorption; Purification;

In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-l-tyrosine-9-aminophenantrene hydrophobic interaction chromatography. SDS polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 901-fold for R isoenzyme and 453-fold for Q isoenzyme. The V max and K M of the purified enzyme were determined for Q isoenzyme 55 EU and 0.599 mM and for R isoenzyme 50 EU and 0.492 mM, respectively. The in vitro effects of some heavy metals (Hg, Cd, Cu, Mn and Ni) were investigated on the purified human serum PON1Q and R isoenzyme, using paraoxon as substrate. Metals were more effective inhibitors on purified human serum PON1R192 activity than PON1Q192 activity. The kinetics of interaction of metals with the purified human serum PON1R192 and PON1Q192 indicated a different inhibition pattern. Kinetic constants K M, V max, and inhibition type were determined.
Keywords: PON1; Hydrophobic interaction chromatography; Purification; Phenotype; Metals; Inhibition;

Investigating the in vitro metabolism of veratridine: Characterization of metabolites and involved cytochrome P450 isoforms by Xuan Ye; Yuguang Wang; Minghui Yang; Qingqing Wang; Qiande Liang; Zengchun Ma; Boli Zhang; Yue Gao (141-148).
Veratridine is a lipid-soluble alkaloid extracted from Veratrum officinale and other species of the family Liliaceae. Veratridine prevents inactivation of Na+ channel via binding the receptor site 2, causes influx of sodium ion and depolarization and induces apoptosis of neuronal cells. In the present study, we investigated the metabolism of veratridine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of veratridine in rat liver microsomes. The metabolites were separated and assayed by liquid-chromatography-electrospray ionization-ion trap tandem mass spectrometry (LC-ESI-QIT-MSn), and further identified by their mass spectra and chromatographic behaviors. Result showed that four CYP isoforms (CYP1A, CYP2B, CYP2E1, CYP3A) were involved in the metabolism of veratridine in vitro and seven metabolites of veratridine were detected incubating with rat liver microsomes. Some of the metabolites were presumed to be potential mediates of neurotoxicity via protein binging. Further research in vivo needs to link the metabolism of veratridine to its toxicity.
Keywords: Veratridine; Cytochrome P450; Metabolites; Microsome;

Rapid quantification of miglustat in human plasma and cerebrospinal fluid by liquid chromatography coupled with tandem mass spectrometry by Jérôme Guitton; Sylvie Coste; Nathalie Guffon-Fouilhoux; Sabine Cohen; Monique Manchon; Marc Guillaumont (149-154).
Miglustat (OGT 918) is an iminosugar recently introduced in therapeutic as potential alternative therapy in disorders found in several diseases such as Tay-Sachs, Gaucher or Niemann-Pick diseases. A highly sensitive liquid-chromatography–electrospray tandem mass spectrometry (LC–MS/MS) assay was developed for the quantification of miglustat in human plasma and cerebrospinal fluid (CSF). The sample preparation consists in a simple protein precipitation with a mixture of acetonitrile/methanol (75/25) which yields 100% recovery. The isocratic separation utilizes an Atlantis Hilic (3 μm, 150 mm × 2.1 mm) column, with a mobile phase of acetonitrile/water/ammonium acetate buffer (75/10/15, v/v/v) delivered at 230 μl/min. Selected reaction monitoring (SRM) mode was used with the transitions m/z 220 → 158 for the miglustat and m/z 208 →  m/z 146 for the miglitol (internal standard). Good linearity was observed in a range from 125 to 2500 ng/ml and from 50 to 1000 ng/ml, for plasma and CSF, respectively. The within-run precision of the assay was less than 6%, and the between-run run precision was less than 6.5%, for six replicates at each of three concentrations and evaluated on three separated days for both plasma and CSF mediums. Assay accuracy was in the range of 98–106.5%. Stability of miglustat was reported under a variety of storage conditions. The miglustat concentrations in two children are presented to demonstrate the clinical interest of this new method.
Keywords: Miglustat; OGT 918; Deoxynojirimycin; Mass spectrometry; Liquid chromatography;

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for determination of terbutaline sulfate has been developed. This method is based on the chemiluminescence reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by terbutaline sulfate. With the peak height as a quantitative parameter applying optimum working conditions, terbutaline sulfate is determined over the range of 7.0 × 10−8 to 3.6 × 10−6  M with a detection limit of 3.0 × 10−8  M. The relative standard deviation (RSD) was 4.6% for 6.0 × 10−7  M terbutaline sulfate (n  = 11). The proposed method has been applied to determination of terbutaline sulfate in commercial terbutaline sulfate drug and spiked in human urine with satisfactory results.
Keywords: Terbutaline sulfate; Chemiluminescence; Capillary electrophoresis;

A liquid chromatography-electrospray ionization–mass spectrometry method has been developed and validated for identification and quantification of four major bioactive saponins in rat plasma after oral administration of extraction of saponins from Flos Lonicerae, i.e., macranthoidin B, macranthoidin A, dipsacoside B, and macranthoside B. Plasma samples were extracted with solid-phase extraction, separated on a Shim-pack CLC-ODS column and detected by MS in negative selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r 2  > 0.999. The method showed the low limit quantification of 7.72, 6.06, 7.16, and 1.43 ng/mL for macranthoidin B, macranthoidin A, dipsacoside B, and macranthoside B, respectively. The inter- and intra-CV precision (R.S.D.) were all within 10% and accuracy (% bias) ranged from −10 to 10%. The overall recovery was more than 70%. This developed method was subsequently successfully applied to pharmacokinetic profiles of the four saponins in rats. After oral administration of extraction of saponins in rats, the concentration–time course was found to be the double peaks of curve.
Keywords: Extraction of saponins; HPLC–MS; Macranthoidin B; Macranthoidin A; Dipsacoside B; Macranthoside B; Pharmacokinetic;

Simultaneous determination of azaperone and azaperol in animal tissues by HPLC with confirmation by electrospray ionization mass spectrometry by Yoichi Aoki; Hideki Hakamata; Yu Igarashi; Kazunari Uchida; Hisato Kobayashi; Norio Hirayama; Akira Kotani; Fumiyo Kusu (166-172).
A simple method is described for the determination of azaperone and its metabolite, azaperol, in animal tissues by high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Chromatography was performed using an ODS column, an acetonitrile–0.025% aqueous diethylamine mixture (2:3, v/v) as a mobile phase and UV detection at 250 nm. Peak heights were found linearly related to the concentrations injected from 0.05 to 2 μg/mL (r  > 0.999). Azaperone and azaperol spiked into several animal tissues were solubilized in 1 mol/L NaOH, extracted with hexane, transferred to 0.1 mol/L H2SO4 and re-extracted with hexane in a mild basic condition. Recoveries of both compounds from 12 types of samples (swine muscle, swine adipose tissue, swine liver, bovine muscle, bovine adipose tissue, bovine liver, poultry muscle, poultry adipose tissue, poultry liver, bovine milk, poultry egg, and salmon muscle) were more than 72%. The lower limit of quantification of was 0.025 μg/g. Azaperone and azaperol at 0.1 μg/g were confirmed by LC/MS. In conclusion, we found this method is both simple and useful for the determination of azaperone and azaperol in a variety of animal tissues for food safety and veterinary applications.
Keywords: Azaperone; Azaperol; Animal tissues; HPLC/UV; LC/MS;

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 ≤4.5% relative standard deviation (R.S.D.) and −8.7 to 5.6% relative error (R.E.) for individual AC and DC enantiomers.
Keywords: Chiral separation; Acebutolol; Diacetolol; Cellobiohydrolase (CBH); Supported liquid extraction (SLE);

Hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-l-tyrosine methyl ester) particles were prepared for the purification of hepatitis B surface antibody from human plasma. N-methacryloyl-l-tyrosine methyl ester was chosen as a complexing agent for hepatitis B surface antibodies. Hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-l-tyrosine methyl ester) particles were characterized by surface area measurements, swelling test, scanning electron microscopy, elemental analysis, and Fourier transform infrared spectroscopy. Ethylene glycol (1.0 M) was used as desorption agent. Adsorption studies were performed from hepatitis B surface antibody and anti-hepatitis A antibody positive human plasma. Effects of antibody concentration, contact time, N-methacryloyl-l-tyrosine methyl ester content and temperature on the adsorption capacity were investigated. The amount of hepatitis B surface antibody adsorbed per unit mass increased with increasing hepatitis B surface antibody concentration, then reached saturation. Maximum hepatitis B surface antibody adsorption amount was 21.4 mIU/mg. Adsorption process reached the equilibrium in 60 min. Competitive adsorption of hepatitis B surface antibody, total anti-hepatitis A antibody and total immunoglobulin E was investigated for showing the selectivity. Hepatitis B surface antibody-imprinted particles could adsorb hepatitis B surface antibody 18.3 times more than anti-hepatitis A antibody and 2.2 times more than immunoglobulin E. It can be concluded that hepatitis B surface antibody-imprinted particles have significant selectivity for hepatitis B surface antibody.
Keywords: Molecular imprinted polymers; Hepatitis B; Affinity beads; Affinity purification;

Suitability of different polymer bags for storage of volatile sulphur compounds relevant to breath analysis by Paweł Mochalski; Beata Wzorek; Ireneusz Śliwka; Anton Amann (189-196).
Suitability of five polymer sampling containers (Nalophan, transparent Tedlar, black layered Tedlar, Teflon and FlexFoil) for sampling and storage of six relevant to breath analysis volatile sulphur compounds: H2S, MeSH, EtSH, COS, DMS and CS2 was studied using solid phase microextraction (SPME) and gas chromatography coupled with mass spectrometry (GC–MS). Investigations were made with respect to the several factors like: recovery, background, influence of light, ageing effect and matrix effects. Additionally, the optimal reusability conditions were established. Findings suggest analyzing the breath VSCs within 6 h after sampling. Flexfoil bags were found to be the best choice for the VSCs storage up to 24 h (recovery about 90% with the exception of DMS). For shorter storing times (6–8 h) transparent Tedlar is a good alternative for Flexfoil (losses up to 10%).
Keywords: Breath analysis; Volatile sulphur compounds; Storage stability; Tedlar; Teflon; Nalophan; SPME;

Validated LC–MS/MS method for determination of Alverine and one of its hydroxy metabolites in human plasma along with its application to a bioequivalence study by Noel A. Gomes; Avdhoot Laud; Ashutosh Pudage; Santosh S. Joshi; Vikas V. Vaidya; Jayram A. Tandel (197-206).
The present research work involves a first of its kind rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method developed and validated for simultaneous analysis of Alverine (ALV) and one of its hydroxy metabolites, para hydroxy Alverine (PHA) in human plasma. The analytes were extracted from the matrix using a simple solid-phase extraction procedure. Mebeverine was used as the internal standard for both analytes. A Kromasil C8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatography conditions and mass spectrometric detection in the positive ionization mode using an API 5000 MS/MS system. The proposed method has been validated with a linear range of 100–10,000 pg/mL for both ALV and PHA. The interrun and intrarun precision values are within 6.3%, 3.7% for ALV and 6.3%, 3.2% for PHA at LOQ levels. The intrarun accuracy in terms of % RE was within the range of −7.0% to −0.1% and −8.1% to −1.7% for ALV and PHA, respectively whereas the interrun accuracy was within the range of −5.1% to −0.5% for ALV and −8.6% to 0.4% for PHA, respectively. The overall recoveries for ALV and PHA were 83.5% and 86.2% respectively. Total elution time was about 4 min which allowed quantitation of more than 150 plasma samples per day. This validated method was used successfully for analysis of real samples from a bioequivalence study.
Keywords: Alverine; Metabolite; Para hydroxy Alverine; Mebeverine; LC–MS/MS; Human plasma;

Development of an LC-ESI–MS/MS method for the determination of histamine: Application to the quantitative measurement of histamine degranulation by KU812 cells by Junko Koyama; Atsuko Takeuchi; Chisato Tode; Maki Shimizu; Izumi Morita; Machiko Nobukawa; Makiko Nobukawa; Norihiro Kobayashi (207-212).
A rapid, simple, and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) method was developed for the identification and quantification of histamine without a previous derivatization step or the addition of general ion-pairing reagents to the mobile phase. This method was used to measure histamine release following degranulation of KU812 human basophilic cells, using pyrazol as an internal standard. Analyses were performed on an LC system employing a Cosmosil 5C18 PAQ column and an isocratic elution with methanol–0.005% trifluoroacetic acid (1:1) at a flow rate of 0.2 mL/min. A triple-quadrupole mass spectrometer, equipped with an electrospray ionization interface was employed, operating in the positive ion mode. The retention time of histamine and the internal standard were 4.0 and 5.0 min, respectively. The relative standard deviations (R.S.D.s) of the retention time and peak area were between 0.47% and 2.03%. Micropipette tip solid-phase extraction (SPE) using LooseTip C18 allowed for not only rapid sample preparation, but also decreased suppression effects, improving peak shape. This method was used to evaluate the anti-allergic effects of compounds contained in Taxus yunnanensis extracts. Four constituents that were isolated from the wood extracts of T. yunnanensis and sodium cromoglicate, which is used as a first line anti-allergic drug, were tested in an in vitro histamine release inhibition assay. Of these compounds, taxiresinol and isotaxiresinol were more inhibitory than sodium cromoglicate.
Keywords: LC-ESI–MS/MS; Histamine; KU812 cell; Micropipette tip SPE; Taxus yunnanensis;

Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes by Hélène Barreteau; Sophie Magnet; Meriem El Ghachi; Thierry Touzé; Michel Arthur; Dominique Mengin-Lecreulx; Didier Blanot (213-220).
Undecaprenyl phosphate is the essential lipid involved in the transport of hydrophilic motifs across the bacterial membranes during the synthesis of cell wall polymers such as peptidoglycan. A HPLC procedure was developed for the quantification of undecaprenyl phosphate and its two derivatives, undecaprenyl pyrophosphate and undecaprenol. During the exponential growth phase, the pools of undecaprenyl phosphate and undecaprenyl pyrophosphate were ca. 75 and 270 nmol/g of cell dry weight, respectively, in Escherichia coli, and ca. 50 and 150 nmol/g, respectively, in Staphylococcus aureus. Undecaprenol was detected in S. aureus (70 nmol/g), but not in E. coli (<1 nmol/g).
Keywords: Bacterial membranes; C55-isoprenoids; High-performance liquid chromatography; Peptidoglycan; Undecaprenyl phosphate;

A precise, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of sertraline (SER) and its primary metabolite, N-desmethyl sertraline (NDS) in human plasma is developed and validated. The analytes and the internal standard-fluoxetine were extracted from 300 μL aliquots of human plasma via liquid–liquid extraction in methyl tert-butyl ether. Chromatographic separation was achieved in a run time of 2.5 min on a Betasil C8 column (100 mm × 2.1 mm, 5 μm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for SER, NDS and IS were m/z 306.2 → 159.0, 292.1 → 159.0 and 310.6 → 148.4, respectively. The method was fully validated for its sensitivity, selectivity, linearity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5–150 ng/mL was established for both the analytes with mean correlation coefficient (r) of 0.9993 and 0.9980, respectively. The intra-batch and inter-batch precision (%CV) across five quality control levels was ≤10.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100 mg sertraline tablet formulation in 32 healthy Indian male subjects under fasting condition.
Keywords: Sertraline; N-Desmethyl sertraline; LC–MS/MS; Multiple reaction monitoring; Human plasma;

Peptide mapping of therapeutic monoclonal antibodies: Improvements for increased speed and fewer artifacts by Lawrence W. Dick; David Mahon; Difei Qiu; Kuang-Chuan Cheng (230-236).
Peptide mapping is a widely utilized technique to characterize monoclonal antibodies for the purpose of product identity and is becoming increasing important as a stability indicating assay. Many conventional peptide-mapping methods are extremely time consuming and yield a map that is wrought with processing artifact peaks such as deamidation, carbamylation, and missed cleavages. Therefore, this work examines the many common individual sample preparation steps of the peptide-mapping procedure for monoclonal antibodies including the steps of denaturing, reduction, sample cleanup, digestion, and HPLC solvent selection. Improvements in each of these steps are demonstrated that greatly help to reduce artifacts and also allow for reduction of overall sample preparation time. After evaluating the many different parameters for increased speed and fewer artifacts, the sample preparation was reduced from days to hours and the resulting peptide map is nearly free of sample or background artifacts. Therefore, this peptide map procedure and optimization scheme is an excellent tool to further examine real sample changes in a shorter amount of time.
Keywords: Peptide mapping; Deamidation; Trypsin digestion; Sample preparation;

A quantitative method was developed and validated to measure the concentration of sulfadimethoxine (SDM) and its major metabolite, 4 N-acetylsulfadimethoxine (AcSDM), in bovine tissues and body fluids. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) gave quantitative results for these two analytes in extracts from bovine plasma, urine, oral fluid, kidney, and liver, using SDM-d4 as internal standard (I.S.). The lower limit of quantitation (LLOQ) for both analytes in these matrices was validated at 2, 100, and 5 ng/mL in plasma, urine, and oral fluid respectively, and 10 ng/g in both kidney (cortex) and liver. The overall accuracy (average of 4 levels) is, for plasma, 104% (SDM) and 95% (AcSDM), with standard deviation of 9% (SDM) and 15% (AcSDM); for urine, 100% (SDM) and 106% (AcSDM), with standard deviation of 5% (SDM) and 6% (AcSDM); for oral fluid, 103% (SDM) and 103% (AcSDM), with standard deviation of 4% (SDM) and 4% (AcSDM); for kidney, 101% (SDM) and 111% (AcSDM), with standard deviation of 7% (SDM) and 6% (AcSDM); and for liver, 99% (SDM) and 115% (AcSDM), with standard deviation of 11% (SDM) and 9% (AcSDM). C18 SPE cartridges were used to clean-up these matrices, except for urine which was diluted directly with buffer before analysis by LC/MS/MS.
Keywords: Sulfadimethoxine; 4 N-acetylsulfadimethoxine; Liquid chromatography/tandem mass spectrometry; Veterinary drug residue; Quantitation; Bovine; Plasma; Urine; Oral fluid; Kidney; Liver;

Kadsurenone is a neolignan with specific antagonistic activity of platelet-activating factor, and is derived from the stems of Piper kadsura. To investigate the mechanism of hepatobiliary excretion of kadsurenone and its association with P-glycoprotein (P-gp), and to explore whether the hepatobiliary excretion of kadsurenone was associated with P-gp, a microdialysis system coupled with HPLC was developed to measure free-form kadsurenone in rat blood and bile. This study design was parallel in the following groups: six rats received kadsurenone alone (20 and 30 mg/kg, i.v.) as control group and the treated-group rats were co-administered with kadsurenone and CsA; P-gp inhibitor. The microdialysis probes were respectively inserted into the jugular vein toward right atrium and bile duct of male Sprague-Dawley rats for blood and bile sampling. CsA (20 mg/kg) was administered 10 min prior to kadsurenone administration through the femoral vein and the collected samples were analyzed by a HPLC system. The analytes were separated by a C18 column (150 × 4.6 mm I.D., 5 μm) with a mobile phase of acetonitrile-water (50:50, v/v) at a flow-rate of 1 mL/min. The UV detection wavelength was set 235 nm. The calibration curve was linear over the concentration range of 0.05–10 μg/mL with the coefficient of determination of 0.997. The inter- and intra-assay accuracy and precision of the method ranged from −9.53% to 6.75%. The limit of detection and the limit of quantification were 0.01 and 0.05 μg/mL, respectively. The hepatobiliary excretion ratio of kadsurenone was defined by dividing the values of the area under the drug concentration curve (AUC) for bile and blood (AUCbile/AUCblood). The results indicated that the hepatobiliary excretion ratio of kadsurenone on the CsA treated-group was 1.2 ± 0.1, which was not significantly different from the group of kadsurenone alone (1.3 ± 0.2). This fact indicates that kadsurenone went through hepatobiliary excretion but might not be regulated by P-gp.
Keywords: Herbal medicine; Kadsurenone; Microdialysis; Pharmacokinetics; Piper Kadsura;

A simple, selective, and sensitive LC–MS/MS method was developed for the simultaneous extraction and determination of eight beta-blockers (atenolol, sotalol, nadolol, pindolol, timolol, metoprolol, betaxolol and propranolol) in various bovine eye tissues including sclera, choroid–RPE, retina, and vitreous. The analytes were extracted by liquid–liquid extraction after samples were alkalinized with 2% NaOH solution in water. The chromatographic separation was performed on a Hypersil-ODS C18 column (100 mm × 2.1 mm, 3.9 μm) using a gradient mixture of (A) 5 mM ammonium formate in water (pH 3.5 adjusted with formic acid) and (B) acetonitrile:methanol (75:25) containing 0.02% triethyl amine (pH 4.0; adjusted with formic acid) as mobile phase at a flow rate of 0.4 ml/min. The compounds were ionized in the positive electrospray ionization (ESI) mode and detected in the multiple reaction monitoring (MRM) mode. The average recoveries in all four eye tissues for all beta-blockers were >82%, except for sotalol (>51%). The matrix effect for beta-blockers ranged from 81 to 110% in the four eye tissues. This analytical method was validated and applied successfully for simultaneous quantification of the beta-blockers in sclera after tissue exposure using cassette dosing method. The calibration curve was linear in the range of 10–2000 ng/ml for all analytes, with the correlation coefficient >0.996. Intra-day and inter-day precision (% CV) was less than 15%, and accuracy ranged from 85 to 110% for all analytes. Scleral uptake was the lowest for sotalol and atenolol, two hydrophilic beta-blockers, and the highest for propranolol, a lipophilic beta-blocker.
Keywords: Beta-blockers; Liquid–liquid extraction; Cassette analysis; LC–MS/MS;

27-Hydroxycholesterol (27OH-Chol) is of potential diagnostic interest due to its role in maintaining whole-body cholesterol homeostasis. Dopant assisted-atmospheric pressure photoionization (DA-APPI) has improved the sensitivity of 27OH-Chol analysis, in comparison to the published LC-APCI–MS method, allowing quantification from a very low amount of sample (≤50 μL plasma). The method was validated for quantification from 50 μL and 15 μL plasma, with the limit of quantification (LOQ) of 10 ng/mL and 40 ng/mL plasma, respectively. A further advantage is that no prior derivatization was needed, unlike the LC-ESI–MS or the standard GC–MS method. The method validation also resulted in good linearity and recovery of 91–106%. The within-day and between-day coefficient of variation were less than 15% while giving the accuracy of 90.9–113.4%. This report summarizes the effects of some critical parameters on the ionization of 27OH-Chol using APPI, as well as the validation of the method. The sensitivity achieved with DA-APPI broadens the usefulness of the LC–MS method in clinical applications.
Keywords: 27-Hydroxycholesterol; Atmospheric pressure photoionization; LC–MS; Plasma;

Liquid chromatography–tandem mass spectrometric assay for sorafenib and sorafenib–glucuronide in mouse plasma and liver homogenate and identification of the glucuronide metabolite by Rolf W. Sparidans; Maria L.H. Vlaming; Jurjen S. Lagas; Afred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (269-276).
The first bioanalytical assay for the simultaneous determination of sorafenib and sorafenib–glucuronide in mouse plasma and liver homogenate was developed and validated. In addition, the structure of the glucuronide metabolite was elucidated. The quantitative assay started with addition of isotopically labeled internal standards to a 20 μl sample volume and protein precipitation with acetonitrile, the supernatant was diluted with water and injected into the chromatographic system. A polar embedded reversed-phase column with gradient elution using formic acid in water–acetonitrile was used. The eluate was transferred into an electrospray interface with positive ionization and the analytes were detected and quantified using triple quadrupole mass spectrometry. The assay was validated in the ranges 10–5000 ng/ml for sorafenib and 1–500 ng/ml for sorafenib–glucuronide, the lowest levels of these ranges (10 and 1 ng/ml) being the lower limits of quantification (LLQ). Within day precisions were 2–8%, between day precisions 2–10% (both excluded the LLQ level of the glucuronide) and accuracies were between 89% and 106%. Both analytes were chemically stable under all relevant conditions. The assay was successfully applied in pilot in vivo pharmacokinetic studies with sorafenib in mice.
Keywords: Sorafenib; Sorafenib–glucuronide; LC/MS/MS; Mouse plasma; Mouse liver homogenate;

Performance of polymeric materials for solid phase extraction prior to chromatographic analysis by Denise Bohrer; Marlei Veiga dos Santos; Adrian G. Ramirez; Paulo Cícero do Nascimento; Leandro M. de Carvalho (277-284).
Synthetic polymeric materials such as polyethylene and polyurethane (PU) were compared to conventional adsorbents for solid phase extraction for cleaning up biological samples. Efficiency in eliminating proteins and other components usually present in biological samples, such as serum, urine, and tissues extracts, was evaluated. The assays consisted of measuring the remaining protein content in serum and tissue homogenates (liver) and collecting the spectra in the UV region for urine samples. Since the analysis of many endogenous and exogenous species in these matrices usually involves chromatographic separation, the efficiency of the clean-up procedures was also evaluated by injecting cleaned samples into a C-18 chromatographic column with UV detection. Among the investigated polymers, polytetrafluorethylene, high density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE) presented the best performance in retaining serum proteins. Proteic components of the liver homogenate were completely retained on polyurethane and polybutadiene (PB). Urine samples were cleaned by crossing columns of polytetrafluorethylene, ultra-high molecular weight polyethylene, high density polyethylene, polyurethane, and polyethylene co-butyl acrylate co-anhydride maleic (PEco), since the spectra collected after column percolation presented no peaks in the region between 190 and 390 nm. SPE cartridges showed different behavior, but along the lines of their usual performance; neither serum proteins nor urine components were retained on the phases and the liver components, though partially retained, were not desorbed with either water or methanol washes, with the exception of SAX. Chromatograms of samples cleaned with high density polyethylene showed that polymeric materials can be satisfactorily used as adsorbent for biological matrix components.
Keywords: Polymers; SPE; Sample clean-up; Serum; Urine; Liver;

Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for determination of isoniazid in human plasma by Liusheng Huang; Florence Marzan; Anura L. Jayewardene; Patricia S. Lizak; Xiaohua Li; Francesca T. Aweeka (285-290).
An LC–MS/MS method for the determination of isoniazid in human plasma was developed and validated. Human plasma aliquots of 100 μL were used for analysis. The assay used nialamide as the internal standard. The calibration curve concentration range was 50–10,000 ng/mL. Sample preparation utilized protein precipitation, and the supernatant was directly injected onto silica column without reconstitution. The recovery was over 90% and matrix effect was negligible. The method is simple and fast, which is advantageous in respect to instability of isoniazid in human plasma and loss on reconstitution due to its low molecular weight.
Keywords: LC–MS/MS; Isoniazid; Nialamide; Human plasma; Method development; Silica column;

This work presents the development and validation of a fully automated quantitative analysis method of melagatran, its prodrug ximelagatran, and its major metabolites for the study of drug behavior in biofluids. The method involves online sample clean-up and enrichment on a C4 capillary column followed by separation on a capillary C18 column. Electrospray ionization tandem mass spectrometric detection in positive ion mode was performed with multiple reactions monitoring of eight different transients, divided into two time segments with four transients each. The structural similarity, the complexity of the matrix (pig liver extract) and the formation of isobaric fragment ions, made efficient chromatographic separation necessary. The analysis method provides valid accuracy (<9%; RSD%), precision (<8%; RSD%), linearity (<1.2 nM–1 μM; R 2  > 0.999), limit of quantitation (<3.6 nM), retention repeatability (<1.2%; RSD%), selectivity, as well as analyte and column stabilities over a wide concentration range.
Keywords: Online; Solid phase extraction; Liquid chromatography; Mass spectrometry; Ximelagatran; Metabolites; Pig liver;

Miniaturized hollow fiber assisted liquid-phase microextraction and gas chromatography–mass spectrometry for determination of benzophenone and derivates in human urine sample by Migaku Kawaguchi; Rie Ito; Hidehiro Honda; Youji Koganei; Noriya Okanouchi; Koichi Saito; Yasuo Seto; Hiroyuki Nakazawa (298-302).
The determination of benzophenones (BPs) in human urine sample by miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) and gas chromatography–mass spectrometry (GC–MS) is described. As analytes, BP, its metabolites benzhydrol (BP-OH) and 2-hydroxybenzophenone (2OH-BP), and its derivatives 2-hydroxy-4-methoxybenzophenone (BP-3) and 2-hydroxy-4-methoxy-4′-methylbenzophenone (BP-10) were selected. The detection limit and the quantification limit of BPs in human urine sample are 5–10 and 20–50 pg mL−1, respectively. The calibration curve for BPs is linear with correlation coefficient higher than 0.99 in the range of 0.02–10 or 0.05–10 ng mL−1. The average recoveries of BPs in human urine samples spiked with 0.5 and 5 ng mL−1 BPs are 89.8–100.2% (RSD: 2.5–9.3%) and 89.3–99.9% (RSD: 2.9–3.7%), respectively. Ten human urine samples were analyzed using the present method. BP-OH and BP-3 were detected in all the samples within the range of 0.24–5.91 and 0.43–5.17 ng mL−1, respectively. This simple, sensitive, and selective analytical method was successfully applied to the determination of trace amounts of BPs in human urine samples.
Keywords: Benzophenone (BP); Miniaturized; Hollow fiber (HF); Liquid-phase microextraction (LPME); GC–MS;

Quantitative measurements of corticosteroids in ex vivo samples using on-line SPE-LC/MS/MS by Lan Gao; William J. Chiou; Heidi S. Camp; David J. Burns; Xueheng Cheng (303-310).
Abnormal elevation of 11β-HSD1 activities in tissues, such as fat and brain, may contribute to the development of the abdominal obesity and Alzheimer disease, and the inhibition of 11β-HSD1 might be beneficial to the management of these diseases. To assess the effects of pharmacologic inhibitors of 11β-HSD1, we developed a fast LC/MS/MS method to quantify corticosteroids in minced tissue samples in the presence of 11β-HSD substrates. The novel on-line SPE-LC/MS/MS method was developed with dual binary gradient and a throughput of 4.5 min/sample. A total of six corticosteroids (cortisol, cortisone, corticosterone, dehydrocorticosterone, dexamethasone, and dehydrodexamethasone) were studied. The lower limit of quantitation from 0.40 to 11.4 fmol and 4.5 orders magnitude of dynamic range were obtained for these six compounds. Three novel enzymatic bi-products, all isomers of cortisol, were observed in the liver or fat samples. Two of them were identified by matching the HPLC retention times and MS/MS spectra with authentic compounds. The potential interferences of these isomers and their removal are discussed.
Keywords: High performance liquid chromatography (HPLC); Solid phase extraction (SPE); Mass spectrometry (MS); Tandem mass spectrometry (MS/MS); Quantitation; Corticosteroid; Steroid; Cortisol; Hydrocortisone; Cortisone; Dexamethasone; Corticosterone; Dehydrocorticosterone; Dehydrodexamethasone; Ex vivo; Endogenous; 11β-Hydroxysteroid dehydrogenase (11β-HSD);

A simple, sensitive and rapid method is presented for the determination of norelgestromin using LC–MS/MS, interfaced via an electrospray ionization (ESI) probe, operating in the positive ion mode with multiple reaction monitoring (MRM). The method was developed and validated over the concentration range of 0.05–20 ng/ml, and showed excellent linearity. The intra- and inter-assay accuracy error and precision were ranging from −2.3% to 6.0% of nominal values and 2.2% to 7.8% over the three concentration levels evaluated. The concentration of formic acid in mobile phase was optimized to achieve satisfactory injection reproducibility and sensitivity, and sample preparation was optimized, with only 1.6 ml organic solvents used in performing the liquid–liquid extraction. The method has been successfully applied to a pharmacokinetic study of the ORTHO EVRA® patch in rabbits.
Keywords: Norelgestromin; Determination; LC–MS/MS; Pharmacokinetic; ORTHO EVRA® patch;

HPLC methods for determination of two novel thiosemicarbazone anti-cancer drugs (N4mT and Dp44mT) in plasma and their application to in vitro plasma stability of these agents by Ján Stariat; Petra Kovaříková; Jiří Klimeš; David B. Lovejoy; Danuta S. Kalinowski; Des R. Richardson (316-322).
The aim of this study was to develop and validate HPLC methods for the determination in plasma of two novel thiosemicarbazone anti-tumour drugs developed in our laboratories (Dp44mT and N4mT). The appropriate separations were achieved using a HS F5 HPLC column with the mobile phase composed of a mixture of either acetate buffer/EDTA or EDTA and acetonitrile (62:38 and 50:50, v/v, respectively). The plasma samples were pretreated with SPE (phenyl and C18, respectively). Furthermore, these methods were successfully applied to in vitro plasma stability experiments. The investigation has clearly shown that both thiosemicarbazones are markedly more stable in plasma than their aroylhydrazone forerunners.
Keywords: Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT); 2-hydroxy-1-naphthylaldehyde-4-methyl-3-thiosemicarbazone (N4mT); Thiosemicarbazone; Stability; HPLC;

Development and validation of a LC/MS/MS method for quantification of nobiliside A in rat plasma by Dan Guo; Yang Xiong; Yue Zhang; Zhongbin Wu; Lili Cui; Jianming Chen (323-327).
A LC/MS/MS method was developed and validated for determination of nobiliside A in rat plasma. Analyses were separated by an Agella Venusil ASB-C18 column and isocratic elution with methanol:water (80:20, v/v) as a mobile phase. A tandem mass spectrometer was used as a detector for quantitative analysis. Calibration curves (R 2  > 0.999) in spiked plasma were linear over the concentration range of 50–5000 ng/mL. The overall accuracy of this method was 93–104% for nobiliside A. Within-day and between-day variability was both less than 9% in plasma. The data indicate that the LC/MS/MS method is an effective method for the pharmacokinetics study of nobiliside A in rat plasma.
Keywords: Nobiliside A; Plasma analysis; LC/MS/MS;

A novel analytical methodology for the estimation of residual ethylene glycol (EG) in ethylene oxide sterilized polymer is reported. The method involves the monitoring of ammonium adduct of EG ions in the presence of 10 mM ammonium acetate buffer and methanol using electrospray ionization liquid chromatography and mass spectrometry (LC–ESI-MS). The method enables the detection and quantification of EG without prior derivatization up to a level of 0.06 μg/ml. The potentiality of the method is demonstrated by estimating EG in ethylene oxide (EtO) sterilized polyethylene terephthalate fabric used in heart valve sewing ring. The method is simple, rapid and can routinely be used for the quantification of residual EG in EtO sterilized medical devices.
Keywords: Ethylene glycol; LC–ESI-MS;

An analytical method for kanamycin A, amikacin and tobramycin of aminoglycoside (AG) antibiotics in milk samples was proposed using capillary zone electrophoresis (CZE) with post-column derivatization and laser-induced fluorescence detection. A simple and convenient homemade coaxial-gap reactor was adopted in the post-column derivatization of the AGs with 1.0 mmol/L naphthalene-2,3-dicarboxaldehyde and 8.0 mmol/L 2-mercaptoethanol in 35 mmol/L sodium tetraborate buffer (pH 10.0) of 30% (v/v) methanol. 50 mmol/L sodium acetate (pH 5.0) containing 0.5 mmol/L cetyltrimethyl ammonium bromide was used as the separation buffer. The linear calibration concentrations and detection limits were from 2.1 × 10−5 to 5.0 × 10−2  g/L and in the range of 7 × 10−6 to 2 × 10−5  g/L, respectively. The recoveries of the AGs in milk samples were from 81.6 to 93.1% (n  = 3).
Keywords: Capillary zone electrophoresis; Post-column derivatization; Laser-induced fluorescence; Aminoglycosides; Kanamycin A; Amikacin; Tobramycin;

Determination of hair nicotine by gas chromatography–mass spectrometry by Che Nin Man; Syazwani Ismail; Gam Lay Harn; Razak Lajis; Rahmat Awang (339-342).
Hair nicotine is a known biomarker for monitoring long-term environmental tobacco smoke (ETS) exposure and smoking status. In general, hair nicotine assay involves alkaline digestion, extraction and instrumental analysis. The gas chromatography–mass spectrometry (GC–MS) assay currently developed has shown to be of high throughput with average ∼100 hair samples being extracted and analyzed per day. This was achieved through simplified extraction procedure and shortened GC analysis time. The extraction was improved by using small volume (0.4 mL) of organic solvent that does not require further evaporation and salting steps prior to GC–MS analysis. Furthermore, the amount of hair utilized in the extraction was very little (5 mg) while the sensitivity and selectivity of the assay is equal, if not better than other established methods. The linearity of the assay (r 2  > 0.995), limit of quantitation (0.04 ng/mg hair), within- and between-assays accuracies and precisions (<11.4%) and mean recovery (92.6%) were within the acceptable range.
Keywords: Nicotine; Hair; Epidemiology; GC–MS; ETS; Chronic exposure;

Miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) and gas chromatography–mass spectrometry (GC–MS) for the determination of progesterone in human serum sample is described. The detection limit and the quantification limit of progesterone in human serum sample are 0.5 and 2 ng mL−1 (ppb), respectively. The calibration curve for progesterone is linear with a correlation coefficient of >0.999 in the range of 2–500 ng mL−1. The average recoveries of progesterone in human serum samples spiked with 5, 50 and 200 ng mL−1 progesterone are 97.4% (R.S.D.: 9.3%), 100.3% (R.S.D.: 4.7%) and 99.8% (R.S.D.: 3.4%), respectively. This simple, accurate, sensitive and selective analytical method can be applicable for the determination of progesterone in human serum samples.
Keywords: Progesterone; Hollow fiber (HF); Liquid-phase microextraction (LPME); Gas chromatography–mass spectrometry (GC–MS);

Dynamic rejection of colloidal particles with generating dextran by enzymatic reaction by Hidetaka Kawakita; Yuko Yoshimura; Kohshi Hamamoto; Hirokazu Seto; Keisuke Ohto; Hiroyuki Harada; Katsutoshi Inoue (347-350).
Dextransucrase forms a complex with dextran during an enzymatic reaction with sucrose. Using its enzymatic character, we performed a continuous and dynamic rejection of colloidal particles by generating dextran with dextransucrase immobilized in an inorganic porous membrane. Inorganic membranes having 1.9 and 3.0 U/g of immobilized dextransucrase, and 4.1 and 9.4 mg/g of generated dextran, respectively, had constant rejection percentages for 55 and 100 nm colloidal particles in permeating solutions. On the other hand, permeating sucrose solutions containing colloidal particles through a dextran-immobilized membrane dynamically increased the rejection percentages of the colloidal particles owing to dextran generation via enzymatic reaction. The dynamic increase was due to the gradually generating dextran dynamically occupying the membrane pore with its steric volume.
Keywords: Dynamic rejection; Dextransucrase; Dextran; Colloidal particle; Porous membrane;

Determination of vitamin K1 in plasma by solid phase extraction and HPLC with fluorescence detection by Rita Paroni; Elena Maria Faioni; Cristina Razzari; Gessica Fontana; Marco Cattaneo (351-354).
We describe a procedure for quantification of vitamin K1 in human plasma by HPLC. Samples, enriched with a vitamin K derivative as internal standard, were deproteinized, purified on polymeric RP-SPE cartridges and injected into HPLC equipped with a post-column on-line zinc metal reactor and a fluorometric detector. Median level in blood donors (n  = 87) was 1.967 nmol/L (0.93–4.01, 5th–95th percentiles), with a significant correlation between plasma levels and age (r  = 0.276, p  = 0.00958) and a lower (not significant) value in women than in men. This method, easy-to-handle and with a high throughput, can be used to identify covert states of vitamin K intake deficiency in patients thus at risk of alterations in blood clotting or bone mineralization.
Keywords: Vitamin K1; HPLC; Reference values; Haemostasis alteration; Bone mineralization;

An analytical method was developed and validated for the quantitative determination of the histone deacetylase inhibitor MS-275 in human plasma. Calibration curves were linear in the concentration range of 1–250 ng/mL. Sample pretreatment involved a liquid–liquid extraction of 0.1 mL aliquots of plasma with methyl tert-butyl ether. MS-275 and the internal standard, benzanilide, were separated on a Zorbax SB-Phenyl column (4.6 mm × 75 mm I.D., 3.5 μm), using a mobile phase composed of methanol and 10 mM ammonium formate (pH 2.9). The column eluent was monitored by mass spectrometry with electrospray ionization. Accuracy and precision of three concentrations of quality control samples ranged from 96.56 to 107.02% and 0.97 to 4.29%, respectively. This method represents an improvement over the previously published analytical assay for this agent, increasing the accuracy and precision (through addition of a suitable internal standard) and expanding the analytical range. The developed method was applied to study the pharmacokinetics of MS-275 in 724 clinical samples.
Keywords: Histone deacetylase inhibitor; LC/MS; MS-275; Benzanilide;