Journal of Chromatography B (v.846, #1-2)

A rapid and sensitive method for determination of sorafenib in human plasma using a liquid chromatography/tandem mass spectrometry assay by Ming Zhao; Michelle A. Rudek; Ping He; Frank-Thorsten Hafner; Martin Radtke; John J. Wright; B. Douglas Smith; Wells A. Messersmith; Manuel Hidalgo; Sharyn D. Baker (1-7).
A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sorafenib in human plasma. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma with 0.5 mL acetonitrile. Analysis of the compounds of interest including the internal standard ([2H3 15N] sorafenib) was achieved on a Waters X-Terra™ C18 (150 mm × 2.1 mm i.d., 3.5 μm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 7.3–7260 ng/mL for the human plasma samples with values for the coefficient of determination of >0.96. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%).
Keywords: Sorafenib; LC/MS/MS; Pharmacokinetics;

Simultaneous determination of fluoroquinolones and tetracyclines in chicken muscle using HPLC with fluorescence detection by Marilyn J. Schneider; Susan E. Braden; Ixchel Reyes-Herrera; Dan J. Donoghue (8-13).
A multiresidue method has been developed which allows for the simultaneous determination of both fluoroquinolones and tetracyclines in chicken muscle. Samples were extracted with a mix of acetonitrile and 0.1 M citrate, 150 mM MgCl2, pH 5.0. After centrifugation and evaporation, the extracts could be analyzed by liquid chromatography with fluorescence detection. Good recoveries (63–95%) were obtained from samples fortified with a mix of five fluoroquinolones and three tetracyclines, with satisfactory relative standard deviations. Limits of detection were 0.5 ng/g (danofloxacin), 1 ng/g (oxytetracycline, ciprofloxacin, enrofloxacin), 1.5 ng/g (tetracycline), 2 ng/g (difloxacin) and 5 ng/g (sarafloxacin, chlortetracycline). Enrofloxacin and its metabolite ciprofloxacin, as well as oxytetracycline were determined in enrofloxacin and oxytetracycline incurred chicken muscle using this method.
Keywords: Fluoroquinolone; tetracycline; antibiotics;

The fabrication and application of a novel electrochemical detection (ED) system with a poly(bromophenol blue) (PBPB) film chemically modified electrode (CME) for high performance liquid chromatography (HPLC) were described. The electrochemical behaviors of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) at this CME were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). It was found that the PBPB CME efficiently exhibited electrocatalytic effect on the current responses of 5-HT and 5-HIAA with relatively high sensitivity, stability and long life of activity. In HPLC–ED, the two analytes had good and stable current responses at the CME and their linear ranges were over four orders of magnitude (R  ≥ 0.9992) with the detection limits being 0.25 nmol L−1 for 5-HT and 0.50 nmol L−1 for 5-HIAA. The application of this method for the determination of 5-HT and 5-HIAA in urine samples from patients with acute appendicitis (AA) was satisfactory.
Keywords: Poly(bromophenol blue) film; Acute appendicitis; 5-Hydroxytryptamine; 5-Hydroxyindoleacetic acid; HPLC–ED;

A simple and sensitive high performance liquid chromatography method with UV detection was described for the determination of tropisetron in human plasma. The prepared sample solution was injected onto BDS-C8 reversed column using a mixture of ammonium acetate (100 mM, PH adjusted to 4.3 with glacial acetic acid) and acetonitrile (80:20, v/v) as mobile phase. The wavelength of UV detector was set at 285 nm. No interference from any endogenous substances was observed during the elution of tropisetron and internal standard (ondansetron hydrochloride). The lower limit of quantification was evaluated to be 1 ng/mL. The method was used in a randomized crossover bioequivalence study of two different tropisetron preparations in 20 healthy volunteers.
Keywords: Tropisetron; High performance liquid chromatography; UV detection; Bioequivalence study;

Distribution of Aβ peptide in whole blood by J. Randall Slemmon; Cory L. Painter; Sashi Nadanaciva; Florentina Catana; Ashley Cook; Ruth Motter; Peter Seubert (24-31).
The measurement of amyloid beta peptides (Aβ) in blood and plasma is expected to be a useful biomarker as potential therapeutics designed to lower Aβ peptide enter clinical trials. Many reports have suggested that Aβ could bind to substances in blood that may influence the recovery of Aβ peptide in plasma, its detection by conventional ELISAs or the actual turnover and half-life of the peptide in blood. In this study we describe a process for analyzing total Aβ in whole blood and plasma using denaturing solid-phase extraction followed by reverse-phase HPLC linked to ELISA. Comparison of total Aβ peptide levels in whole blood and plasma from the same bleed showed that most of the Aβ peptide is captured in the plasma if the samples are first denatured. In contrast, plasma that was assayed without denaturation could show greater than 70% reduction in apparent total Aβ peptide. This suggested that there was a pool of Aβ peptide in non-denatured plasma that is occluded from detection by ELISA, perhaps by binding to plasma proteins.
Keywords: Aβ peptides; ELISA; HPLC; Blood; Plasma; Denaturation;

Development of the fingerprints for the quality of the roots of Salvia miltiorrhiza and its related preparations by HPLC-DAD and LC–MS n by Ai-Hua Liu; Yan-Hua Lin; Min Yang; Hui Guo; Shu-Hong Guan; Jiang-Hao Sun; De-An Guo (32-41).
High-performance liquid chromatographic (HPLC) fingerprints were developed for identification of both lipophilic and hydrophilic components of the roots of Salvia miltiorrhiza and four related preparations. These samples were separated with an Agilent Zorbax Extend C18 reserved-phase column (5 μm, 250 mm × 4.6 mm) by linear gradient elution using water-phosphoric acid (100:0.026, v/v) and acetonitrile as mobile phase. The flow rate was 0.8 ml/min and the detector wavelength was set at 280 nm. Mean chromatograms and correlation coefficients of samples were calculated by the software “Similarity Evaluation System for Chromatographic Fingerprint of TCM”. The correlation coefficients of Danshen and Fufang Danshen tablets (FDT) samples were in the range of 0.352–0.993 and 0.768–0.987, respectively. The correlation coefficients of Compound Danshen dripping pills (CDDP), Danshen injection (DSI) and Xiangdan injection (XDI) samples were higher than 0.928, 0.850 and 0.960, respectively. It was the first time to identify 34 peaks by comparing with standard compounds and using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS n ) technique. All results indicated that the developed fingerprint assay could be readily utilized as a quality control method for S. miltiorrhiza and its related preparations.
Keywords: Salvia miltiorrhiza; Salvianolic acids; Tanshinones; Fingerprint; HPLC-DAD; LC–MS n ;

Isotope dilution LC/MS/MS for the detection of nerve agent exposure in urine by Frederic L. Ciner; Carla E. McCord; Roy W. Plunkett; Michael F. Martin; Timothy R. Croley (42-50).
Organophosphorus nerve agents (OPNA), chemically related to and derived from organophosphate insecticides, constitute a clear and present threat to both military and civilian targets. Military regimes and terrorist organizations have demonstrated the will and ability to produce mass casualties by dispersing organophosphorus nerve agents, which, in turn could terrorize populations and overwhelm healthcare systems. A high throughput, robust and sensitive analytical protocol has been developed for the quantitation of the urinary metabolites of sarin (GB), soman (GD), VX, Russian VX (RVX) and cyclohexylsarin (GF) utilizing solid phase extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)–isotope dilution tandem mass spectrometry (LC/MS/MS). The method has demonstrated linearity and reproducibility (1–200 ng/mL) for all analytes and has a Limit of Quantitation (LOQ) ≤ 0.5 ng/mL for all analytes (S/N ≥ 10/1). The method was validated by performing 20 individual analyses over 10 days by five scientists with all values falling within two standard deviations of the mean.
Keywords: Nerve agents; LC/MS/MS; Method development;

Purification by expanded bed adsorption and characterization of an α-amylases FORILASE NTL® from A. niger by A.L. Toledo; J.B. Severo; R.R. Souza; E.S. Campos; J.C.C. Santana; E.B. Tambourgi (51-56).
In this work the purification and biochemistry characterization of α-amylases from Aspergillus niger (FORILASE NTL®) were studied. The effects of expansion degree of resin bed on enzyme purification by expanded bed adsorption (EBA) have also been studied. Residence time distributions (RTD) studies were done to achieve the optimal conditions of the amylases recovery on ion-exchange resin, and glucose solution was used as a new tracer. Results showed that height equivalent of the theoretical plates (HETP), axial dispersion and the Prandt number increased with bed height, bed voidage and linear velocity. The adsorption capacity of α-amylases, on the resin, increased with bed height and the best condition was at four-expansion degree. α-Amylase characterization showed that this enzyme has high affinity with soluble starch, good hydrolysis potential and molecular weight of 116 kDa.
Keywords: Aspergillus niger; α-Amylase; Expanded bed adsorption; Expansion degree; Purification; Biochemistry characterization;

Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry by Wenkui Li; Suyi Luo; Shaoyong Li; Lawrence Athill; Amy Wu; Tapan Ray; Wei Zhou; June Ke; Harold T. Smith; Francis L.S. Tse (57-68).
For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC–MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10–5000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC–MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm × 2.1 mm, 5 μm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid (A) and 90% acetonitrile (containing 0.5% formic acid (B). The flow rate was 0.4–0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were <9.0% relative standard deviation (R.S.D.) and 10.8% bias for ribavirin, and 10.3% R.S.D. and 11.3% bias for ribavirin base. The current specific, accurate and precise assay is useful in support of the toxicokinetic and pharmacokinetic studies of these compounds.
Keywords: Ribavirin; Ribavirin base; LC–MS/MS; Simultaneous analysis;

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine by Takaaki Goto; Khin Than Myint; Koichi Sato; Osamu Wada; Genta Kakiyama; Takashi Iida; Takanori Hishinuma; Nariyasu Mano; Junichi Goto (69-77).
We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3β,12α-dihydroxy-5β-cholanoic acid 3-sulfate.
Keywords: Bile acid; Sulfate; Liquid chromatography/tandem mass spectrometry; Electrospray ionization; Human; Urine;

A new method for determination of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair based on alkaline hair hydrolysis, extraction by iso-octane, combined derivatization with N,O-bis-(trimethylsilyl)-trifluoroacetamide and headspace solid phase microextraction of the extract residue, and gas chromatography–mass spectrometry was developed and evaluated. The limits of detection of the three compounds were 0.01–0.02 ng/mg. The method was routinely applied to more than 250 hair samples. In 77 positive samples, the concentrations ranged from LOD to 4.2 ng/mg for THC (mean 0.49 ng/mg), to 12.1 ng/mg for CBD (mean 0.37 ng/mg) and to 0.85 ng/mg for CBN (mean 0.12 ng/mg) using a sample amount of 30 mg. The frequently observed increase of the segmental drug concentrations from proximal to distal is explained by progressive accumulation in the hair shaft from sebum or side stream smoke.
Keywords: Cannabidiol in hair; Cannabinol in hair; Gas chromatography–mass spectrometry; Hair analysis; Headspace solid phase microextraction; Δ9-Tetrahydrocannabinol in hair;

The composition of human scent collected from the hands is of interest to the medical community as a mechanism to diagnose disease and the forensic community as a means to investigate canine scent discriminations. An extensive survey of the volatile organic compounds (VOCs) identified in the headspace of hand odor samples utilizing solid phase micro-extraction gas chromatography/mass spectrometry (SPME-GC/MS) has been conducted to determine the constituents of the human base odor profile. Sixty-three compounds were extracted from the collected odor samples. The composition included acids, alcohols, aldehydes, hydrocarbons, esters, ketones and nitrogen-containing compounds. The majority of the compounds detected (79.4%) were present in less than one third of the individuals sampled. Spearman correlation coefficient comparisons at a match/no-match threshold of 0.9 produced a distinguish ability of 99.67% across the population.
Keywords: Human scent; Hand odor; VOCs; Canine; Human scent line-up;

A bioanalytical method for determination of eflornithine (DFMO) in 1000 μL human plasma has been developed and validated. DFMO and the internal standard (IS) were analysed by liquid chromatography with evaporative light-scattering detection (ELSD). Separation was performed on a Chirobiotic TAG (250 mm × 4.6 mm) column with ethanol (99.5%):0.01 mol/L acetic acid-triethylamine buffer at the rate of 25:75% (v/v) with flow rate of 1.0 mL/min. For d-DFMO in plasma the inter-assay precision was 6.5% at 75 μmol/L, 6.6% at 375 μmol/L and 5.8% at 750 μmol/L. For l-DFMO in plasma the inter-assay precision was 10.4% at 75 μmol/L, 6.5% at 375 μmol/L and 5.0% at 750 μmol/L. The lower limit of quantification (LLOQ) was determined to 25 μmol/L where the precision was 4.3% and 5.7%, respectively.
Keywords: Eflornithine; DFMO; 2-Fluoromethyl-dl-ornithine; Chiral chromatography; Chirobiotic TAG; Human African Trypanosomiasis; Evaporative light-scattering detection; ELSD; HPLC; Chirality;

A sensitive, simple and highly selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and evaluated to determine simultaneously the concentrations of pseudoephedrine and cetirizine in human plasma. The chief benefit of the present method is the minimal sample preparation, as the procedure is only one-step protein precipitation. Two drugs were separated on a C8 column and analyzed by LC/MS/MS using positive electrospray ionisation (ESI). The method had a chromatographic run time of 12.0 min and a linear calibration curve over the concentration range of 1.0–800 ng/ml for pseudoephedrine and 1.0–400 ng/ml for cetirizine, respectively. The lower limit of quantification of the two drugs was 1.0 ng/ml, respectively. The intra- and inter-batch precisions were less than 9.7%. The method described herein has been first used to reveal the pharmacokinetic characters in healthy Chinese volunteers treated with oral administration of different dosages of cetirizine dihydrochloride and controlled-released pseudoephedrine hydrochloride compound tablet, and approached the influence of a standard meal on the extent and rate of absorption of the combination tablet.
Keywords: Pseudoephedrine; Cetirizine; HPLC–MS/MS; Human plasma; Pharmacokinetics;

A high-performance liquid chromatography-atmospheric pressure chemical ionization–mass spectrometry (HPLC-APCI–MS) method was established for the determination of gambogic acid (GA) in human plasma using ursolic acid as the internal standard (I.S.). Plasma samples were extracted with ethyl acetate and separated on a Hanbon Lichrospher 5-C18 column with a mobile phase of acetonitrile–tetrahydrofuran–water (70:23:7, v/v). Gambogic acid was determined by using atmospheric pressure chemical ionization (APCI) in a single quadrupole mass spectrometer. HPLC-APCI–MS was performed in the selected ion monitoring (SIM) mode using target ions at [M−H] m/z 627.4 for gambogic acid and [M−H] m/z 455.4 for the I.S. Calibration curve was linear over the range of 3.108–4144 μg/L. The lower limit of quantification was 3.108 μg/L. The intra- and inter-run precisions were less than 12.3 and 14.1%, respectively. The method has been successfully applied to study the pharmacokinetics of gambogic acid in patients with malignant tumour.
Keywords: Gambogic acid; HPLC-APCI–MS; Pharmacokinetics;

Chiral separation of Frovatriptan isomers by HPLC using amylose based chiral stationary phase by Muzaffar Khan; Balaji Viswanathan; D. Sreenivas Rao; Rajasekhar Reddy (119-123).
A stereospecific HPLC method for separation of Frovatriptan enantiomers in bulk drug and pharmaceutical formulations was developed and validated on a normal-phase amylose derivertized chiral column. The effects of the organic modifiers namely 2-propanol, ethanol and diethyl amine (DEA) in the mobile phase were optimized to obtain the best enantiomeric separation. Calibration curves were linear over the range of 200–6150 ng/mL, with a regression coefficient (R 2) of 0.9998. The limit of detection (LOD) and limit of quantification (LOQ) were 65 ng/mL and 200 ng/mL, respectively. The method was accurate and precise and suitable for the intended purpose. Analysis results were compared with the results obtained by using a validated chiral CE method and found to be in very good agreement. This method can be successfully applied to the enantiomeric purity analysis of Frovatriptan in pharmaceutical bulk drug samples and formulations.
Keywords: Frovatriptan; Enantiomeric separation; HPLC; Amylose based stationary phase and validation;

Exploiting heparin-binding properties of MoMLV-based retroviral vectors for affinity chromatography by María de las Mercedes Segura; Amine Kamen; Marie-Claude Lavoie; Alain Garnier (124-131).
Chromatography is deemed the most promising technology for large-scale purification of viral vectors. The authors have previously shown that heparin affinity chromatography could be successfully employed for the purification of VSV-G pseudotyped Moloney murine leukemia virus (MoMLV)-derived vectors giving excellent results in terms of recovery of active particles, reproducibility and selectivity. In this study, the authors examined whether the ability of retrovirus particles to specifically bind to heparin ligands is restricted to VSV-G pseudotypes produced by 293-based packaging cells. It is shown that VSV-G deficient retrovirus particles are captured by a heparin chromatography column as efficiently as VSV-G containing particles. Most strikingly, RD114 pseudotyped retrovirus particles derived from a HT1080-based cell line were found to bind heparin with the same affinity as 293-derived VSV-G pseudotypes. RD114 pseudotyped retrovirus particles were successfully isolated using heparin affinity chromatography obtaining good recoveries of functional particles (43%). These results indicate that heparin affinity chromatography can be extended to the purification of retroviral vectors produced by different packaging cell lines independently of the Env-protein used for pseudotyping.
Keywords: Retrovirus; Gene therapy vectors; Purification; Heparin affinity chromatography; Pseudotypes;

We describe a sensitive determination of aspirin (ASA) and its three metabolites (salicylic acid [SA], 2,3-dihydroxybenzoic acid [2,3-DHBA], and 2,5-dihydroxybenzoic acid [gentisic acid (GA)]) in rat plasma. Analysis was carried out by on-line solid-phase extraction (SPE) using a methylcellulose-immobilized-strong anion-exchanger (MC-SAX), followed by liquid chromatography (LC) coupled with UV detection. The lower limits of quantitation for ASA and SA were 60 ng/mL in 100 μL of plasma, respectively. This method was validated with respect to intra- and inter-day precision, accuracy, and linearity up to concentrations of 20,000 ng/mL for ASA, SA, 2,3-DHBA and gentisic acid, respectively. The method was successfully applied to an analysis of the pharmacokinetics of ASA and SA in rats.
Keywords: Restricted-access media; Plasma; Anion-exchanger; Solid-phase extraction; Aspirin; Pharmacokinetics;

To standardize and control herbal medicines, a feasible approach and control system is necessary. In this paper, a high-performance liquid chromatography with a coulometric electrode array detector (HPLC-CEAD) system was applied to fingerprint Salvia miltiorrhiza Bunge (S. miltiorrhiza Bunge), a popular herbal medicine, for the first time. pH of mobile phase, working potentials and sample preparation were included in our research. Twenty-five common peaks were obtained from extracts of S. miltiorrhiza Bunge (Shandong province), more than that obtained in previous report. Fingerprints of S. miltiorrhiza Bunge from different locations were also studied. The content of main components varied in different samples. Overlapping ratio of peaks (ORP) in 10 batches of S. miltiorrhiza Bunge (Shandong province) was not less than 72.46%. In method validation, relative standard deviation (RSD) of relative retention times and relative peak areas were of not more than 3%. It was concluded that HPLC-CEAD system can be applied in fingerprinting herbal medicines.
Keywords: HPLC; Coulometric electrode array; Fingerprint; Salvia miltiorrhiza Bunge;

Development of an at-line method for the identification of angiotensin-I inhibiting peptides in protein hydrolysates by Chris J. van Platerink; Hans-Gerd M. Janssen; Johan Haverkamp (147-154).
A fast at-line method was developed for the identification of ACE inhibiting (ACEI) peptides in protein hydrolysates. The method consists of activity measurements of fractions collected from a two-dimensional HPLC fractionation of the peptide mixture followed by MS identification of the peptides in the inhibiting fractions. The inhibition assay is based on the inhibiting effect of ACEI peptides on the hydrolytic scission of the substrate Hippuric acid-His-Leu (HHL) during the ACE-catalysed hydrolysis reaction. A fast LC method was developed for the quantification of Hippuric acid (H) and Hippuric acid-Histidine-Leucine (HHL), allowing a large number of fractions to be analysed within a reasonable time period. The method is sensitive and uses only standard laboratory equipment. The limit of detection is 0.34 μM for the known ACEI peptide IPP. This is sufficiently sensitive for the identification of only moderately active peptides and/or ACEI peptides present at low concentrations. The relative standard deviation of the inhibition assay was 12% measured over a time period of 2 months. The IC50 value of IPP measured with the assay was 5.6 μM, which is comparable to the values of 5 μM and 5.15 μM reported in literature for the standard Matsui method. The assay was successfully applied in the identification of ACEI peptides in enzymatically hydrolysed caseinate samples. Two new, not earlier published ACEI peptides were identified; MAP (β-casein f102-104) and ITP (α-s2-casein f119-121) with IC50 values of 3.8 μM and 50 μM, respectively.
Keywords: ACE; Peptides; Assay; IPP; HPLC; MS; HHL; Hydrolysed caseinate;

Quantification of fluorotelomer-based chemicals in mammalian matrices by monitoring perfluoroalkyl chain fragments with GC/MS by W. Matthew Henderson; Eric J. Weber; Stephen E. Duirk; John W. Washington; Mary Alice Smith (155-161).
Perfluorocarboxylic acids (PFCAs), namely perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA), have been identified as persistent, bioaccumulative and potentially toxic compounds. The structural analog, 8-2 fluorotelomer alcohol (8-2 fTOH) is considered the probable precursor of these stable metabolites. Because simultaneous quantification is needed for volatile and non-volatile perfluorinated chemicals (PFCs) in complex matrices, a GC/MS method was developed and tested based on selected ion monitoring of perfluorinated alkyl parent chain fragment ions. Although the method requires a derivatization step, combined GC/MS analysis of PFCA-me's and FTOHs increases analytical efficiency and decreases sample analysis time. The method instrument detection limits are between 7.1 and 24.5 ng/mL extract (MTBE), and the method quantification limits are below 50 ng/mL serum or ng/g liver for all PFCs investigated. Recoveries from mouse serum and liver homogenates, which were spiked with FTOHs and PFCAs at levels of 25 and 200 ng/mL or ng/g, ranged from 81 to 101%. Finally, the utility of the method was demonstrated by dosing male CD-1 mice with 30 mg/kg-BW of 8-2 fTOH and quantifying PFCs 6 h post-treatment. The advantages of this method are (1) the simultaneous detection of both volatile and non-volatile fluorotelomer-based chemicals in complex matrices, such as mammalian tissues, (2) as a confirmatory method to LC–MS/MS, and (3) as an alternative method of analysis for laboratories without access to LC–MS/MS.
Keywords: Perfluorinated chemicals; Fluorotelomer alcohols; PFOA; Method validation; Biological tissue analysis; GC/MS;

Development and validation of a liquid chromatography–tandem mass spectrometry for the determination of BPR0L075, a novel antimicrotuble agent, in rat plasma: Application to a pharmacokinetic study by Yi-Wei Chang; Wei-Cheng Chen; Ke-Ta Lin; Ling Chang; Hsien-Tsung Yao; Hsing-Pang Hsieh; Shih-Jung Lan; Chiung-Tong Chen; Yu-Sheng Chao; Teng-Kuang Yeh (162-168).
A rapid and sensitive liquid chromatography–tandem mass spectrometric method (LC–MS/MS) had been developed and validated to determine the concentrations of BPR0L075 in rat plasma. After a simple protein precipitation of plasma samples by acetonitrile, BPR0L075 was analyzed on a C8 column at a flow rate of 0.5 mL/min. The mobile phase consisted of a mixture of 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile (20:80, v/v). Both BPR0L075 (analyte) and the internal standard (BPR0L092) were determined using electro-spray ionization and the MS data acquisition was via multiple reactions monitoring (MRM) in positive scanning model. The MS/MS ion transitions monitored are m/z 342.2/195.2 and 312.5/165.2 for BPR0L075 and BPR0L092, respectively. The low limit of quantitation was 0.5 ng/mL. Each plasma sample was chromatographed within 5 min. The method was validated with respect to linearity, accuracy, precision, recovery, and stability. A good linear relationship was observed over the concentration range of 0.5–1000 ng/mL (r  > 0.9994). Absolute recoveries ranged from 63.45 to 68.34% in plasma at the concentrations of 2, 40, 400, and 800 ng/mL. The intra- and inter-day accuracy ranged from 92.04 to 111.80%. Intra- and inter-day relative standard deviations were 1.08–3.29% and 1.96–5.46%, respectively. This developed and validated assay method had been successfully applied to a pharmacokinetic study after intravenous injection of BPR0L075 in rats at a dose of 5 mg/kg.
Keywords: BPR0L075; LC–MS/MS; Bioanalysis; Validation; Pharmacokinetics;

A rapid, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric method (HPLC–MS–MS) has been developed and validated for the determination of soyasaponins Ba and Bb in human serum using glycyrrhizin as internal standard (I.S.). Soyasaponins Ba and Bb were extracted from human serum by liquid–liquid extraction and cleaned up by C18 solid-phase extraction (SPE), followed by separation on a C18 reversed-phase column using acetonitrile/water containing 0.025% acetic acid as a mobile phase for gradient elution. Soyasaponins Ba and Bb, and I.S. were ionized by negative ion pneumatically assisted electrospray and detected by HPLC–MS–MS in the multiple-reaction monitoring (MRM) mode using precursor → product ion combinations at m/z 958 → 940, 942 → 924 and 822 → 351, respectively. The calibration curves were linear (r 2  > 0.991) in the concentration range of 0.5–100.0 ng/mL, with lower limits of quantification of 0.5 and 0.3 ng/mL for soyasaponins Ba and Bb, respectively, in human serum. Intra-day and inter-day relative standard deviations (R.S.D.) were less than 7.9 and 11.3%, respectively. The mean recoveries of soyasaponins Ba and Bb ranged from 92 to 101% and from 85 to 94%, respectively.
Keywords: Soyasaponin; Human serum; HPLC–MS–MS; MRM; ESI-MS;

On-line sample extraction and enrichment of non-steroidal anti-inflammatory drugs by pre-column in capillary liquid chromatography mass spectrometry by Koichi Suenami; Lee Wah Lim; Toyohide Takeuchi; Yasuhide Sasajima; Kiyohito Sato; Yuji Takekoshi; Susumu Kanno (176-183).
A rapid and sensitive analytical method has been developed for the simultaneous determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma by capillary liquid chromatography (LC) and quadrupole mass spectrometry with electrospray ionization operated in the negative ion mode. The sample clean-up and enrichment on a pre-column were accomplished on-line to improve the sensitivity. This method greatly reduced sample preparation time and sample volume compared with off-line sample extraction methods and conventional LC methods, respectively. The recoveries of NSAIDs from human plasma were 56.7–96.9%. The total analytical time for a single analytical run was approximately 15 min. The detection limits of NSAIDs were 0.001–0.075 μg ml−1 using a selected ion monitoring mode.
Keywords: Non-steroidal anti-inflammatory drugs; Capillary column; Liquid chromatography-mass spectrometry; On-line enrichment;

Bacterially expressed and refolded envelope protein (domain III) of dengue virus type-4 binds heparan sulfate by Priyabrata Pattnaik; J. Pradeep Babu; Shailendra Kumar Verma; Vijay Tak; P.V. Lakshmana Rao (184-194).
An arboviral infection like dengue fever/dengue hemorrhagic fever (DHF) with high morbidity and mortality rate are extensively prevalent in several parts of the world. Global efforts have been directed towards development of vaccine for prevention of dengue. However, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. Here we report molecular interaction of domain III of envelope protein of dengue virus type-4 with heparan sulfate. A codon optimized synthetic gene encoding domain III of dengue virus type-4 envelope protein was expressed in Escherichia coli and purified under denaturing conditions, refolded and purified to homogeneity. Refolded Den4-DIII was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified protein was recognized in Western analyses by monoclonal antibody specific for the 6× His tag as well as the H241 monoclonal antibody. The in vitro refolded recombinant protein preparation was biologically functional and found to bind cell free heparan sulfate. This is the first report providing molecular evidence on binding of dengue-4 envelope protein to heparan sulfate. We developed a homology model of dengue-4 envelope protein (domain III) and mapped the possible amino acid residues critical for binding to heparan sulfate. Domain III envelope protein of dengue virus is a lead vaccine candidate. Our findings further the understanding on biology of dengue virus and will help in development of bioassay for the proposed vaccine candidate.
Keywords: Dengue virus; Envelope protein; Domain III; Expression; Purification; Refolding; Heparan sulfate;

A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the quantification of dehydroepiandrosterone (DHEA) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC–MS–MS. The derivatization with HMP was very effective for increasing the detectability of DHEA in the positive-ESI-MS. Quantification was based on the selected reaction monitoring and androsterone was used as an internal standard. This method allowed the reproducible and accurate quantification of the salivary DHEA using a 200-μl sample and the limit of quantitation for DHEA was 25 pg/ml. No significant matrix effect or change in the measured value by freeze/thaw repetition was observed. The developed method was applied to clinical studies, and produced satisfactory results.
Keywords: Dehydroepiandrosterone; Saliva; Liquid chromatography–electrospray ionization-tandem mass spectrometry; Derivatization; Clinical study;

A method based on hollow fiber supported liquid membrane extraction coupled with a gas chromatograph equipped with flame ionization detector (GC-FID) was developed for the determination of six short-chain fatty acids including acetic acid, propionic acid, i-butyric acid, n-butyric acid, i-valeric acid and n-valeric acid in serum. Hollow fiber supported liquid membrane extraction was employed for preconcentration and clean-up of the samples. The fatty acids were extracted from the acidic donor (diluted serum) into a liquid membrane formed in the wall of the hollow fiber with 10% tri-n-octylphoshphine oxide (TOPO) in di-n-hexyl ether, and then extracted back into a basic acceptor solution filled in the lumen of the hollow fiber. After being acidified with HCl, the acceptor was directly analyzed by GC-FID. The acceptor concentration, donor pH, membrane liquid and extracting time were optimized giving an enrichment factor up to 155 times. The good linearity (r 2  > 0.980), reasonable recovery (87.2–121%), and satisfactory intra-assay (8.2–11.5%) and inter-assay (6.1–11.6%) precision illustrated the good performance of the present method. Limits of detection (LOD) ranged from 0.04 to 0.24 μM and limits of quantification (LOQ) varied from 0.13 to 0.80 μM.
Keywords: Hollow fiber supported liquid membrane extraction; Short-chain fatty acid (SCFA); Serum; Gas chromatography; Determination;

A liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) assay for the determination of bencycloquidium bromide (BCQB) in rat plasma was firstly developed and validated. After addition of 1-ethyl-bencycloquidium bromide as an internal standard (I.S.), the plasma samples were deproteinized with methanol and the supernatant was assayed by LC–ESI-MS. Chromatographic separation was achieved with a Hanbon Lichrospher 5-C18 column. The mobile phase consisted of methanol–40 mM ammonium acetate buffer–formic acid (75:25:0.25, v/v/v) and delivered at the flow rate of 1.0 ml/min. LC–ESI-MS was carried out on a single quadrupole mass spectrometer using electrospray ionization (ESI) and positive selected-ion monitoring (SIM). Target ions were monitored at [M]+ m/z 330.2 for BCQB and [M] + m/z 344.2 for I.S. Calibration curve was linear over the range of 3–1500 ng/ml. The lower limit of quantification (LLOQ) was 3.0 ng/ml. The intra- and inter-run relative standard deviations (R.S.D.%) of the assay were less than 7.1 and 12.3%, respectively. The accuracy determined at the concentrations of 3.0, 100.0, 500.0 and 1500 ng/ml for BCQB were within ±15.0%. The established method has been applied successfully to study the pharmacokinetics of BCQB in rats after intranasal administration.
Keywords: Bencycloquidium bromide; LC–MS; Pharmacokinetics;

A simple, rapid, precise and accurate isocratic reversed-phase stability-indicating HPLC method was developed and validated for the simultaneous determination of atorvastatin (AT) and amlodipine (AM) in commercial tablets. The method has shown adequate separation for AM, AT from their associated main impurities and their degradation products. Separation was achieved on a Perfectsil® Target ODS-3, 5 μm, 250 mm × 4.6 mm i.d. column using a mobile phase consisting of acetonitrile–0.025 M NaH2PO4 buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The linearity of the proposed method was investigated in the range of 2–30 μg/ml (r  = 0.9994) for AT and 1–20 μg/ml (r  = 0.9993) for AM. The limits of detection were 0.65 μg/ml and 0.35 μg/ml for AT and AM, respectively. The limits of quantitation were 2 μg/ml and 1 μg/ml for AT and AM, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of AT and AM and the assay can thus be considered stability-indicating.
Keywords: Atorvastatin; Amlodipine; Stability-indicating; Simultaneous HPLC-UV;

A simple and fast method intended for large-scale bioequivalence studies for the determination of glibenclamide in plasma samples is presented. The chromatographic separation was achieved on a monolithic octadecyl chemically modified silicagel column and a mobile phase containing 42% aqueous 0.1% HCOOH solution (v/v) and 58% acetonitrile, at a flow rate of 1 mL/min, in isocratic conditions. Preparation of plasma samples was based on protein precipitation with acetonitrile. Gliquidone was used as internal standard. The target analytes were transferred into an ion trap mass analyzer via an atmospheric pressure chemical ionization interface. The precursor ions with mass 494 a.m.u. for glibenclamide and 528 a.m.u. for gliquidone were isolated, while in the second MS stage product ions 369 a.m.u. and 403 a.m.u., respectively, were monitored. The analytical process was characterized by a low limit of quantitation of 1.5 ng/mL. The mean recovery for glibenclamide was 98.1 ± 2.8% over a concentration interval ranging from 1 to 500 ng/mL. Intra-day and inter-day precision calculated over 2–400 ng/mL concentration interval ranged from 15.4% to 3.4%. Inter-sequence accuracy expressed as % bias from theoretical concentration values over the concentration interval of 10–400 ng/mL fall within −13.9% and +14.6%. The method was applied for evaluation of the bioequivalence between two formulations containing 3.5 mg glibenclamide per dose.
Keywords: Glibenclamide; Bioequivalence; Human plasma; HPLC; APCI; MS/MS; Method development; Method validation; Pharmacokinetic parameters;

Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes by Chi-Chung Chou; Show-Ping Lin; Kuo-Ming Lee; Cheng-Teng Hsu; Thomas W. Vickroy; Jyh-Myng Zen (230-239).
A simple, rapid and reliable method based on high-performance liquid chromatography with electrochemical detection was developed to routinely differentiate among meat products from fifteen food animal species. Samples from cattle, pigs, goats, deer, horses, chickens, ducks, ostriches, salmon, cod, shrimp, crabs, scallops, bullfrogs and alligators each exhibited unique electrochemical profiles. Species-specific markers exhibited reproducible peak retention times with coefficients of variation less then 6% across different runs, body regions and subjects. The method requires no derivatization or extraction steps and may be applicable to fresh or cooked meats. Incubation of fresh beef, pork or chicken at room temperature for 24 h or repeated freezing and thawing changed the intensity but not the pattern of species-specific peaks. In conclusion, this method appears suitable for rapid differentiation of meats from various food animal species and demonstrates the utility of electrochemical detection to supplement existing immunochemical and molecular biological methods. The possibility of using this method to detect adulteration and degradative changes of meat proteins is discussed.
Keywords: Meat differentiation; Protein degradation; HPLC-EC; Copper nanoparticle electrode; Adulteration;

Acetic acid improves the sensitivity of theophylline analysis by gas chromatography–mass spectrometry by Kanju Saka; Koichi Uemura; Kaori Shintani-Ishida; Ken-ichi Yoshida (240-244).
In the analysis of theophylline by gas chromatography–mass spectrometry (GC–MS), we found that the addition of acetic acid to the solvent (ethyl acetate) decreased the adsorption of theophylline to the glass wool packed into the inlet liner. The addition of acetic acid to ethyl acetate improved the sensitivity for theophylline (optimum concentration of 3%). This simple and sensitive method without derivatization can be applied to the quantification of theophylline in serum samples in clinical and toxicological practice.
Keywords: Theophylline; Acetic acid; Gas chromatography–mass spectrometry;

Analysis of creatinine in mouse and rat serum by ion exchange high performance liquid chromatography for in vivo studies of renal function by Kenneth J. Fountain; Alla Kloss; Ilya Garibyan; Bella Blitshteyn; Alexander Brezzani; Sirkka Kyostio-Moore; Anna Zuk; Robert Sacchiero; Aharon S. Cohen (245-251).
An ion exchange high performance liquid chromatography method was developed for determining creatinine levels in both mouse and rat serum samples. Separation of creatinine from other serum components was achieved in 10 min using a 100 × 4.1-mm, 10 μm strong cation exchange column following acetonitrile precipitation of serum proteins. Incorporation of a guard cartridge placed in-line prior to the analytical column was employed to prevent interference from compounds used in renal disease animal trials. Creatinine levels in normal and diseased animals were accurately determined in the 0.01–10 mg/dL range, and average recovery of the method was approximately 85% for both mouse and rat serum. Addition of 0.5–1.0% acetic acid to the acetonitrile used for protein precipitation significantly improved creatinine recovery to above 97% in mouse serum. The method was used for routine preclinical diagnosis of rat and mouse model renal function, and for the evaluation of renal disease treatment efficacy.
Keywords: Creatinine; Renal disease; Acute renal failure; Cisplatin; Ion exchange; High performance liquid chromatography; Mouse serum; Cation exchange;

An analytical method comprised of automated solid-phase extraction and determination using gas chromatography mass spectrometry (single quadrupole) has been developed for the determination of 12 polybrominated diphenyl ethers (PBDEs), 26 polychlorinated biphenyls (PCBs), two organochlorine compounds (OCs) (hexachlorobenzene and octachlorostyrene) and two brominated phenols (pentabromophenol, and tetrabromobisphenol-A (TBBP-A)). The analytes were extracted using a sorbent of polystyrene-divinylbenzene and an additional clean-up was performed on a sulphuric acid–silica column to remove lipids. The method has been validated by spiking horse serum at five levels. The mean accuracy given as recovery relative to internal standards was 95%, 99%, 93% and 109% for the PBDEs PCBs, OCs and brominated phenols, respectively. The mean repeatability given as RSDs was respectively 6.9%, 8.7%, 7.5% and 15%. Estimated limits of detection (S/N = 3) were in the range 0.2–1.8 pg/g serum for the PBDEs and phenols, and from 0.1 pg/g to 56 pg/g serum for the PCBs and OCs. The validated method has been used to investigate the levels of PBDEs and PCBs in 21 pooled serum samples from the general Norwegian population. In serum from men (age 40–50 years) the sum of seven PBDE congeners (IUPAC No. 28, 47, 99, 100, 153, 154 and 183) increased from 1977 (0.5 ng/g lipids) to 1998 (4.8 ng/g lipids). From 1999 to 2003 the concentration of PBDEs seems to have stabilised. On the other hand, the sum of five PCBs (IUPAC No. 101, 118, 138, 153 and 180) in these samples decreased steadily from 1977 (666 ng/g lipids) to 2003 (176 ng/g lipids). Tetrabromobisphenol-A and BDE-209 were detected in almost all samples, but no similar temporal trends to that seen for the PBDEs were observed for these compounds, which might be due to the short half-lives of these brominated flame retardants (FR) in humans.
Keywords: PBDEs; PCBs; Automated solid-phase extraction; Method development; Validation; Human serum levels;

The method development and validation characteristics are described of a simple gas chromatographic–mass spectrometric (GC–MS) analytical procedure to determine residual fentanyl in used Durogesic® reservoir patches and Durogesic® D-Trans® matrix technology based systems to estimate the actual rate of transdermal fentanyl delivered in individual patients. The sample preparation protocol constituting a saline based extraction of sets of new patches of each nominal dose available, resulted in fentanyl extraction recoveries to increase steadily as a function of increasing extraction time. For the reservoir type transdermal therapeutic system (TTS), fentanyl extraction efficiencies at equilibrium (16 h) ranged from approximately 60% (100-μg/h TTS) to 95% (25-μg/h TTS), whereas for the matrix type system considerable lower recoveries were demonstrated for the highest nominal dose rates (35%–52%), while reaching 90% for the 25-μg/h system. For the latter type of fentanyl TTS, an optimized methanol based extraction protocol yielded virtually quantitative fentanyl recoveries for each matrix patch nominal dose level at substantially shorter extraction periods (15 min). The GC–MS analytical method using selected ion monitoring (SIM) and deuterated fentanyl as internal standard was shown to be adequately selective with regard to the presence of other compounds in the Durogesic® patches. It was further demonstrated that the developed analytical protocols provided highly reproducible and accurate estimates of the initial fentanyl content of each patch type at all available nominal doses, with coefficients of variation and relative errors generally below 10%. These advantageous assay validation characteristics can be further transposed to the application of residual fentanyl level estimates in used patches, provided that with each batch of samples also a set of new TTSs with equal dose is assayed to perfectly mimic extraction phenomena. Finally, the presented GC–MS analytical protocol was successfully applied for the determination of residual fentanyl in a subset of 57 reservoir type patches obtained from four palliative patients.
Keywords: Fentanyl; Transdermal patch; Reservoir type; Matrix type; Method validation; GC–MS;

Simultaneous determination of the antipsychotic drugs levomepromazine and clozapine and their main metabolites in human plasma by a HPLC-UV method with solid-phase extraction by Laura Mercolini; Francesca Bugamelli; Ernst Kenndler; Giancarlo Boncompagni; Livia Franchini; Maria Augusta Raggi (273-280).
A HPLC method with UV detection has been developed for the simultaneous determination of levomepromazine, clozapine and their main metabolites: N-desmethyl-levomepromazine, levomepromazine sulphoxide, O-desmethyl-levomepromazine, N-desmethylclozapine and clozapine N-oxide. The analytes were separated on a C8 reversed-phase column using a mobile phase composed of acetonitrile and a pH 2.0, 34 mM phosphate buffer containing 0.3% triethylamine (29:71, v/v). Loxapine was used as the internal standard. A reliable biological sample pre-treatment procedure by means of solid-phase extraction on C1 cartridges was implemented, which allows to obtain good extraction yields (>91%) for all analytes and appropriate sample purification from endogenous interference. The method was validated in terms of extraction yield, precision and accuracy. These assays gave RSD% values for precision always lower than 4.9% and mean accuracy values higher than 92%. The method is suitable for the therapeutic drug monitoring (TDM) of patients undergoing polypharmacy with levomepromazine and clozapine.
Keywords: Levomepromazine; Clozapine; Metabolites; Human plasma; Liquid chromatography; Solid-phase extraction;

A fast, convenient capillary electrophoresis (CE) method was developed for monitoring the enzymatic reaction of herpes simplex virus type 1 thymidine kinase (HSV-1 TK). The reaction was performed in a test tube followed by quantitative analysis of the products. The optimized CE conditions were as follows: polyacrylamide-coated capillary (20 cm effective length × 50 μm), electrokinetic injection for 30 s, 50 mM phosphate buffer at pH 6.5, constant current of −60 μA, UV detection at 210 nm, UMP or cAMP were used as internal standards. Phosphorylated products eluted within less than 7 min. The limits of detection were 0.36 μM for dTMP and 0.86 μM for GMP. The method was used to study enzyme kinetics, and to investigate alternative substrates and inhibitors.
Keywords: Capillary electrophoresis; Herpes simplex virus type 1; K cat value; Michaelis-Menten analysis; Thymidine kinase assay;

Polymethoxyflavones (PMFs) from citrus genus are of particular interest because of their broad spectrum of biological activities, such as anti-inflammatory, anti-carcinogenic, and anti-atherogenic properties. Recently, the exploration into the beneficial health properties of PMFs in citrus fruits has dramatically increased. However, the supply of pure PMFs in the in vivo study is a limiting factor due to the difficulties in large-scale isolation of the interested PMFs. Therefore, the development of an efficient and a scalable separation method of PMFs is necessary and significant. In this paper, we report a newly developed method for efficient and relatively large-scale isolation of four PMFs from sweet orange (Citrus sinensis) peel by employing supercritical chromatography (SFC): nobiletin, tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone and 5,6,7,4′-tetramethoxyflavone.
Keywords: Sweet orange peel (Citrus sinensis); Polymethoxyflavones; Isolation and characterization; Supercritical fluid chromatography;

A simple procedure for the determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum using headspace solid-phase microextraction (HS-SPME) was developed. The analysis was carried out by gas chromatography (GC) equipped with electron capture detector (ECD). A 27–4 Plackett–Burman reduced factorial design for screening and a central composite design for optimizing the significant variables were applied. A 100 μm PDMS fiber, 3/5 headspace ratio (3 ml in 5 ml vial), 85 °C extraction temperature, 50 min extraction time, and 1 ml of acidic solution (pH 3) added to 1 ml of diluted serum (1:1) were chosen for the best response in HS extraction mode. The detection limits found were from 1 pg/ml (PCB 167) to 52 pg/ml (β-HCH), the relative standard deviation for the procedure varied from 3% (PCB 52) to 12% (PCB 189) and the accuracy was checked by using validated solid-phase extraction (SPE) procedure. The method that avoids the use of clean-up steps and the hazardous solvents enabled reliable determinations of the OCPs and the PCBs except β-HCH. The method was applied to the analysis of 33 human serum samples. The most abundant target compound was p-p′-DDE (range, 0.3–8.0 ng/ml; median value, 2.1 ng/ml). Among the PCBs the prevalent congeners were 138, 153 and 180.
Keywords: Organochlorine pesticides (OCPs); Polychlorinated biphenyls (PCBs); Headspace solid-phase microextraction; GC-ECD; Plackett–Burman design; Central composite design; Human serum;

A simple, rapid, specific, precise, accurate and sensitive method for determination of WCK 771 in human serum has been developed. The method uses high performance liquid chromatography with tandem mass spectrometric detection. Sample preparation involves protein precipitation method by addition of acetonitrile. Gatifloxacin was used as internal standard. The response was found to be linear from 0.312 to 40 μg/ml of serum with correlation coefficient greater than 0.99. Limit of detection and lower limit of quantification for WCK 771 was found to be 0.078 μg/ml and 0.312 μg/ml, respectively. The intra-day precision and accuracy from analysis of quality control (QC) samples at four concentrations was in the range of 2.36–2.58% and from 96.71 to 103.2%, respectively. The inter-day precision and accuracy from analysis of quality control samples at four concentrations was in the range of 3.14–6.82% and from 96.84 to 105.76%, respectively. WCK 771 was found to be stable for 24 h at auto-injector environment. WCK 771 was also found to be stable for 2 h in serum at 25 ± 3 °C and for 3 months at −20 °C. Mean absolute recovery at four different concentrations was 86.92% with standard deviation of 1.79. Throughput of the method is approximately one sample every 4 min. The method was also reproduced with monkey serum. The method was employed for estimation of drug serum levels during pre-clinical and clinical trials.
Keywords: WCK 771; Liquid chromatography–tandem mass spectrometry; Human serum;

A reversed-phase HPLC-UV method, involving simple instrumental setup and mobile phase without ion-pairing reagent, was developed and validated for direct simultaneous quantification of free mycophenolic acid (MPA) and its major metabolite MPA-glucuronide (MPAG) in human plasma. Both free MPA and MPAG were isolated from plasma samples using ultrafiltration prior to analysis. Each chromatographic run was completed within 13 min. The optimized method showed good performance in terms of specificity, linearity (r 2  = 0.9999), sensitivity (limit of quantitation (LOQ): 0.005 mg/L for MPA; 1 mg/L for MPAG), and intra- and inter-day precision (R.S.D. < 7%). This assay was successfully applied to free MPA and MPAG measurements in clinical samples.
Keywords: Mycophenolic acid; Mycophenolic acid glucuronide; Reversed-phase HPLC; Free drug; Human plasma;

Development of a gradient reversed-phase HPLC method for the determination of sodium ferulate in beagle dog plasma by Feng-Qian Li; Shu Xu; Hua Su; Jia-Xin Deng; Ji-Yong Liu; Jin-Hong Hu (319-322).
A gradient reversed-phase HPLC assay has been developed to determine sodium ferulate (SF) in beagle dog plasma with tinidazole as an internal standard. Chromatographic separation was made on a C18 column using 0.5% acetic acid and acetonitrile (80:20, v/v) as mobile phase. UV detection was performed at 320 nm. The calibration curve for SF was linear in the range of 0.05–10 μg/ml, and the achieved limit of quantification (LOQ) was 51.4 ng/ml. The results of linearity, within- and between-day precision, and accuracy demonstrate that this method is reliable, sensitive and sufficient for in vivo beagle dog pharmacokinetic (PK) studies of SF.
Keywords: Sodium ferulate; HPLC;

A novel, selective and sensitive high performance liquid chromatography–mass spectrometric (HPLC–MS) method has been developed for the determination of isosorbide 5-mononitrate (5-ISMN) in human plasma. With acetaminophen as internal standard, sample pretreatment involved one-step extraction with diethyl ether of 0.5 mL plasma. Analysis was performed on an ACQUITY UPLC™ BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with mobile phase consisting of acetonitrile–water (20:80, v/v). The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode with selected ion recording (SIR). Standard curves were linear (r 2  ≥ 0.99) over the concentration range of 1.04–1040 ng/mL. The lower limit of quantification (LLOQ) was 1.04 ng/mL. The intra- and inter-day precisions (RSDs) were less than 8.6% and 13.4%, respectively, and the accuracy (RE) was within ±0.45%. The method herein described was fully validated and successfully applied to the pharmacokinetic study of 5-ISMN in compound extended-release tablets in 18 healthy male volunteers after oral administration.
Keywords: 5-ISMN; HPLC–ESI–MS; Human plasma; Pharmacokinetics;

QRAR models for cardiovascular system drugs using biopartitioning micellar chromatography by Sumin Wang; Gengliang Yang; Hua Zhang; Haiyan Liu; Zhiwei Li (329-333).
The capability of biopartitioning micellar chromatography (BMC) to describe and estimate pharmacological parameters of cardiovascular system drugs has been studied. The retention of cardiovascular system drugs was studied using different pH of Brij-35 as micellar mobile phase in modified C18 stationary phase. Quantitative retention–activity relationships (QRAR) in BMC were investigated for these compounds. An adequate correlation between the retention factors (log k) and the toxicity (LD50) of cardiovascular system drugs was obtained.
Keywords: Biopartitioning micellar chromatography (BMC); Cardiovascular system drugs; QRAR model;

Salting-out thin-layer chromatography of several chosen sulphonamides on silica gel has been examined with aqueous solutions of salts: sulphates, chlorides, nitrates, phosphates, acetates, thiocyanates. It was established that applied salts have different effects on retention of sulphonamides accordingly to Hofmeister's clasification (e.g. kosmotropes, chaotropes and neutral).The parameters of the linear regression analysis of dependences between the R M values and concentration of the salt in the eluent system were correlated with QSAR ones. It appeared that chromatographic parameters obtained by SOTLC method reflect not only physico-chemical properties of examined compounds but also they include information about their activity. 3D graph revealing pharmacological properties of analytes was constructed. Universal character of this method for predicting and classification of drug containing sulphonamide group was confirmed by localisation of additional compounds structurally similar but acting antagonistically towards sulphonamides.
Keywords: Salting-out thin-layer chromatography; Sulphonamides; Quantitative structure–retention relationships; Cluster analysis;

Simultaneous determination of AMN107 and Imatinib (Gleevec®, Glivec®, STI571) in cultured tumour cells using an isocratic high-performance liquid chromatography procedure with UV detection by Gunther Guetens; Hans Prenen; Gert De Boeck; Allan van Oosterom; Patrick Schöffski; Martin Highley; Ernst A. de Bruijn (341-345).
A reversed phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the simultaneous determination of imatinib (Gleevec®, Glivec®, STI571) and AMN107 in cultured tumour cells, using clozapine as an internal standard. The compounds of interest were extracted by liquid–liquid extraction using TOXI-TUBES® A extraction tubes. Chromatographic separation was performed on a Phenomenex Gemini C18 reversed phase column (150 mm × 2.0 mm, 5 μm particle size), using a mixture of 65% CH3OH (methanol) and 35% NH4Ac (Ammonium acetate) buffer (20 mM, pH 10). Separation was achieved under isocratic conditions at a flow rate of 0.5 ml/min. Imatinib, clozapine and AMN107 are detected by UV detection at 260 nm. Calibration curves were linear from 50 to 7500 ng/ml with correlation coefficients (r 2) better than 0.998. The limit of quantitation (LOD) was 50 ng/ml. The method has been successfully applied to a cellular kinetics study.
Keywords: Imatinib; AMN107; HPLC; Cell culture;

A rapid, selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS–MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid–liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 μl specimen of plasma, HPLC separation was achieved on a Nova-Pak C18 column, using acetonitrile–water–formic acid–10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5 →  m/z 279.1 for ranolazine and m/z 344.3 →  m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5–4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within ±3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.
Keywords: Ranolazine; Liquid chromatography; Tandem mass spectrometry;

Sensitive quantification of atomoxetine in human plasma by HPLC with fluorescence detection using 4-(4,5-diphenyl-1H-imidazole-2-yl) benzoyl chloride derivatization by Hao-Jie Zhu; Jun-Sheng Wang; Jennifer L. Donovan; C. Lindsay DeVane; Bryan B. Gibson; John S. Markowitz (351-354).
The first HPLC-fluorescence method for the determination of atomoxetine in human plasma was developed and validated. Atomoxetine was derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) under mild conditions, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (λex: 318 nm, λem: 448 nm). A linear calibration curve was obtained over the concentration range 1–1000 ng/mL (r  = 0.999). The limit of detection (S/N = 3) was 0.3 ng/mL. The relative standard deviations of intra-day and inter-day variations were ≤8.30% and 7.47%, respectively. This method is rapid, sensitive, and suitable for both basic and clinical studies of atomoxetine.
Keywords: Atomoxetine; 4-(4,5-Diphenyl-1H-imidazol-2-yl) benzoyl chloride;

The applicability of capillary zone electrophoresis (CZE) for analysis of cephalosporin antibiotics has been studied in bronchial secretion as highly viscous, thick and non-homogeneous samples. The lyophilization was found to be a simple but effective pretreatment of these samples to bring them into a form which is suitable for injection to CE capillary. The obtained good recovery data prove that the lyophilization/dissolution of bronchial secretion samples can be reproducibly performed.
Keywords: Cephalosporins; Capillary electrophoresis; Bronchial secretion;

The ability to selectively measure serine enantiomer concentrations in rat brain microdialysate is essential during drug discovery to study the interaction of d-serine with the N-methyl-d-aspartate (NMDA) subtype of the glutamate receptor. NMDA receptor-stimulating agents, such as d-serine, have been shown to reduce the negative symptoms and cognitive dysfunction in individuals with schizophrenia when added to conventional or atypical antipsychotic drug regimens. In the work presented here, an LC/MS/MS assay was developed and validated to simultaneously measure d-serine and l-serine concentrations in rat brain microdialysate. Reverse phase chromatographic resolution of the enantiomers was obtained through derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (Marfey's reagent). The assay was validated to determine concentrations over the range of 10–7500 ng/mL using electrospray ionization and multiple reaction monitoring (MRM). Both intra- and inter-day precision and accuracy were less than 16.5% (RE) and 7% (CV) for both analytes, respectively, and assay throughput was increased significantly relative to existing methodologies.
Keywords: d-Serine; l-Serine; Marfey's reagent; LC/MS/MS; Schizophrenia;

A partial filling micellar electrokinetic capillary chromatography (PF-MEKC) method with a mixed micelle system composed of a zwitterionic surfactant named 3-(N,N-dimethylhexadecylammonium)propanesulfonate (PAPS) and a nonionic surfactant polyethylene glycol dodecyl ether (Brij 35) for peptide mapping is described. The method was demonstrated by the separation of tryptic digestion of bovine serum albumin (BSA). The optimal mixed micelle solution was 50 mM NH4OH–HCOOH buffer (pH 2.0) containing 32 mM PAPS and 0.6% (m/v) Brij 35. It was found that the mixed micelle system permitted a highly selective separation of the tryptic digestion. The high separation selectivity was probably due to the ion-pairing interaction between the zwitterionic surfactant molecules and the peptides.
Keywords: Mixed micelle; Micellar electrokinetic capillary chromatography; Peptide mapping;

An improved high-performance liquid chromatographic (HPLC) method for the separation of zwitterionic detergents is described. It is based on a reversed-phase liquid chromatography with evaporative light-scattering detection (ELSD). The method was shown to be highly specific, allowing the separation of three detergents of the alkyl sulfobetaine family: 3-(N-dodecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB12), 3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14) and 3-(N-hexadecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB16). It was further used to develop a quantitation method for SB14, which was validated for linearity, precision, robustness, limits of detection and quantitation, specificity and accuracy. Linearity was found in the range of 50–500 μg/ml with a correlation coefficient of 0.9938 ± 0.0029. The mean value of slope and intercept were 1.567 ± 0.06 and 0.1541 ± 0.0271, respectively. The limits of detection (LOD) and quantitation (LOQ) were 2 and 10 μg/ml, respectively. The validated method was used to determine the concentration of SB14 in different biological samples, specially in bulks of a recombinant membrane protein, the Klebsiella pneumoniae outer membrane protein A, which is produced at the pilot scale for human clinical studies.
Keywords: High-performance liquid chromatographic (HPLC); Evaporative light-scattering detection (ELSD); Detergent; Zwitterionic; Alkyl sulfobetaine; Surfactant;

The phenethylamine-derived designer drug 4-bromo-2,5-dimethoxy-β-phenethylamine (2C-B) is known to be extensively metabolized in various species including humans. In rat urine, 2C-B was found to be excreted mainly via its metabolites. In the current study, the toxicological detection of these metabolites in the authors’ systematic toxicological analysis (STA) procedure was examined. The STA procedure using full-scan GC–MS allowed proving an intake of a common drug abusers’ dose of 2C-B by detection of the O-demethyl deaminohydroxy and two isomers of the O-demethyl metabolites in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-B in human urine.
Keywords: 4-Bromo-2,5-dimethoxy-β-phenethylamine; 2C-B; Designer drug; Metabolism; GC–MS;

Identification of flavonols in leaves of Maytenus ilicifolia and M. aquifolium (Celastraceae) by LC/UV/MS analysis by Luciana A. Tiberti; Janete H. Yariwake; Karine Ndjoko; Kurt Hostettmann (378-384).
A comparative analysis of the flavonoid components of the leaves of two medicinal plants known in Brazil as “espinheira santa”, namely, Maytenus ilicifolia Mart. ex Reiss. and M. aquifolium Mart. (Celastraceae), and a hybrid plant, M. aquifolium  ×  M. ilicifolia, has been carried out using high-performance liquid chromatography coupled with photodiode array UV detection and mass spectrometry. One methoxyflavonoid glycoside and 18 flavonol-3-O-glycosides were identified in the extracts on the basis of their on-line UV spectra (measured in the absence and presence of shift reagents) and multiple stage mass spectral data. Fingerprint analysis of the flavonoid extracts revealed significant differences in the profiles of the two Maytenus species, while the hybrid plant contained flavonoids found in both parent species.
Keywords: LC/UV/MS; Flavonols; Phytomedicines; Maytenus aquifolium; Maytenus ilicifolia;

An improved HPLC method for the analysis of citrus limonoids in culture media by Qingguo Tian; Edward G. Miller; G.K. Jayaprakasha; Bhimanagouda S. Patil (385-390).
Recent studies have shown that citrus limonoids have potential health benefits. However, information on the absorption and metabolism of limonoids in human gastrointestinal (GI) tract is limited. In the present study we have investigated the metabolism of limonin glucoside (LG), the predominant limonoid in citrus by four microorganisms (Enterococcus fecalis, Escherichia coli, Lactobacillus salivarius, and Candida albican) widely present in the human lower GI tract. LG and metabolites in the culture medium were purified using solid phase extraction and analyzed using HPLC using UV detection at 210 nm. The identity of LG was further confirmed by electrospray ionization mass spectrometry (ESI-MS). Significant metabolic activity of Escherichia coli and Candida albican on LG was observed. Several unidentified metabolites were also found in the medium. The results of the present study indicated that LG may be metabolized in the intestine by some microbes. Further studies are needed to establish the possible route of LG metabolism in the human system.
Keywords: Lower GI tract; Limonoids; HPLC;