Journal of Chromatography B (v.877, #27)

Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS by Delphine Carli; Mylène Honorat; Sabine Cohen; Mehdi Megherbi; Bruno Vignal; Charles Dumontet; Léa Payen; Jérôme Guitton (2937-2944).
To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography–tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2′-deoxyuridine-5′-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2′-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2′-deoxyuridine-5′-monophosphate (dUMP)/thymidine-5′-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5 μm, (100 mm × 2.1 mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2 min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5–150 ng mL−1 according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at −20 °C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.
Keywords: 5-FU; Mass spectrometry; Intracellular; 5-FdUMP; Metabolite;

A rapid, simple and high sensitive method is described here for extraction of hippuric acid (HA) from human urine samples by using an automated micro solid phase extraction system (μ-SPE). However in order to increase sensitivity of gas chromatography with flame ionization detector, a post derivatization procedure was developed. In this work, a polypyrrole was synthesized by chemical oxidation of the pyrrole monomer in non-aqueous solution and applied as an excellent and efficient sorbent for μ-SPE. The calibration curves were linear in the range of 0.018–8.95 μg mL−1 for HA, in both water and urine samples with correlation coefficients 0.9973 and 0.9946, respectively; limits of detections were 12.1 ng mL−1 and 16.5 ng mL−1, respectively. This method was successfully used to analyze trace amounts of HA in human urine samples without any interference from coexisting substances.
Keywords: Gas chromatography; Derivatization; Hippuric acid; Micro solid phase extraction; Urine sample;

Assessment of sampling strategies for gas chromatography–mass spectrometry (GC–MS) based metabolomics of cyanobacteria by Leonard Krall; Jan Huege; Gareth Catchpole; Dirk Steinhauser; Lothar Willmitzer (2952-2960).
Metabolomics is the comprehensive analysis of the small molecules that compose an organism's metabolism. The main limiting step in microbial metabolomics is the requirement for fast and efficient separation of microbes from the culture medium under conditions in which metabolism is rapidly halted. In this article we compare three different sampling strategies, quenching, filtering, and centrifugation, for arresting the metabolic activities of two morphologically diverse cyanobacteria, the unicellular Synechocystis sp. PCC 6803 and the filamentous Nostoc sp. PCC 7120 for GC–MS analysis. We demonstrate that each sampling technique produces internally consistent and reproducible data, however, cold methanol–water quenching caused leakage and substantial loss of metabolites from various compound classes, while fast filtering and centrifugation produced quite similar metabolite pool sizes, even for metabolites with predicted high turnover. This indicates that cyanobacterial metabolic pools, as measured by GC–MS, do not show high turnover under standard growing conditions. As well, using stable 13C labeling we show the biological origin of some of the consistently observed unknown analytes. With the development of these techniques, we establish the basis for broad scale comparative metabolite profiling of cyanobacteria.
Keywords: Metabolite profiling; Cyanobacteria; Metabolite quenching; Fast filtering; GC–MS;

Increasing concerns on public health safety have led researchers to develop efficient methods to characterize veterinary drugs and pesticides residues simultaneously in animal products. This investigation presents a simple and rapid method to determine five types of fluoroquinolones (FQs), organophosphorus (OP) and N-methyl carbamates (NMCs) in porcine tissue simultaneously with the use of matrix solid-phase dispersion (MSPD), high performance liquid chromatography (HPLC) and diode array detection (DAD). The results show a recovery ratio of 60.1–107.7% with satisfactory relative standard deviations. The limits of detection (LOD) in porcine tissue are between 9 and 22 μg/kg. The applicability of the method to the multiresidue analysis in porcine tissue is reported in this contribution.
Keywords: Matrix solid-phase dispersion; High performance liquid chromatography; Fluoroquinolones; Organophosphorus; N-methyl carbamates; Multiresidue;

Nitrogen-containing bisphosphonates (N-BPs) are shown to inhibit a key enzyme of intracellular mevalonate pathway, FPP synthase, leading to intracellular accumulation of pathway metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In our previous studies we have shown that a new type of ATP analog, ApppI (triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester), is also formed in addition to IPP and DMAPP accumulation. ApppI has cytotoxic effects leading to direct apoptosis of various cancer cells. In this study we present a validated method based on ion-pair LC–MS2 for the analysis of isomeric mevalonate pathway metabolites and ATP analogs in cell culture samples. Limit of quantitation for IPP and DMAPP was 0.030 μM (1.35 fmol on-column) and for ApppI and ApppD 0.020 μM (0.9 fmol on-column). Acceptable accuracies and precision were also obtained for quality control samples in low and high concentrations of the calibration curve. In addition, we present a new method for quantitation of each coeluting isomer utilizing the peak intensity ratios of two characteristic fragment ions of each compound. For IPP and DMAPP, fragment ions m/z 177 and m/z 159 in the MS2 were monitored, whereas for ATP analogs, ApppI and ApppD (triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-2-enyl) ester), the same fragments in the MS3 spectra were followed. IPP and DMAPP accumulation as well as ApppI and ApppD formation was demonstrated using MCF-7 breast cancer cells. Cells were treated with 25 μM zoledronic acid (an N-BP) for 24 h, conditions found to induce significant production of the metabolites. We found that the total amount of IPP and DMAPP was 2.4 nmol/mg of protein and amount of ApppI and ApppD was 1.1 nmol/mg protein. Relative portions of the isomers were approximately 1:4 IPP:DMAPP and 3:7 ApppI:ApppD. Untreated control samples did not contain IPP, DMAPP, ApppI or ApppD.
Keywords: ATP analogs; Bisphosphonates; Mevalonate pathway; Zoledronic acid;

Many lipidomic approaches focus on investigating aspects of sphingolipid metabolism. Special emphasis is put on neutral sphingolipids and cholesterol and their interaction. Such an interest is attributed to the fact that those lipids are altered in a series of serious disorders including various sphingolipidoses. High performance thin-layer chromatography (HPTLC) has become a widely used technique for lipid analysis. However, mass spectrometric profiling is irreplaceable for gaining an overview about the various molecular species within a lipid class. In this work we have developed a sensitive method based on a gradient normal phase high performance liquid chromatography (HPLC) coupled to quadrupole time of flight (QTOF) atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in positive mode, which for the first time enables separation, on-line detection, and mass spectrometric profiling of multiple neutral sphingolipids including ceramide, glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin as well as cholesterol within less than 15 min. An important advantage of the presented HPLC/APCI-MS approach is that the separation pattern emulates the one obtained by an optimized HPTLC method with a multiple stage development. Thus, the lipid classes previously separated and quantified by HPTLC can be easily screened regarding their mass spectrometric profiles by HPLC/APCI-MS. In addition, the selected ionization conditions enable in-source fragmentation providing useful structural information. The methods (HPLC/APCI-MS and the optimized HPTLC) were applied for the analysis of the mentioned lipids in human fibroblasts. This approach is aimed basically at investigators who perform studies based on genetic modifications or treatment with pharmacological agents leading to changes in the biochemical pathways of neutral sphingolipids and cholesterol. In addition, it can be of interest for research on disorders related to impairments of sphingolipid metabolism.
Keywords: Lipidomics; Sphingolipidoses; Neutral sphingolipids; Cholesterol; HPTLC; HPLC/MS; APCI-MS; Q-TOF;

Determination of arotinoid acid in human plasma by liquid chromatography–tandem mass spectrometry by Pan Deng; Xiaoyan Chen; Yunbiao Tang; Yongqing Wang; Hongwen Zhang; Dafang Zhong (2983-2988).
Arotinoid acid (Ro 13-7410) is the third generation of synthetic retinoid, which was developed for the treatment of psoriasis and other hyperkeratotic skin disorders. The therapeutically active dose is less than 0.5 μg/kg body weight/day. To investigate the pharmacokinetics of arotinoid acid, a sensitive and selective liquid chromatographic–tandem mass spectrometric method (LC–MS/MS) for the determination of arotinoid acid in human plasma was developed and validated. The sample processing was carried out in the dark to minimize photodegradation of the analytes. Arotinoid acid and the internal standard (IS), acitretin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was achieved on a Zorbax Extend C18 column (150 mm × 4.6 mm, i.d., 5 μm) using methanol:acetonitrile:5 mM ammonium acetate (48:32:20, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. The detection was carried out in multiple reaction monitoring (MRM) mode via negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 37.1 pg/mL with intra-day and inter-day precision (RSD) of 8.7% and 8.5%, and accuracy (RE) of 2.0%. Inter-day and intra-day RSD for three quality control levels (QCs) across validation runs were less than 11.0% and the accuracy ranged from 1.9% to 3.3%. The validated LC–MS/MS method was applied to a phase I clinical pharmacokinetic study after a single oral administration of 40 μg arotinoid trometamol to healthy subjects.
Keywords: Retinoid; Ro 13-7410; Arotinoid acid; Arotinoid trometamol; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics;

Simultaneous determination of OZ277, a synthetic 1,2,4-trioxolane antimalarial, and its polar metabolites in rat plasma using hydrophilic interaction chromatography by Ravi K. Bhamidipati; Julia Morizzi; Francis C.K. Chiu; David M. Shackleford; Susan A. Charman (2989-2995).
OZ277 is a synthetic 1,2,4-trioxolane antimalarial currently being evaluated in clinical trails. Biotransformation of OZ277 in rats results in the generation of metabolites with large differences in polarity which complicates the development of a method for the simultaneous analysis of all species. A simple, sensitive and selective hydrophilic interaction liquid chromatography–mass spectroscopy (HILIC/MS) method for simultaneous determination of OZ277 and its major metabolites in rat plasma was developed and validated. The method involves protein precipitation with acetonitrile followed by separation on a Waters Atlantis™ HILIC Silica column using gradient elution. The analytes were monitored using a positive electrospray ionization interface in selected ion monitoring mode. The calibration range for all of the analytes was 5–10,000 ng/mL and the lower limit of quantitation was 5 ng/mL using a 50 μL rat plasma sample. The inter- and intra-day accuracy and precision was within 12%. The recovery of all analytes from rat plasma over a wide concentration range was 90% or greater. The method has been successfully used for quantifying OZ277 and its metabolites in plasma following intravenous administration to rats.
Keywords: OZ277; RBx11160; 1,2,4-trioxolane; Rat plasma; HILIC/MS;

HPLC and TLC characterisation of ecdysteroid alkyl ethers by Silvia Lapenna; Laurence Dinan (2996-3002).
Semi-synthetic ecdysteroid alkyl ethers have increased potential over natural ecdysteroids as actuators of ligand-inducible gene-expression systems based on the ecdysteroid receptor for in vivo applications. However, a scalable synthesis of these compounds has yet to be developed. We report a set of reversed-phase (RP; C18 and C6) and normal-phase (NP; diol) HPLC systems which can be used to analyse and separate ecdysteroid ethers with single or multiple O-methyl substitutions at the 2α-, 3β-, 14α-, 22- and 25-positions. The elution order of methyl ether analogues of the prototypical ecdysteroid 20-hydroxyecdysone (20E) was 3-methyl < 2-methyl < 14-methyl < 25-methyl < 22-methyl with both C18- and C6-RP-HPLC, when eluted with methanol/water mixtures. Further, the elution order of 20E 22-O-alkyl ethers was methyl < ethyl < allyl <  n-propyl < benzyl <  n-butyl with both C18- and C6-RP-HPLC. Moreover, the ecdysteroid alkyl ethers can also be adequately resolved by NP-HPLC and silica HPTLC. On the latter, detection of ecdysteroid O-alkyl ethers with the p-anisaldehyde/sulphuric acid reagent distinguishes 22-O-alkyl ethers from non-22-O-alkyl ether analogues by the colour of the resulting spot.
Keywords: HPLC; TLC; Ecdysteroids; Alkyl ethers; Gene switches;

A method for identifying the metabolites of sibutramine 1-(4(chlorophenyl)-N,N-dimethyl-α-(2-methylpropyl))cyclobutanemethanamine) in urine, utilizing a double derivatization strategy, with N-methyl-N-(trimethylsilyl)-trifluoroacetamide and N-methyl-bis-(trifluoroacetamide), in gas chromatography/mass spectrometry is proposed. This methodology results in mass spectra with at least three fragments in abundance superior to 20%, attending the World Anti-Doping Agency identification criteria for qualitative assays. The characterization of the derivatives was obtained through two ionization modes: Chemical Ionization and Electron Impact ionization, both in full scan mode. Sibutramine was administered to 5 (five) volunteers and the excretion profile followed for 92 h. Routine analytical, hydroxy-cyclobutane-bis-nor-sibutramine which becomes the more abundant metabolite in the first 10 h and hydroxy-isopropyl-bis-nor-sibutramine which becomes the most abundant after 40 h, were proposed for doping monitoring.
Keywords: Doping; Sibutramine; GC-MS; WADA; MSTFA/MBTFA;

A high throughput screening assay was developed to determine the total dimer level in antibody samples. This method utilizes high speed microchip electrophoresis separation following chemical cross-linking. Upon reacting with homobifunctional N-hydroxysuccinimide-esters (NHS-esters), covalent linkages can be established between the primary amines of two neighboring antibody molecules. The reaction conditions are optimized to achieve quantitative cross-linking of only physically associated monomers within an antibody dimer. The resulting cross-linked dimers, originating from either covalent or non-covalent antibody dimers, can then be separated from monomers by SDS electrophoresis. A commercial microchip electrophoresis instrument is used for high speed separation, allowing each sample to be analyzed in about 1 min. This approach was applied to crude mammalian cell culture samples. Using a 96-well gel filtration spin column format, interfering species in the cell culture media were efficiently removed from the samples. This method is well suited to the purpose of high throughput antibody dimer quantitation during cell culture expression, including clone selection and cell culture development. The total dimer content, both covalent and non-covalent, can be determined for hundreds of crude samples in a few hours. The effects of different cross-linking conditions on the determined dimer levels, as well as of different antibody pI values, are discussed.
Keywords: Cross-linking; Microchip; Monoclonal antibody; Dimer screening; Aggregation; High throughput analysis;

We report the development of an analytical method combining cell membrane chromatography (CMC) with gas chromatography/mass spectrometry (GC/MS). This was applied to the purification and identification of anti-inflammatory components from traditional Chinese medicines. The stationary phase of the CMC employed mouse peritoneal macrophage (PM) cell membranes. We investigated the performance of the PM/CMC-offline-GC/MS method using hydrocortisone (HC) and dexamethasone (DM) as standards. The method was then applied to the identification of anti-inflammatory components in extracts of Rhizoma Atractylodes macrocephala (RAM) and Rhizoma Atractylodes lancea Thunb DC (RALD). The major components from both species retained by CMC were identified as atractylenolide I (AO-I) by GC/MS. Competition experiments’ results showed that AO-I and lipopolysaccharide (LPS) bound competitively to cell surface receptors while AO-I and HC had only partly overlapping binding sites on the PM membrane. In vitro experiments revealed that AO-I was able to inhibit LPS-induction of TNF-α, IL-1β and NO production in a dose-dependent manner. IC50 values were 5.3 μg/mL, 5.1 μg/mL and 7.5 μg/mL, respectively. The PM/CMC-offline-GC/MS method is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples.
Keywords: Peritoneal macrophage; Cell membrane chromatography; Gas chromatography/mass spectrometry; Atractylenolide I; Anti-inflammatory;

Perhexiline is a drug that is used for treatment of moderate to severe angina pectoris that has not responded to other treatment. It has a low therapeutic index, and saturable metabolism that is also subject to genetic polymorphism (CYP2D6). Concentration monitoring of the parent drug and its major metabolite is considered necessary to optimise efficacy and reduce the risk of hepatotoxicity and neuropathy. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was developed for the determination of perhexiline and its metabolite cis-hydroxyperhexiline in human plasma. After proteins were precipitated with acetonitrile, perhexiline, the major metabolite cis-hydroxyperhexiline and nordoxepin as the internal standard were resolved on a phenyl-hexyl column using gradient elution of 0.05% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 10–2000 μg/L (r  > 0.999), bias was ≤±10%, intra- and inter-day coefficients of variation (imprecision) were ≤8.1%, and the limit of quantification was 10 μg/L for both perhexiline and hydroxyperhexiline. The assay is being used successfully in clinical practice to enhance the safe and effective use of perhexiline.
Keywords: Perhexiline; Hydroxyperhexiline; Plasma; LC–MS/MS;

Use of response surface methodology to evaluate the extraction of Debaryomyces hansenii xylose reductase by aqueous two-phase system by Janaína Teles de Faria; Fábio Coelho Sampaio; Attilio Converti; Flávia M. Lopes Passos; Valéria Paula Rodrigues Minim; Luis Antônio Minim (3031-3037).
Xylose reductase (XR) from Debaryomyces hansenii was extracted by partitioning in aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) 4000 in the presence of different salts, specifically sodium sulfate, lithium sulfate and potassium phosphate. Batch extractions were carried out under different conditions of temperature (25–45 °C) and tie-line length (TLL) for each system, according to a central composite design face-centered of 36 tests, and the response surface methodology was used to evaluate the results. Quadratic polynomial models were adjusted to the data to predict the behavior of four responses, namely the XR partition coefficient (K XR ), the selectivity (S), the purification factor (PF T ) and the activity yield (Y T ) in the top phase. The optimal extraction conditions were found using the PEG 4000/sodium sulfate system at 45 °C and TLL = 25.1, which ensured PF T  = 3.1 and Y T  = 131%. The ATPS proved effective for partial purification of D. hansenii xylose reductase in cell-free crude extract, and the response surface methodology revealed to be an appropriate and powerful tool to determine the best dominion of temperature and ATPS composition.
Keywords: Enzyme extraction; Xylose reductase; Partition; Aqueous two-phase system; Response surface methodology;

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of Irinotecan (CPT-11) and its metabolites in human plasma has been developed. Samples were prepared after protein precipitation and analyzed on a C18 column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.05% formic acid), using gradient procedure. The analytes and internal standard camptothecin were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 10.0–2000.0 ng/ml for CPT-11 and 0.5–200.0 ng/ml for 7-ethyl-10-hydroxycamptothecin (SN-38), respectively. The lower limit of quantification (LLOQ) was 10 ng/ml for CPT-11 and 0.5 ng/ml for SN-38. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 10.6%. The accuracy determined at three concentrations was within ±11.4% in terms of relative error. Due to the unavailability of standard for 7-ethyl-10-O-glucuronyl-camptothecin (SN-38G) and the importance of knowing the concentration of this metabolite, we developed a method for analysis SN-38G by taking advantage of the quantitive conversion of SN-38G to SN-38 using glucuronidase. This enzymatic method of identification and quantitation of gluconated compound can be widely used when the standard for phase II glucuronide metabolites are not available.
Keywords: Irinotecan; LC–MS-MS; Metabolites; Pharmacokinetics;

Validation of a fast liquid chromatography–UV method for the analysis of drugs used in combined cardiovascular therapy in human plasma by Gorka Iriarte; Oskar Gonzalez; Nerea Ferreirós; Miren Itxaso Maguregui; Rosa Maria Alonso; Rosa Maria Jiménez (3045-3053).
Ultra-performance liquid chromatography (UPLC) was investigated as a faster alternative to high-performance liquid chromatography (HPLC) for the simultaneous analysis of drugs usually prescribed in cardiovascular therapy. Upon a previously developed and validated solid phase extraction (SPE)–HPLC–photodiode array (PDA)–fluorescence (FLR) method, separation of chlorthalidone (CLTD; diuretic), valsartan and its metabolite (VAL and VAL-M1 respectively; angiotensin II receptor antagonist drugs) and fluvastatin (FLUV; statin) was performed in human plasma using an RP C18 column (50 mm × 2.1 mm, 1.7 μm, Waters Acquity UPLC™ (BEH)) and a tunable UV–vis (TUV) detector. After method transfer, different system variables were modulated to study the evolution of responses of the analytes and the endogenous interferences. The improved method was fully validated and the results were compared with its precursor HPLC method relating to analysis time, efficiency and sensitivity. The studied compounds were separated in less than 8 min and the method showed good linearity (20–3000 μg/L for chlorthalidone, 110–1100 μg/L for valsartan-M1, 67–1900 μg/L for valsartan and 48–1100 μg/L for fluvastatin), precision and accuracy. The proposed method was found to be reproducible (RSD < 10%), accurate (RE < 15%), robust and suitable for quantitative analysis of the studied drugs in plasma obtained from patients under combined cardiovascular treatment.
Keywords: Metabolic syndrome; Human plasma; Fast liquid chromatography; Bioanalytical method validation;

Rapid determination of cyanide in human plasma and urine by gas chromatography–mass spectrometry with two-step derivatization by Guojie Liu; Junting Liu; Kenji Hara; Yanfang Wang; Yanping Yu; Lina Gao; Ling Li (3054-3058).
Cyanide (CN) is a powerful poison and rapidly toxic agent. Because of its wide availability and high toxicity, quantification of CN in blood and urine is frequently required in clinical and forensic practice. We present a sensitive and less time consuming method based on solid-supported liquid–liquid extraction (SLE) and gas chromatography–mass spectrometry (GC–MS) with two-step derivatization for determination of CN in plasma and urine. Buffer solution, 1,3,5-tribromobenzene (internal standard) and benzaidehyde were added to sample to complete the first-step derivatization. Then the analytes were poured onto the column of diatomaceous earth, eluted with n-hexane containing 0.4% of heptafluorobutyryl chloride (HFB-Cl) to complete the second-step derivatization forming the final analyte, heptafluoro-butyric acid-alpha-cyanobenzyl ester. This method was linear (r 2  = 0.9988, 0.9993), reproducible (intra-day RSD = 4.37–7.24%, 3.19–5.74%; inter-day RSD = 5.13–7.63%, 4.31–6.69%), accurate (recoveries = 90.58–115.56%, 93.01–114.6%) and sensitive (LOD = 0.04, 0.01 μg/mL) for plasma and urine, respectively. The total time was about 25 min. This method was successfully applied to the analysis of blood sample and urine sample collected from a victim who died as a result of ingestion of potassium cyanide.
Keywords: Cyanide; Plasma; Urine; SLE; Two-step derivatization; GC–MS;

Amino acid analysis that is based on the use of hydrophilic interaction liquid chromatography (HILIC) coupled with isotope dilution mass spectrometry (IDMS) has been developed for the accurate quantification of underivatized amino acids from hydrolyzed protein/peptide. Sufficient separation of amino acids on a zwitterion chromatography (ZIC)-HILIC column was achieved after removal of chloride ions in the hydrolyzate. The detection limits and quantification limits as concentration of the four amino acids ranged from 0.003 to 0.04 pmol μL−1 and from 0.01 to 0.1 pmol μL−1, respectively. The analytical results for the certified reference materials, angiotensin I and bovine serum albumin (BSA), were satisfactory. Furthermore, the quantitative results by this method were compared with those by the commercially available precolumn method, derivatizd with aminoquinolylhydroxysuccinimidyl carbamate (AQC method), and better recovery and more precise data were obtained with this method.
Keywords: Amino acid analysis; Hydrophilic interaction liquid chromatography (HILIC); Isotope dilution mass spectrometry (IDMS); Protein/peptide; Accuracy;

A liquid chromatography mass spectrometric selected reaction monitoring mode (SRM) method for methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine (BE), 6-acetylmorphine, morphine and codeine quantification in human umbilical cord was developed and fully validated. Analytes were extracted from homogenized tissue (1 g) by solid phase extraction. Linearity was 2.5–500 ng/g, except for methadone (10–2000 ng/g). Method imprecision was <12.7%CV with analytical recovery 85.9–112.7%, extraction efficiency >59.2%, matrix effect 4.5–39.5%, process efficiency 48.6–92.6% and stability >84.6%. Analysis of an umbilical cord following controlled methadone administration and illicit drug use contained in ng/g, 40.3 morphine, 3.6 codeine, 442 BE, 186 methadone and 45.9 EDDP.
Keywords: Methadone; Cocaine; Opiates; Umbilical cord; LCMS; In utero;

A liquid chromatography–tandem mass spectrometry assay for simultaneous determination of the plasma concentration of 11 antiretroviral agents (nevirapine, indinavir, atazanavir, amprenavir, saquinavir, ritonavir, lopinavir, efavirenz, tipranavir, darunavir and maraviroc) has been developed. Sample pre-treatment is limited to protein precipitation with a mixture of methanol and zinc sulfate. After centrifugation the supernatant is injected in the chromatographic system, which consists of on-line solid phase extraction followed by separation on a phenyl–hexyl column. This method, with its simple sample preparation provides sensitive, accurate and precise quantification of the plasma concentration of antiretroviral drugs and can be used for therapeutic drug monitoring in patients infected with HIV.
Keywords: Antiretroviral drugs; Mass spectrometry;

Enantioselective analysis of praziquantel and trans-4-hydroxypraziquantel in human plasma by chiral LC–MS/MS: Application to pharmacokinetics by Renata Monteiro Lima; Maria Augusta Drago Ferreira; Teresa Maria de Jesus Ponte; Maria Paula Marques; Osvaldo Massaiti Takayanagui; Hector Hugo Garcia; Eduardo Barbosa Coelho; Pierina Sueli Bonato; Vera Lucia Lanchote (3083-3088).
A simple enantioselective method for the determination of praziquantel (PZQ) and trans-4-hydroxypraziquantel (4-OHPZQ) in human plasma was developed and validated by high-performance liquid chromatography/mass spectrometry. The plasma samples were prepared by liquid–liquid extraction using a mixture of methyl-tert-butylether/dichloromethane (2:1, v/v) as extraction solvent. The direct resolution of PZQ and 4-OHPZQ enantiomers was performed on a Chiralpak AD column using hexane–isopropanol (75:25, v/v) as the mobile phase. Diazepam was used as internal standard. The method described here is simple and reproducible. The quantitation limit of 1.25 ng/ml for each PZQ enantiomer and of 12.5 ng/ml for each 4-OHPZQ enantiomer permits the use of the method in studies investigating the kinetic disposition of a single dose of 1.5 g racemic PZQ. Enantioselectivity in the kinetic disposition of PZQ and 4-OHPZQ was observed in the clinical study, with the demonstration of a higher proportion of the (+)-(S)-PZQ and (−)-(R)-4-OHPZQ enantiomers in plasma.
Keywords: Praziquantel; Enantiomers; Pharmacokinetics; Metabolism;

Assays for the quantification of melphalan and its hydrolysis products in human plasma by liquid chromatography–tandem mass spectrometry by Asmae Mirkou; Bruno Vignal; Sabine Cohen; Marc Guillaumont; Olivier Glehen; Jérôme Guitton (3089-3096).
Two high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assays are described for the quantification of melphalan in human plasma. N-phenyldiethanolamine was tested as internal standard. The first assay consisted of a protein precipitation by cold methanol and a reversed-phase HPLC whereas the second one was based on a solid phase extraction and a hydrophilic interaction chromatography. Both provided a very satisfactory mean extraction yield with a small volume of sample. The first method was simple, rapid and used as a routine assay. The second one was developed in order to determine melphalan hydrolysis products and to avoid scarce cases when interferences from biological matrix alter the quantification of melphalan using the first method. The two assays were linear and sensitive in the range of 1–500 ng/mL for the first one and in a range of 25–2000 ng/mL for the second one. Concentrations out of the range fixed with the first method were also validated. The procedure was reliable with precision and accuracy below 10%. All compounds were detected after positive mode electrospray ionization in selected reaction monitoring mode. These new analytical procedures were developed for melphalan pharmacokinetic studies or therapeutic drug monitoring.
Keywords: Melphalan; Mass spectrometry; Hydrolysis products;

A sample preparation method including protein extraction by an aqueous buffer system, precipitation with trichloroacetic acid, washing with acetone, and desalting by dialysis was developed for 2D gel electrophoresis of mature leaves from Tropaeolum majus, a plant species with a high content of glucosinolates. By the optimized method, 1D- and 2D-gels could also be produced from Festuca rubra leaves and Helianthus annuus seeds. A strong influence of the varied protein preparation parameters on arsenic and zinc bindings was observed. Microwave-digestion with subsequent atomic spectroscopy analysis of protein fractions revealed the highest arsenic binding capacity of 76.2 ± 1.7% for proteins from sunflower seeds spiked with arsenite. After spiking of T. majus extracts with different arsenic species and zinc salts to 100 μg As or Zn in 10 mL, 9.5 ± 0.97%, 0.95 ± 0.39%, 0.24 ± 0.02%, 0.20 ± 0.09%, 0.02%, 0.83 ± 0.02%, 2.21 ± 1.64%, and 1.45 ± 0.69% were recovered in the final protein fraction for phenylarsine oxide, arsenite, arsenate, monomethylarsonate, dimethylarsinate, zinc chloride, zinc sulfate, and zinc acetate, respectively. The cultivation of T. majus under arsenic exposure resulted in a highly elevated arsenic-binding capacity of the proteins that was also dependent on the kind of arsenic species in the following order: arsenite (14.9%) > monomethylarsonate (12.4%) > arsenate (10.8%) > dimethylarsinate (0.32%).
Keywords: Arsenic species; Zinc salts; Plant proteins; Sample preparation; Tropaeolum majus; Two-dimensional gel electrophoresis;

Purification process development for HER1 extracellular domain as a potential therapeutic vaccine by Dania León; Yadira Prieto; Eutimio G. Fernández; Noemí Pérez; José A. Montero; Julio Palacios; Dubhe Bulté; Kathya R. de la Luz; Vladimir Peña; Williams Ferro; Belinda Sánchez; Rodolfo Valdés; Adolfo Castillo (3105-3110).
HER1 is a tumor associated antigen emerging as an attractive target for cancer therapy. In the present study we demonstrated for first time that HER1 extracellular domain can be purified by a downstream process at pilot scale based on immunoaffinity chromatography from bioreactor supernatant of HEK 293 transfectomes. Filtered supernatant was applied to CNBr-activated Sepharose CL-4B with monoclonal antibody anti-human EGF immobilized, followed by three additional chromatographic polishing steps. HER1 extracellular domain was obtained with high purity (>95%), low DNA content, and biological activity.
Keywords: HER1 ECD; Immunoaffinity chromatography; Protein purification; Monoclonal antibody; HEK 293;

Metabolomic study for diagnostic model of oesophageal cancer using gas chromatography/mass spectrometry by Hao Wu; Ruyi Xue; Chunlai Lu; Chunhui Deng; Taotao Liu; Huazong Zeng; Qun Wang; Xizhong Shen (3111-3117).
The prognosis for oesophageal cancer is poor. Attempts have been made for the identification of biomarkers for early diagnosis. Metabolomic panel has been evaluated as potential candidate biomarkers. With gas chromatography/mass spectrometry (GC/MS) as a sensitive modality for metabolomics, various tissue metabolites can be detected and identified. We hypothesized that tissue metabolomic biomarkers may be identifiable and diagnostically useful for oesophageal cancer. We present a metabolomic method of chemical derivatization followed by GC/MS to analyze the metabolic difference in biopsied specimens between oesophageal cancer and corresponding normal mucosae obtained from 20 oesophageal cancer patients. The GC/MS data was analyzed using a two sample t-test to explore the potential metabolic biomarkers for oesophageal cancer. A diagnostic model was constructed to discriminate normal from malignant samples, using principal component analysis (PCA) and receiver–operating characteristic (ROC) curves. t-Test showed a total of 20 marker metabolites detected were found to be different with statistical significance (P  < 0.05). The multivariate logistic analysis yielded a complete distinction between the two groups. The diagnostic model could discriminate tumors from normal mucosae with an area under the curve (AUC) value of 1. Our findings suggest that this assay may potentially provide a new metabolomic biomarker for oesophageal cancer.
Keywords: Metabolomic profiling; Oesophageal cancer; Biomarker; Gas chromatography/mass spectrometry;

E-3-(4′-Hydroxy-3′-adamantylbiphenyl-4-yl) acrylic acid (ST1926) is a novel oral synthetic adamantyl retinoid derivative, now under early clinical investigation in patients with ovarian cancer. To investigate the pharmacokinetics of this new antitumor agent in human plasma, we developed and validated a high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method based on the addition of ST2222 as internal standard and simple protein precipitation with methanol. The method requires a small volume of sample (100 μL); it is rapid and selective, allowing a good resolution of peaks from the plasma matrix in 9 min. The method offers high recovery (>90%), it is sensitive, precise and accurate with overall precision expressed as CV% less than 8.2%. We set the lower limit of quantitation at 20.0 ng/mL and validated the assay up to the concentration of 1000.0 ng/mL. The present method has been successfully applied to study ST1926 pharmacokinetics in patients with advanced ovarian cancer in a Phase I trial, administering the drug orally for five consecutive days. During analysis of the study samples, it became evident the presence of glucuroconjugates of the parent compound, established by LC-Orbitrap MS. Preliminary results show low and variable drug absorption in patients, with extensive glucuroconjugation influencing the bioavailability of ST1926.
Keywords: ST1926; Adamantyl retinoid derivative; HPLC–MS/MS method development; Pharmacokinetics; Phase I study;

A label-free nano-liquid chromatography–mass spectrometry approach for quantitative serum peptidomics in Crohn's disease patients by Paolo Nanni; Fredrik Levander; Giulia Roda; Alessandra Caponi; Peter James; Aldo Roda (3127-3136).
The identification of serum biomarkers for the diagnosis of inflammatory bowel diseases able to reduce the need for invasive tests represents a major goal in their therapy and follow-up. We report here a methodological approach for the evaluation of specific changes in the serum peptides abundance in healthy (H) and Crohn's disease (CD) subjects, based on a label-free LC ESI/Q-TOF differential mass spectrometry (MS) approach combined with targeted MS/MS analysis. The low molecular weight serum proteins were separated by RP nano-LC ESI/Q-TOF MS and the resulting datasets were aligned with msInspect software. The differently abundant peptides, evaluated using Proteios Software Environment, were identified by MS/MS analysis and database search. The identification of clusters of peptides resulting from proteins (such as fibrinogen-α) commonly involved in physiological processes lead to the evaluation of a possible role in CD of specific serum exoproteases. An assay based on synthetic peptides spiked into H, CD and ulcerative colitis (UC) serum samples as substrate, followed by MALDI MS and chemometric analysis of the metabolite patterns has been developed achieving a 100% discrimination between CD, UC and H subjects. The results are promising for the application of this approach as a simple tool for diagnostic aims and biomarker discovery in CD.
Keywords: Crohn's disease; Exoproteases; Label-free liquid chromatography–mass spectrometry; Protein profling; Proteomics;

A rapid, specific and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method has been established for simultaneous quantitation of psoralen and isopsoralen in rat plasma. Plasma samples were pretreated by direct protein precipitation with acetonitrile. Chromatographic separations were performed on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, i.d., 1.7 μm) at 35 °C with a linear gradient of acetonitrile and 0.1% formic acid in water at a flow rate of 0.3 mL/min. The two isomers were satisfactorily separated (R  = 1.7) with a runtime of 4 min. Psoralen, isopsoralen, and the internal standard (IS) furazolidone were ionized with an APCI source operated in positive ion mode. The MS/MS transitions used for monitoring were at m/z 187.0 → 130.9 for psoralen and isopsoralen, and m/z 225.9 → 121.9 for IS. Calibration curve was linear over the concentration range of 1–500 ng/mL with the lower limit of quantitation of 1 ng/mL for both isomers. The mean extraction recoveries were 78.5 ± 6.7% and 81.9 ± 8.0% for psoralen and isopsoralen, respectively. The intra- and inter-day precisions were less than 5.6% and 5.2%, and the accuracy was within ±2.1% for both isomers. No matrix effect was observed in this method. Psoralen and isopsoralen were stable during all storage, pretreatment and analytical periods. The validated method has been successfully applied to a pharmacokinetic study of psoralen and isopsoralen after oral administration of Haigou Pill to rats.
Keywords: Psoralen; Isopsoralen; Isomers; UPLC/MS/MS; Pharmacokinetics;

In this paper, a fast method for the study on the interactions of a series of drugs used in the treatment of hypertension with human serum albumin (HSA) by flow injection-capillary electrophoresis (FI-CE) was developed based on the principle of frontal analysis (FA). The binding parameters were determined by FI-CE-FA from Scatchard equation and compared with results obtained by non-CE methods and literature values. A multiple linear regression (MLR) model between the drug–protein binding constants (K) and structural descriptors of drugs was constructed. L-tryptophan (L-try) and phenylbutazone (PB) were used as displacement reagents to investigate the binding sites of a series of drugs on HSA. The binding synergism effect between drugs and the effects of many metal ions existing in human plasma on protein binding were also investigated systematically.
Keywords: Drug–protein interactions; Flow injection-capillary electrophoresis; Frontal analysis; Human serum albumin; QSAR;

Application of the high-performance liquid chromatography method with coulometric detection for determination of vitamin B6 in human plasma and serum by Marcin L. Marszałł; Anna Lebiedzińska; Wojciech Czarnowski; Ryszard Makarowski; Mateusz Kłos; Piotr Szefer (3151-3158).
A reversed-phase high-performance liquid chromatography method (HPLC) with coulometric electrochemical detection has been developed and validated for the simultaneous analysis of pyridoxamine-5′-phosphate (PMP), pyridoxamine (PM), pyridoxal 5′-phosphate (PLP), pyridoxal (PL), pyridoxine (PN) and 4-pyridoxic acid (4-PA) in human plasma and serum. The isocratic separation was achieved on C18 column (250 mm × 4.6 mm, I.D., 5 μm) with a mobile phase consisted of 35 mM sodium phosphate containing 2.5 mM heptanesulfonic acid, adjusted to pH 3.2 with 85% orthophosphoric acid and 12% methanol (v/v). Within-run and between-run precisions expressed by the relative standard deviations were less than 2.7% and 7.7% for all the analysed vitamins and 4-PA, respectively. The limits of detection (LOD) and quantification (LOQ) were: 0.8 and 2.6 nM, 1.1 and 3.8 nM, 1.5 and 4.5 nM, 1.3 and 4.2 nM, 1.1 and 3.7 nM, 2.1 and 6.3 nM for PMP, PM, PLP, PL, PN and 4-PA, respectively. The recoveries ranged from 90.4% to 98.4%. Stability of vitamins was checked under a variety of storage conditions. The developed application demonstrated acceptable sensitivity, precision, accuracy, stability, and linearity over the physiological concentration range. The major advantage of the proposed method is its great simplicity.
Keywords: Vitamin B6; Pyridoxic acid; HPLC; Coulometric detection; Plasma; Serum;

High-throughput HPLC-MS/MS method to determine ibandronate in human plasma for pharmacokinetic applications by Isabela Tarcomnicu; Mihaela Cristina Gheorghe; Luigi Silvestro; Simona Rizea Savu; Irina Boaru; Ariana Tudoroniu (3159-3168).
A sensitive high-throughput liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of ibandronate in human plasma. In a previous study, we have analyzed alendronate in urine samples of subjects treated at therapeutic dosages, using a derivatization approach; a similar derivatization was adapted and improved to determine ibandronate in plasma. The bisphosphonate was isolated from the biological matrix by liquid–liquid extraction, and derivatized with trimethylsilyldiazomethane prior to separation on a reversed-phase column (Supelco Discovery HSC18) and detection on a quadrupole-linear ion trap mass spectrometer (API 4000 QTrap). Various parameters of extraction and derivatization were optimized in order to get adequate recovery, high derivatization yield and minimal ion suppression; a deuterated analogue, d3-ibandronate, was used as internal standard. The transitions 376.1 → 114.2 and 379.1 → 61.0 were acquired to monitor ibandronate and d3-ibandronate derivatives, respectively. A multiplexing LC system made possible the overlapping of two chromatographic runs, thus the interval between injections being reduced to only 2 min, a very short analysis time for compounds of this class. The method was fully validated over the quantification range 0.2–175.0 ng/ml, allowing an appropriate evaluation of the plasma concentrations of ibandronate, expected at therapeutic dosage, as proved by application to a pharmacokinetic study. A good linearity over the selected range (r  > 0.99), accuracy and precision within ±15% of the target values and a recovery over 50% were obtained.
Keywords: Ibandronate; Bisphosphonates; Liquid chromatography; Tandem mass spectrometry; Derivatization; Plasma; Pharmacokinetics;

Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography–tandem mass spectrometry to affinity purification followed by enzyme immunoassay by Michael Armstrong; Andrew H. Liu; Ronald Harbeck; Rick Reisdorph; Nathan Rabinovitch; Nichole Reisdorph (3169-3174).
A new analytical method suitable for high throughput measurements of LTE4 in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500 pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE4 from healthy adults and children are reported.
Keywords: LTE4; Leukotriene; Asthma; Liquid chromatography; Mass spectrometry;

Juvenile hormone (JH) III is responsible for control of a variety of insect physiological and developmental states, including caste differentiation of the Formosan subterranean termite (Coptotermes formosanus Shiraki). We report here a simplified, efficient sample preparation and an optimized LC–ESI-MS method for quantifying JH III in whole body extracts. Sample preparation comprises hexane extraction (from termite whole bodies) and C18 cartridge purification. Previous LC–ESI-MS protocols exhibited the following two problems: (1) ion fragmentation differed when comparing spectra from insect samples and authentic JH III and (2) a JH III monitoring ion was not resolved from other unknown compounds in whole body samples from termites. To overcome these problems, we used a pentafluorophenyl LC column and water/acetonitrile containing ammonium acetate as solvent. In a mass chromatogram (m/z 235) of termite samples, a peak was detected at the retention time of authentic JH III, and MS2 of this peak confirmed that the ion is a fragment of JH III, [M–CH3OH+H]+, being the base peak in both termites and authentic JH III samples. The protocol enables quantification of JH III in a single termite with signal/noise >10:1 and the limit of quantification is 21 pg.
Keywords: Juvenile hormone III; LC–ESI-MS; Formosan subterranean termite; Pentafluorophenyl; Ammonium acetate;

PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. It is converted systemically to the corresponding alcohol, PR-104A, which is activated by nitroreduction to the hydroxylamine (PR-104H) and amine (PR-104M). PR-104A is also metabolised to the O-glucuronide (PR-104G), and by oxidative debromoethylation to the semi-mustard PR-104S. We now report a validated ultra-high-pressure liquid chromatography and tandem mass spectrometry (UHPLC–MS/MS) method for the determination of these metabolites in human plasma. Plasma proteins were precipitated with acidified methanol and the supernatant diluted into water. Aliquots were analysed by UHPLC–MS/MS using a Zorbax Eclipse XDB-C18 Rapid Resolution HT (50 mm × 2.1 mm, 1.8 μm) column and gradient of acetonitrile and 0.01% formic acid with a 6 min run time. The method had a linear range of 0.1–50 μM for PR-104, PR-104A and PR-104G, 0.05–5 μM for PR-104H, 0.025–2.5 μM for PR-104M and 0.01–1 μM for PR-104S. The intra-day and inter-day precision and accuracy were within 14%. The extraction recovery of all analytes was over 87%. The validated method was illustrated by using it to study the pharmacokinetics of PR-104 and its metabolites in a human patient.
Keywords: LC–MS/MS; PR-104; Nitrogen mustards; Bioreductive prodrugs; Metabolites; Pharmacokinetics;

Metal ion mediated synthesis of molecularly imprinted polymers targeting tetracyclines in aqueous samples by Guorun Qu; Sulian Zheng; Yumin Liu; Wei Xie; Aibo Wu; Dabing Zhang (3187-3193).
Molecularly imprinted polymers (MIPs) prepared in water-containing systems are more appropriate as adsorption materials in analyte extraction from biological samples. However, water as a polar solvent involved in the synthesis of MIPs frequently disrupts non-covalent interactions, and causes non-specific binding. In this study Fe2+ was used as mediator to prepare MIPs, targeting tetracyclines (TCs) of tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC), with TC as template molecule and methacrylic acid (MAA) as functional monomer. The subsequent binding assay indicated that Fe2+ was responsible for substantially improved specific binding in recognition of TCs by decreasing the non-specific binding. Spectrophotometric analysis suggested the existence of the strong interactions among TC, metal ions and MAA in the mixture of methanol and water. Moreover, mass spectrometric measurements verified that Fe2+ could bridge between TC and MAA to form a ternary complex of one TC, one Fe2+ and four MAAs with a mass of 844.857. Furthermore, combined with molecularly imprinted solid-phase extraction (MISPE) for sample pretreatment, HPLC–UV analysis data revealed good performance of the obtained MIPs as adsorbents. The recoveries of TC, OTC and CTC in urine samples were 80.1–91.6%, 78.4–89.3% and 78.2–86.2%, respectively. This research strategy provides an example for preparation of desirable water-compatible MIPs extracting target drugs from aqueous samples by introducing metal ion as mediator into conventional polymerization system.
Keywords: Molecular imprinting; Water-containing system; Metal ion; Tetracyclines; Extraction;

Leuprolide is a synthetic nonapeptide that has two basic amino acids, arginine and histidine, in its structure. By selection of an appropriate analytical column and optimizing the mobile phase composition, an improved analytical method has been developed and validated to determine leuprolide concentrations in human serum. After methanol-induced protein precipitation of serum samples and Oasis® HLB cartridge solid-phase extraction, leuprolide and an internal standard (alarelin) were analyzed on a C18 column interfaced with a triple quadrupole tandem mass spectrometer with positive electrospray ionization. The mobile phase consisted of acetonitrile–water–propionic acid (20:80:0.05). The analyte and internal standard were both detected in the selective reaction monitoring mode. The method exhibited a linear range of 0.018–45.2 ng/mL for leuprolide. The intra- and inter-assay precisions were 11.5% or less relative standard deviation (R.S.D.), and accuracy was within ±2.8% relative error (RE). The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.018 ng/mL, with acceptable precision and accuracy. The validated LC–MS/MS method was tested to a clinical pharmacokinetic study of leuprolide after a single subcutaneous injection of 1 mg leuprolide acetate in healthy male Chinese volunteers.
Keywords: Liquid chromatography–tandem mass spectrometry; Leuprolide; Pharmacokinetics;

Internal standard (IS) responses can directly impact the accuracy of reported concentrations in bioanalysis as the majority of LC–MS/MS methods are based on analyte/IS response ratios for quantitation. Due to the complexity of incurred sample matrices and drug formulation, variable IS responses are quite common upon applying a validated method to the analysis of incurred samples. To maintain the integrity of a study and to avoid economic losses, it is therefore extremely important to monitor IS response variations during bioanalysis and to quickly identify the root causes if variations are observed. Presented in this article are twelve trouble-shooting examples from the analyses of incurred samples by a wide variety of bioanalytical methods, including human error, malfunctioning equipment/instruments, wrong material, matrix effect and inherent issues with a bioanalytical method. Insightful ideas for how to trouble-shoot and how to develop more reliable bioanalytical methods can be drawn from these practical examples.
Keywords: Internal standard; Variation; LC–MS/MS; Incurred sample; Matrix effect; Bioanalysis; Trouble-shooting;

Two solid phase microextraction modes were investigated and compared for their performance on the determination of selenites in various biological liquids like human urine and saliva and various types of milk. Using sodium tetraethylborate (NaBEt4) as ethylating reagent, selenites are converted in situ to volatile diethylselenides (DESe) in aqueous medium. The derivative is collected in situ by solid phase microextraction (SPME) using a silica fiber coated with poly(dimethylsiloxane) (PDMS) either from the headspace (HS-SPME) or directly from the liquid phase (LP-SPME) and finally determined by capillary GC/MS. Under optimum conditions of SPME, the GC separation was also optimized. Between the two examined microextraction techniques, direct immersion of the PDMS fiber in the liquid phase was proved less satisfactory. In contrast, the headspace procedure appears to be more efficient. The quantification of selenites was achieved in SIM mode with good analytical performance. A non-fat milk powder certified reference material was analyzed to evaluate the accuracy of the method. The overall precision of the method was ranged between 6.2% and 9.7%. Detection limits achieved were 0.05 μg L−1 for human urine, 0.08 μg L−1 for saliva and 0.03–0.06 μg L−1 in various milk matrices.
Keywords: Selenite; Headspace solid phase microextraction; Gas chromatography mass spectrometry; Urine; Saliva; Milk;

In this manuscript, we present a simple and reliable method for the quantitation of para-aminohippuric acid (PAH; 2-(4-aminobenzamido)acetic acid) in human plasma and urine using liquid chromatography coupled to electrospray ionisation low-energy collision-induced dissociation tandem mass spectrometry (HPLC–ESI-CID-MS/MS) analysis (negative ion mode) via multiple reaction monitoring (MRM). Sample preparation involved protein precipitation of plasma samples with acetonitrile and subsequent dilution of urine samples with the mobile phase. The internal standard (IS) adopted was para-aminosalicylic acid (PAS; 4-amino-2-hydroxybenzoic acid). Chromatographic separation was achieved on a Cosmosil HILIC column using an isocratic mobile phase consisting of ammonium acetate buffer (20 mM) and acetonitrile (45:55, v/v) at a flow rate of 200 μl/min. The transactions monitored were m/z 192.9 → 149.1 for PAH and m/z 152.1 → 108.1 for IS. Linear calibration curves were generated over the PAH concentration range of 0.2–100 mg/L in human plasma and urine. The method was validated for selectivity, accuracy, precision, recovery and stability according to USFDA criteria, and has been successfully applied to a pharmacokinetic study in healthy volunteers administered an intravenous dose of 440 mg PAH.
Keywords: para-Aminohippuric acid; High-performance liquid chromatography; Mass spectrometry; Human plasma; Human urine;

A sensitive and rapid liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of deserpidine in human plasma. The plasma samples were prepared using liquid–liquid extraction (LLE) with ethyl ether–dichloromethane (3:2, v/v). Chromatographic separation was accomplished on an Ultimate XB-C18 column. The mobile phase consisted of methanol–5 mM ammonium acetate–formic acid (72:28:0.036, v/v/v). Detection of deserpidine and the internal standard tropisetron was achieved by tandem mass spectrometry with an electrospray ionization interface in positive ion mode. The lower limit of quantification was 4.0 pg/ml. The linear range of the method was from 4.0 to 2000 pg/ml. The intra- and inter-day precisions were lower than 14.7% in terms of relative standard deviation (RSD), and the accuracy was within ±8.7% in terms of relative error (RE). This validated method was successfully applied for the evaluation of pharmacokinetics of deserpidine after a single oral administration dose of 0.25 mg deserpidine to 22 healthy volunteers.
Keywords: Deserpidine; Reserpine; LC/MS/MS; Pharmacokinetics;

Warfarin is a well-known anticoagulant agent that occurs in two enantiomers, (R)-(+)-warfarin and (S)-(−)-warfarin. A new liquid chromatography method for the determination of both enantiomers was developed, validated and applied in in vitro studies with the aim of evaluating the accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line. OptiMEM cell cultivation medium samples and cellular lysates were purified using Waters Oasis® MAX extraction cartridges. The chiral separation of warfarin and the internal standard p-chlorowarfarin enantiomers was performed on an Astec Chirobiotic™ V2 column at a flow rate of 1.2 mL/min. The mobile phase was composed of 31% acetonitrile, 5% of methanol and 64% of ammonium acetate buffer (10 mmol/L, pH 4.1). The enantiomers were quantified using a fluorescence detector (λ excit  = 320 nm, λ emiss  = 415 nm). The limit of detection was found to be 0.121 μmol/L of (S)-warfarin and 0.109 μmol/L of (R)-warfarin. The range of applicability and linearity was estimated from 0.25 to 100 μmol/L. The precision ranged from 1.3% to 12.2% of the relative standard deviation, and the accuracy reached acceptable values from 95.5% to 108.4%. The new bioanalytical method confirmed the same accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line.
Keywords: High-performance liquid chromatography; Chiral separation; Macrocyclic glycopeptides; (R)-warfarin; (S)-warfarin;

A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC–IDMS by Chiara Cipollina; Angela ten Pierick; André B. Canelas; Reza M. Seifar; Antonius J.A. van Maris; Jan C. van Dam; Joseph J. Heijnen (3231-3236).
A gas chromatography isotope dilution mass spectrometry (GC–IDMS) method was developed for the quantification of the metabolites of the non-oxidative part of pentose phosphate pathway (PPP). A mid-polar GC column (Zebron ZB-AAA, 10 m, film composition 50% phenyl 50% dimethyl polysiloxane) was used for the chromatographic separation of the intermediates. The optimized GC–MS procedure resulted in improved separation performances and higher sensitivities compared to previous methods. Furthermore, the use of 13C-labeled cell extracts as internal standards improved the data quality and eliminated the need to perform a recovery check for each metabolite. The applicability of the new method was demonstrated by analyzing intracellular metabolite levels in samples derived from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae at steady state as well as following a short-term glucose pulse. The major achievements of the proposed quantitative method are the independent quantification of the epimers ribulose-5-phosphate and xylulose-5-posphate and the measurement of compounds present at very low concentrations in biological samples such as erythrose-4-phosphate and glyceraldehyde-3-phosphate.
Keywords: Methoxime-trimethylsilyl (MOX-TMS) derivatives; GC–MS; Isotope dilution mass spectrometry; Metabolomics; Pentose phosphate pathway; Saccharomyces cerevisiae;

Enhancement of chemical derivatization of steroids by gas chromatography/mass spectrometry (GC/MS) by John A. Bowden; Dominic M. Colosi; Diana C. Mora-Montero; Timothy J. Garrett; Richard A. Yost (3237-3242).
Steroid derivatization was investigated by varying the experimental parameters (reagent, reaction time, and reaction temperature) to determine the optimal conditions for individual steroids, and for larger subsets. Three methods of derivatization enhancement were also investigated: the use of sonication, the use of a microwave heating, and the addition of solvents to the reaction mixture. On a comprehensive level, derivatization using N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) was most efficient, while the application of solvent addition and microwave heating, in several cases, provided a clear enhancement. In addition, generalized rules for steroid derivatization are described.
Keywords: Chemical derivatization; Derivatization enhancement; Gas chromatography/mass spectrometry; Microwave-accelerated derivatization; Silylation; Steroid profiling; Steroids;

Quantification of prochlorperazine maleate in human plasma by liquid chromatography–mass spectrometry: Application to a bioequivalence study by Miao Yan; Yun-Gui Zhu; Huan-De Li; Ben-Mei Chen; Ning Ma; Yan-Qing Lei; Yi-Ping Liu (3243-3247).
A sensitive and specific method using a one-step liquid–liquid extraction with dichloromethane followed by liquid chromatographic–electrospray ionization-mass spectrometric was developed and validated to determine prochlorperazine maleate in human plasma using amitriptyline hydrochloride as an internal standard. The samples were separated using a Thermo Hypersil-Hypurity C18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm). A mobile phase containing 10 mM ammonium acetate (pH 3.6)–methanol–acetonitrile (27:68:5, v/v/v) was used isocratically eluting at a flow rate of 0.22 ml/min. The average extraction recovery of prochlorperazine and internal standard were 81.8 ± 2.2% and 79.5 ± 3.7%, respectively. Prochlorperazine maleate and internal standard were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 0.20 to 6.40 ng/ml with r 2  = 0.9989. The limit of quantification for prochlorperazine maleate in the plasma was 0.20 ng/ml. The established method has been successfully applied to a bioequivalence study of two prochlorperazine maleate formulations in 18 healthy male Chinese volunteers.
Keywords: Prochlorperazine maleate; Amitriptyline hydrochloride; LC–MS; Electrospray ionization; Bioequivalence;

This study presented a capillary zone electrophoresis (CZE) method for the analysis of trace amount of neurotransmitters glutamate (Glu) and aspartate (Asp) in the microdialysates of rat periaqueductal gray matter (PAG). Glu and Asp were derivatized with the fluorescent agent 5-carboxyfluorescein N-succinimidyl ester (CFSE) for the first time and detected by laser-induced fluorescence (LIF) after separation by CZE. The concentration detection limits (S/N = 2) were 6.9 × 10−10  M and 8.1 × 10−10  M for Glu and Asp, respectively. The repeatability (expressed as RSD) of the migration times of CFSE–amino acid were better than 0.5%, and not higher than 1.5% even over the period of 1 month. This method was applied to quantify Glu and Asp in rat PAG microdialysates with the treatment of formalin injection, and the measured basal concentrations of Glu and Asp were (22.4 ± 1.6) × 10−6  M and (0.9 ± 0.1) × 10−6  M, respectively.
Keywords: Microdialysis; Capillary electrophoresis; Laser-induced fluorescence; 5-Carboxyfluorescein N-succinimidyl ester; Glutamate; Aspartate;

Matrine determination and pharmacokinetics in human plasma using LC/MS/MS by Xiao-Lin Zhang; Hong-Rong Xu; Wei-Li Chen; Nan-Nan Chu; Xue-Ning Li; Gang-Yi Liu; Chen Yu (3253-3256).
A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of matrine in human plasma extracted by isopropanol:ethyl acetate (v/v, 5:95). Rapid chromatographic separation was achieved in the mobile phase composition of 5-mM aqueous ammonium acetate and acetonitrile (v/v, 70:30) with a flow rate of 0.20 ml/min. Detection was carried out using positive-ion electrospray tandem mass spectrometry on a Sciex API3000. The method was accurate, specific and sensitive for the analysis of matrine in human plasma in the concentration range of 5–2000 ng/ml, when huperzine A was used as internal standard. The method facilitated a clinical pharmacokinetic study after oral administration of a single dose of matrine soft gelatin capsules (100, 200 and 400 mg) in a three-period crossover design. Dose-related linear trends were observed for the AUC0−t and the C max of matrine. The t 1/2 and the T max of matrine were independent of the administered doses.
Keywords: LC/MS/MS; Matrine; Pharmacokinetics;

The use of plasmid DNA-based therapeutics relies on procedures that efficiently purify the supercoiled plasmid isoform. The present study describes a new strategy that uses a lysine ligand in affinity chromatography to efficiently separate supercoiled and open circular plasmid DNA isoforms. To achieve higher specificity in this chromatography it is essential to characterize the behaviour of binding/elution of supercoiled isoforms. The results show that the lysine support promotes complex interactions with supercoiled isoform, according to ionic strength and temperature manipulation.
Keywords: Affinity chromatography; Lysine support; Supercoiled plasmid DNA;

Keywords: ADMA; LC–MS/MS; Quantification; Validation; Ion enhancement; Ion suppression; Matrix effects;

Liquid chromatography: Is it essential for the determination of arginine and methylated arginines by tandem mass spectrometry? by Gary Weaving; Bernard F. Rocks; Michael P. Bailey; Michael A. Titheradge (3267-3269).
Keywords: Asymmetric dimethylarginine; Mass spectrometry; Chromatography; Internal standards;