Journal of Chromatography B (v.875, #2)

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d 6 as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.
Keywords: Phospholipids; LC/MS/MS; Matrix effects; Ion suppression;

GC–MS analysis of breath odor compounds in liver patients by Sandra Van den Velde; Frederik Nevens; Paul Van hee; Daniel van Steenberghe; Marc Quirynen (344-348).
Liver diseases can cause a sweet, musty aroma of the breath, called fetor hepaticus. Even in a stage of cirrhosis, the disease can be asymptomatic for many years. Breath analysis might be helpful to detect occult liver pathology.This study examined whether specific breath odor compounds can be found in liver patients, suffering from cirrhosis, which might be useful for diagnosis.Fifty-two liver patients and 50 healthy volunteers were enrolled. Alveolar air was analyzed by gas chromatography–mass spectrometry. Using discriminant analysis a model for liver disease was built.Dimethyl sulfide, acetone, 2-butanone and 2-pentanone were increased in breath of liver patients, while indole and dimethyl selenide were decreased. Sensitivity and specificity of the model were respectively 100% and 70%.Fetor hepaticus is caused by dimethyl sulfide and to a lower extent by ketones in alveolar air. Breath analysis by GC–MS makes it possible to discriminate patients with breath malodor related to hepatic pathologies.
Keywords: VOCs; Liver disease; Alveolar air; GC–MS; Halitosis;

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3 → 100.0, m/z 411.3 → 100.0 and m/z 268.3 → 116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1–100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.
Keywords: Tiloronoxim; Tilorone; LC–MS/MS; Urine;

Simultaneous determination method of N-acetyl-l-aspartyl-l-glutamate (NAAG), an endogenous agonist at type3 metabotropic glutamate receptor, and its degradation product, N-acetyl-l-aspartate (NAA) was developed by using reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. The detection limits of NAAG and NAA were approximately 12 and 34 fmol on the column, respectively (signal to noise ratio 3). The proposed HPLC method was applied to determine NAAG and NAA simultaneously in the rat brain homogenate. Both concentrations of NAAG and NAA in the male rat cerebrum (13 weeks old) were 5.7 ± 0.30 and 2.1 × 102  ± 9.2 nmol/mg protein, respectively (n  = 6), while those in the hippocampus were 6.8 ± 0.48 and 1.9 × 102  ± 8.5 nmol/mg protein, respectively (n  = 5). Hippocampal NAA concentration was significantly increased in the ketamine-treated rats as compared to the control rats (p  < 0.01).
Keywords: N-Acetyl-l-aspartyl-l-glutamate; N-Acetyl-l-aspartate; HPLC-fluorescence detection; 4-N,N-Dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole; Rat brain;

A simple and specific HPLC assay for simultaneous determination of two major active components (−) epigallocatechin-3-gallate (EGCG), and (−) epicatechin-3-gallate (ECG) of tea polyphenols (TP) in rat plasma was developed and validated. Following addition of resorcinol as internal standard (IS) the analytes were isolated from rat plasma by liquid–liquid extraction with ethyl acetate. The chromatographic separation was achieved on a reversed-phase C18 column using an isocratic mobile phase consisting of 0.1% citric acid + CH3CN (86:14, v/v) running at flow rate of 1.5 mL/min. The effluent was monitored at a wavelength of 280 nm. EGCG, ECG and IS were well separated from each other and free from interference from blank plasma and other components in TP as well as metabolites post-dosing. The calibration curve was constructed by plotting peak area ratio of analytes to IS vs. concentration. The method showed good linearity over range of 0.5–300 μg/mL for EGCG and 0.1–60 μg/mL for ECG (r  > 0.999). The intra- and inter-day precision (R.S.D.) was better than 6 and 12%, respectively. Assay accuracy was better than 94.78% for both compounds. Extraction recovery at QC samples was between 85.73 and 91.93% for EGCG and 79.08 and 86.51% for ECG. The developed method was successfully used to simultaneously measure plasma concentrations of EGCG and ECG after intravenous administration of TP to rats and yielded two typical biexponential decay concentration–time curves.
Keywords: Tea polyphenols; EGCG; ECG; HPLC;

N-[2-(Dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide (SN 28049) is a potent topoisomerase II poison being developed to treat solid tumours. A reliable and sensitive LC–MS method has been developed and validated for the determination of SN 28049 in plasma using a structurally similar internal standard. This method had acceptable intra- and inter-assay accuracy (95–105%) and precision (R.S.D. < 6.5%) over the range 0.062–2.5 μM (using a 100 μl sample), and had a lower limit of quantitation of 0.062 μM. Both aqueous and plasma solutions of SN 28049 were stable during short-term (24 h at room temperature or 4 °C) and long-term storage (8 months at −80 °C), and following freezing and thawing (three cycles). The method was applied to study the pharmacokinetics of SN 28049 in mice after iv administration (8.9 mg/kg; n  = 3 mice per time point). The maximum plasma concentration achieved was 1.22 ± 0.05 μM, and concentrations were measurable up to 12 h post-administration. A bi-exponential concentration-time curve was observed with an elimination half-life of 2.3 ± 0.2 h (mean ± S.E.), a volume of distribution of 34.5 ± 2.2 l/kg, and a plasma clearance of 12 ± 0.5 l/h/kg.
Keywords: SN 28049; LC–MS; Pharmacokinetics; Cancer; Topoisomerase poison;

An LC/MS/MS method was developed to quantify carboplatin and eribulin mesylate (E7389) in human plasma and urine. For carboplatin, sample clean-up by protein precipitation and supernatant injection into a Waters Spherisorb® S5 SCX column was used. Liquid-phase extraction and reverse-phase chromatography on a Polaris® C18 column were used for eribulin. Quantitation involved LC/MS/MS with positive electrospray ionization. Accuracy, precision, linearity, range, specificity, recovery and stability were also evaluated. Both compounds were stable in human plasma (≥80 days at −80 °C), at room temperature (≥4 h), following three freeze–thaw cycles and in 50/50 methanol/H2O (<4 °C for ≥252 days).
Keywords: Eribulin; Carboplatin; Human plasma; LC/MS/MS;

In vitro oxidized and glycated human low-density lipoprotein particles characterized by capillary zone electrophoresis by Jing-Huei Chiu; Yu-Nong Peng; Ying-Ling Yang; Ming-Hua Tsai; Yu-Ling Ho; Chung-Yu Wu; Mine-Yine Liu (383-391).
A simple capillary zone electrophoresis (CZE) method was used to determine native, in vitro Cu2+ and glucose modified low-density lipoprotein (LDL) particles for four healthy subjects. The LDL electropherograms are highly reproducible with good precisions of effective mobility and peak area. The native LDL capillary electrophoresis (CE) profile shows a major peak with lower mobility and two minor peaks with higher mobilities. For three-hour Cu2+ oxidation, one major peak with mobility close to that of the native major peak, and one minor peak with mobility extending to −47 × 10−5  cm2  V−1  s−1 appear. For eighteen-hour Cu2+ oxidation, one major peak with mobility much higher than that of the native major peak appears. As the reaction time for LDL and Cu2+ increases from 0 to 24 h, effective mobility of the LDL major peak increases, suggesting that LDL particles become more negatively charged and oxidized as the time increases. The in vitro glycated LDL particles are characterized by a major peak and two minor peaks. Mobility of the major peak is close to that of native major peak, but the second minor peak is much more negatively charged with mobility extending to −53 × 10−5  cm2  V−1  s−1. Native, oxidized and glycated LDL particles show distinctive differences in their CZE profiles. Agarose electrophoresis shows that the charge to mass ratios of native, three-hour Cu2+ and glucose modified LDL particles are similar, but that of eighteen-hour Cu2+ oxidized LDL particles is higher.
Keywords: Capillary zone electrophoresis; Low-density lipoprotein; In vitro oxidation; In vitro glycation;

Headspace solid phase microextraction/gas chromatography–mass spectrometry combined to chemometric analysis for volatile organic compounds determination in canine hair: A new tool to detect dog contamination by visceral leishmaniasis by Lidia S. de Oliveira; Frederico de M. Rodrigues; Fabio S. de Oliveira; Paulo R.R. Mesquita; Danielle C. Leal; Adriano C. Alcântara; Barbara M. Souza; Carlos R. Franke; Pedro. A. de P. Pereira; Jailson B. de Andrade (392-398).
A new analytical methodology using HS-SPME/GC–MS was optimized in order to attain maximum sensitivity, using multivariate strategies. The proposed method was employed to evaluate the VOC profile exhaled from canine hair samples collected from 8 healthy dogs and from 16 dogs infected by Leishmania infantum. 274 VOCs were detected, which could be identified as aldehydes, ketones and hydrocarbons. After application of the Soft Independent Modeling of Class Analogy (SIMCA) and Principal Component Analysis (PCA) healthy and infected dogs, with similar VOCs profiles, could be separately grouped, based on compounds such as 2-hexanone, benzaldehyde, and 2,4-nonadienal. The proposed method is non-invasive, painless, readily accepted by dog owners and could be useful to identify several biomarkers with applications in the diagnosis of diseases.
Keywords: Canine hair; VOCs; HS-SPME/GC–MS; Multivariate optimization; PCA; Diagnosis; Visceral leishmaniasis;

A reliable and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) confirmation method has been developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in chicken muscle. Samples were extracted with basic ethyl acetate, defatted with hexane, and cleaned up on Oasis MCX cartridges. LC separation was achieved on a XTerra C18 column with gradient elution using a mobile phase composed of acetonitrile and water at a flow rate of 0.20 mL/min. The analysis was carried out on a triple–quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via electrospray interface operated in the positive and negative ionization modes, with deuterated chloramphenicol-d5 (d5-CAP) as the internal standard. The method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for each analyte with one precursor ion and two product ions. Limits of detection (LODs) were 0.1 μg/kg for CAP, 0.2 μg/kg for FF and 1 μg/kg for TAP and FFA in chicken muscle. Linear calibration curves were obtained over concentration ranges of 0.3–20 μg/kg for CAP, 0.5–20 μg/kg for FF and 3–100 μg/kg for TAP and FFA in tissues. Mean recoveries of the 4 analytes ranged from 95.1% to 107.3%, with the corresponding intra- and inter-day variation (relative standard deviation, R.S.D.) less than 10.9% and 10.6%, respectively. The decision limit (CCα) and detection capability (CCβ) of the method were also reported.
Keywords: Chloramphenicol; Thiamphenicol; Florfenicol; Florfenicol amine; Liquid chromatography–tandem mass spectrometry; Chicken muscle;

Reason: The method described in this paper is exclusively an intellectual property of the Dr. Margarete Fischer-Bosch, Institute of Clinical Pharmacology, Stuttgart, Germany and has been published already in two papers on pharmacokinetics of digoxin (1. Becquemont L, Glaeser H, Drescher S, Hitzl M, Simon N, Murdter TE, Heinkele G, Hofmann U, Schaefer C, Burk O, Verstuyft C, Eichelbaum M, Fromm MF. Effects of ursodeoxycholic acid on P-glycoprotein and cytochrome P450 3A4-dependent pharmacokinetics in humans. Clin. Pharmacol. Ther., 79 (2006) 449–460; and 2. Igel S, Drescher S, Mürdter T, Hofmann U, Heinkele G, Tegude H, Glaeser H, Brenner SS, Somogyi AA, Omari T, Schäfer C, Eichelbaum M, Fromm MF. Increased absorption of digoxin from the human jejunum due to inhibition of intestinal transporter-mediated efflux. Clin. Pharmacokinet., 46 (2007) 777–785).

Development of a high-performance liquid chromatographic method for the determination of a new potent radioiodinated melanoma imaging and therapeutic agent by Delphine Denoyer; Pierre Labarre; Janine Papon; Elisabeth Miot-Noirault; Marie-Josephe Galmier; Jean-Claude Madelmont; Jean-Michel Chezal; Nicole Moins (411-418).
N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carbamide (ICF 01012) is a new melanoma imaging agent showing promising properties for application in internal radionuclide therapy. We developed an analytical protocol for detection of ICF 01012 in biological samples using HPLC. The proposed method was first validated using standard of ICF 01012 and four potent metabolites of this compound and then applied to follow the metabolic fate of [125I]ICF 01012 after injection in melanoma-bearing mice. The results demonstrate that this method exhibits a good linearity (r 2  = 0.9947), specificity and acceptable accuracy. This simple method appears convenient and sufficient for pharmacokinetic studies on [125I]ICF 01012.
Keywords: Radiopharmaceutical targeting melanoma; N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide; ICF 01012; HPLC analysis;

Environmental tobacco smoke is a major factor influencing the indoor air quality. Various toxic compounds emitted during tobacco smoking into the environment have a significant influence on the chemical composition of human biological fluids. The thiocyanate concentration in saliva is a biochemical measure, frequently used as an objective indicator of tobacco consumption. The goal of this study was to find significant relationships between salivary thiocyanates and other inorganic ions, which are constituents of natural saliva (Na+, K+, Mg2+, Ca2+, Cl, PO4 3−) and to present the effectiveness of the proposed sample preparation procedure combined with ion chromatography technique for the determination of inorganic ions in human saliva samples collected from passive, moderate and heavy smokers.
Keywords: Environmental tobacco smoke (ETS); Active and passive smokers; Thiocyanate; Ion chromatography (IC); Human saliva;

Liquid chromatography–tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat by Zhanzhong Zhao; Lifang Zhang; Feiqun Xue; Xiaoyang Wang; Wenli Zheng; Tao Zhang; Chenzhong Fei; Keyu Zhang; Minqi Qiu; Ruixiang Xin; Fengkun Yang (427-436).
A rapid, sensitive and specific liquid chromatography–electrospray ionization (ESI) tandem mass spectrometry (LC–MS–MS) method has been developed for the identification of nitazoxanide metabolites in goat plasma and urine. The purified samples was separated using an XTerra MS C8 column with the mobile phase consisted of acetonitrile and 10-mM ammonium acetate buffer (pH 2.5) followed a linear gradient elution, and detected by MS–MS. Identification and structural elucidation of the metabolites were performed by comparing their retention-times, full scan, product ion scan, precursor ion scan and neutral loss scan MS–MS spectra with those of the parent drug or other available standard. Four metabolites (tizoxanide, tizoxanide glucuronide, tizoxanide sulfate and hydroxylated tizoxanide sulfate) were found and identified in goat after single oral administration of 200 mg/kg dose of nitazoxanide. In addition, the possible metabolic pathway was proposed for the first time. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active metabolites responsible for pharmacological effects of nitazoxanide and to better understand its in vivo metabolism.
Keywords: Nitazoxanide; Metabolites; LC–MS–MS; Goat;

A method was developed for the simultaneous determination of 15 phenylurea herbicides (fenuron, tebuthiuron, metoxuron, monuron, chlortoluron, fluometuron, isoproturon, diuron, monolinuron, metobromuron, buturon, siduron, linuron, chlorbromuron, and neburon) in rice and corn samples by HPLC with fluorescence detection combined with UV decomposition and post-column derivatization. After extraction with acetonitrile and evaporation, the herbicides were redissolved in n-hexane and purified on a Florisil solid-phase extraction column. HPLC separation was carried out on a C18 column with water–acetonitrile gradient elution. UV decomposition was carried out under a 254-nm UV lamp. The method was evaluated in terms of the limits of detection and quantification. The linearity was satisfactory, with a correlation coefficient of >0.9980. Precision and recovery studies were evaluated at three concentration levels for each matrix. Good precision was obtained, with relative standard deviation in the range 1.5–9.6% for spiked rice samples and 0.9–9.9% for spiked corn samples. Recovery (n  = 6) ranged between 75.3% and 104.3% for rice and between 75.0% and 105.1% for corn. The intra-day precision (n  = 5) for the 15 herbicides in rice and corn samples spiked at an intermediate level was between 1.5% and 7.1%, and the inter-day precision over 10 days (n  = 10) was between 6.4% and 15.6%.
Keywords: Phenylurea herbicides; Solid-phase extraction; UV decomposition; Post-column derivatization;

A method was developed for the determination of the monoterpene alcohols verbenol, myrtenol, perillyl alcohol, α-terpineol, Δ3-carene-10-ol, thymol and p-α,α-trimethylbenzylalcohol in urine samples. After an enzymatic cleavage of their glucuronide- and sulfate conjugates the monoterpene alcohols were converted in the urine matrix with 7-diethylaminocoumarin-3-carbonylazide into monoterpene-[7-(diethylamino)-coumarin-3-yl]-carbamate derivates prior to analyses. Enrichment of the monoterpene alcohols from the urine matrix was achieved by online-solid phase extraction (SPE) with restricted-access material (RAM). After removal of excess derivatization reagent and urine matrix components, the monoterpene derivatives were separated by high-performance liquid chromatography (HPLC) in combination with fluorescence (FLD) detection and simultaneous mass spectrometric (MS) identification. Detection limits (LOD) for studied monoterpene alcohols ranged between 22 and 197 ng/L. The method was validated and successfully applied to urine samples from human subjects orally exposed to monoterpenes trough an intake of cough medication containing monoterpenes as active medicinal ingredients.
Keywords: Derivatization; 7-Diethylaminocoumarin-3-carbonylazide; Monoterpene alcohols; Fluorescence detection; Urine; Restricted-access material; HPLC–FLD–ESI-MS;

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein–membrane interactions, protein–protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.
Keywords: Palmitoylation; Peptide; MEKC; Capillary electrophoresis;

Previous studies have shown that plasma 1,5-anhydroglucitol (1,5-AG) is markedly reduced among diabetic patients and therefore serves as a sensitive marker for short-term glycemic control. The current study describes the development of the liquid chromatography negative ion electrospray tandem mass spectrometry (LC–MS/MS) method to measure 1,5-AG in human plasma. The samples were pre-treated with protein precipitation and an isotope-labeled internal standard was used. Chromatographic separation was achieved on amide column (150 mm × 2.0 mm i.d., 5 μm) followed by detection with multiple reaction monitoring mode. Linearity, accuracy, precision, recovery, matrix effect, and stability were evaluated during method validation over the range of 1–50 μg/mL. The validated method has been clinically applied among 159 type 2 diabetic patients and 290 control subjects. A marked reduction in 1,5-AG levels among the diabetic patients and significant between-gender difference in nondiabetic subjects were observed.
Keywords: 1,5-Anhydroglucitol; LC–MS/MS; Glycemic monitoring marker; Human plasma;

Simultaneous analysis of THC and its metabolites in blood using liquid chromatography–tandem mass spectrometry by Maria del Mar Ramirez Fernandez; Gert De Boeck; Michelle Wood; Manuel Lopez-Rivadulla; Nele Samyn (465-470).
Cannabis is considered to be the most widely abused illicit drug in Europe. Consequently, sensitive and specific analytical methods are needed for forensic purposes and for cannabinoid pharmacokinetic and pharmacodynamic studies. A simple, rapid and highly sensitive and specific method for the extraction and quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy- Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy- Δ9-tetrahydrocannabinol (THC-COOH) in blood is presented. The method was fully validated according to international guidelines and comprises simultaneous liquid–liquid extraction (LLE) of the three analytes with hexane:ethyl acetate (90:10, v/v) into a single eluant followed by separation and quantification using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Chromatographic separation was achieved using a XBridge C18 column eluted isocratically with methanol:0.1% formic acid (80:20, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor–product ion transitions. The use of the LLE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using 250 μL of blood. The method was linear over the range investigated (0.5–40 μg/L for THC, 1–40 μg/L for 11-OH-THC, and 2–160 μg/L for THC-COOH) with excellent intra-assay and inter-assay precision; relative standard deviations (RSDs) were <12% for THC and 11-OH-THC and <8% for THC-COOH for certified quality control samples. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. No instability was observed after repeated freezing and thawing or in processed samples. The method was subsequently applied to 63 authentic blood samples obtained from toxicology cases. The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well suited for routine analysis.
Keywords: THC; Blood; LLE; LC–MS/MS;

A novel analytical method was developed and validated for the rapid and simultaneous analysis of five toxic alkaloids: Brucine, Strychnine, Ephedrine, Aconitine and Colchicine, in blood and urine using high-performance liquid chromatography–electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (HPLC–ESI-MRM) mode. The linear range was 0.05–50.0 ng mL−1 for Brucine, 0.1–50.0 ng mL−1 for Strychnine and Ephedrine, 0.01–10.0 ng mL−1 for Aconitine and Colchicine. The limits of quantification for Brucine, Strychnine, Ephedrine, Aconitine and Colchicine were found to be 0.03, 0.05, 0.20, 0.05, 0.01 ng mL−1, respectively. The average extraction recoveries in urine ranged from 96.0 to 114.0% and in whole blood were 94.0 to 113.0%. The intra-day and inter-day RSDs were less than 8.3 and 10.6%, respectively. The five alkaloids could be well separated within 7 min in a single run. The established method should be suitable for the determination of trace alkaloids in body fluids.
Keywords: Alkaloids; Solid-phase extraction; High-performance liquid chromatography–electrospray ionization tandem mass spectrometry; Multiple reaction monitoring; Blood; Urine;

In an effort to optimize reverse-phase liquid chromatography (RPLC) for proteomics, we studied the impact of composition of the sample injection solution on protein on-column selection and retention. All the proteins studied were retained on-column when injections were made in 50% formic acid, 0.1% TFA or 8.3 M urea. When formic acid was increased to 80%, the superoxide dismutase standard (MW 26,159) and 58 mouse microsomal proteins that possessed low-range molecular weights, high pIs or basic amino acid clusters were non-retained, resulting in retention selectivity during sample injection. Introducing to the 80% formic acid injection solution an organic solvent such as acetonitrile or acetonitrile–DMSO induced further retention selectivity, and increasing levels of organic solvents reduced on-column retention. The proteome was split into the proteins that were retained on-column which eluted at higher retention times (RTs), vs the proteins that collected in the injection flow-through which normally eluted at lower RTs. This protein selectivity was confirmed after fraction collection, 1D-GE and nano-LC–MS/MS. The significance of this procedure is that it can be exploited for fast extraction of small basic proteins from the bulk of the proteome and for on-column enrichment of hydrophobic proteins.
Keywords: Proteomic profiling; Microsomal proteins; Cytochrome P450; Reverse-phase liquid chromatography; Fractionation; Sample injection; Flow-through; Selectivity; DMSO; 1D gel electrophoresis; SDS PAGE; Nanospray LC–MS/MS; Mass spectrometry;

A reverse-phase high performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of doxifluridine and its active metabolite 5-fluorouracil in monkey serum. A liquid/liquid extraction with ethyl acetate (90%) and isopropyl alcohol (10%) was used to extract simultaneously doxifluridine and 5-FU which have considerable difference in the polarity. Optimum chromatographic separation was achieved on a Agilent Zorbax C18 (100 mm × 2.1 mm, 3.5 μm) column with a mobile phase of methanol–water (20:80, v/v). The flow rate was 0.2 mL/min with total cycle time of 5 min. The lower limit of quantification (LLOQ) was validated at 10.0 ng/mL of serum for both doxifluridine and 5-FU. Accuracy and precision of quality control (QC) samples for both compounds met FDA Guidance criteria of ±15% with average QC accuracy of 95.5–105.0% and coefficients of variation of 1.1–9.5% in the 10–2000 ng/mL concentration range. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability to support the analysis of monkey serum samples.
Keywords: Doxifluridine; 5-FU; LC/MS/MS; Monkey; Serum;

Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag by Maozhou Yang; Xinli Wang; Liang Zhang; Chiyang Yu; Bingbing Zhang; William Cole; Greg Cavey; Paula Davidson; Gary Gibson (493-501).
Like other members of the transforming growth factor beta (TGF-β) family of growth factors, the biological activity of TGF-β2 is believed to be regulated by the formation and dissociation of multiprotein complexes. To isolate the molecular complex formed by TGF-β2 secreted by hypertrophic chondrocytes we have used expression of TGF-β2 fused with the humanized, tandem affinity purification (hTAP) tag and mass spectrometry for the identification of interacting proteins. The hTAP synthetic gene was assembled by systematically replacing the rare codons of the original TAP tag with codons most preferred in highly expressed human genes to circumvent the poor translation efficiency of the original TAP tag in animal cells. TGF-β2 was shown to interact with Type X collagen and this interaction confirmed using V5 tagged TGF-β2. Functional interaction was suggested by the inhibition of TGF-β2 activity by type X collagen in culture and the influence of a mutation in type X collagen on the distribution of TGF-β2 in growth cartilage.
Keywords: Transforming growth factor beta 2; Type X collagen; Tandem affinity purification; Skeletal growth;

Porous silicon affinity chips for biomarker detection by MALDI-TOF–MS by Ya-Qing Chen; Feng Bi; Shi-Quan Wang; Shou-Jun Xiao; Jian-Ning Liu (502-508).
B-type natriuretic peptide (BNP) is an important biomarker in early diagnosis of congestive heart failure. Many efforts have been made previously to evaluate the BNP level in human plasma. We developed a porous silicon (PSi) affinity chip to detect BNP present at low concentrations in human plasma by matrix assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF–MS) directly. The PSi surface immobilized with antibodies captured and concentrated BNP through antibody–antigen interaction specifically and sensitively. A detection limit as low as 10 pg/mL BNP in human plasma was demonstrated by mass analysis. This effective on-chip recognition, enrichment, and detection strategy could be employed in identification of biomarkers in complex body fluids in diagnoses.
Keywords: Biomarker; BNP; Affinity chip; Porous silicon; MALDI-TOF–MS;

Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography–mass spectrometry by Megumi Motojyuku; Takeshi Saito; Kazuki Akieda; Hiroyuki Otsuka; Isotoshi Yamamoto; Sadaki Inokuchi (509-514).
This paper describes an assay for the determination of glyphosate (GLYP), glyphosate metabolites [(aminomethyl) phosphonic acid] (AMPA), and glufosinate (GLUF) in human serum. After protein precipitation using acetonitrile and solid-phase extraction, serum samples were derivatized and analyzed by gas chromatography–mass spectrometry (GC–MS). The assay was linear over a concentration range of 3–100.0 μg/ml for GLYP, AMPA, and GLUF. The overall recoveries for the three compounds were >73%. The intra- and inter-day variations were <15%. Precision and accuracy were 6.4–10.6% and 88.2–103.7%, respectively. The validated method was applied to quantify the GLYP and AMPA content in the serum of a GLYP-poisoned patient. In conclusion, the method was successfully applied for the determination of GLYP and its metabolite AMPA in serum obtained from patient of GLYP-poisoning.
Keywords: Glyphosate; Serum; Gas chromatography–mass spectrometry;

Rasagiline is a highly potent, selective and irreversible second-generation monoamine oxidase inhibitor with selectivity for type B of the enzyme (MAO-B). The present studies aimed at developing and validating a rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for determination of rasagiline in human plasma and urine. LC–MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI+) and selected reaction monitoring (SRM). The assay for rasagiline was linear over the range of 0.01–40 ng/mL in plasma and 0.025–40 ng/mL in urine. It took 5.5 min to analyze a sample. The average recoveries in plasma and urine samples were both >85%. The RSD of precision and bias of accuracy were less than 15% and 10%, respectively, of their nominal values based on the intra- and inter-day analysis. The developed method was proved to be suitable for use in clinical pharmacokinetic study after single oral administration of 0.5, 1 and 2 mg rasagiline mesylate tablets in healthy Chinese volunteers.
Keywords: Rasagiline; Human pharmacokinetics; Urine excretion; LC–MS/MS;

A sensitive, rapid LC–MS/MS assay has been developed and validated for the simultaneous quantification of CPT-11 and its two principal metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38), and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxy-camptothecin (APC) in human liver microsomal fractions and plasma. The method was linear over the ranges of 1.56–100 ng/mL, 3.13–150 ng/mL, and 0.78–100 ng/mL for CPT-11, SN-38, and APC, respectively. The total run time was 7.0 min. This assay offers advantages in terms of expediency, recovery of analytes, and suitability for the analysis of CPT-11 and its metabolites in various biological fluids.
Keywords: Irinotecan; Tandem mass spectrometry; Drug metabolites;

The aim of this study was the development of a method for the determination of 12 urinary monohydroxy metabolites of PAHs, namely 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 9-hydroxyfluorene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene, and 3-hydroxybenzo[a]pyrene. Analytes were determined in the presence of deuterated analogues as internal standards, by GC/MS operating in the electron impact mode. Sample preparation was performed by enzymatic hydrolysis of glucoronate and sulphate conjugates of hydroxy metabolites of PAHs, liquid–liquid extraction with n-hexane, and derivatization with a silylating reagent. The method is very specific, limits of quantification are in the range 0.1–1.4 μg/l, and range of linearity is from limit of detection to 208 μg/l. Within- and between-run precision, expressed as coefficient of variation, are <20%; accuracy for most analytes is within 20% of the theoretical value. An application of the proposed method to the analysis of 10 urine samples from coke-oven workers shows that 1-hydroxynaphthalene and 2-hydroxyfluorene were the most abundant compounds (median 61.4 and 69.7 μg/l, respectively), while 6-hydroxychrysene, and 3-hydroxybenzo[a]pyrene were always below the quantification limit.
Keywords: Polycyclic aromatic hydrocarbons; Urinary metabolites; GC/MS; Biological monitoring;

HPLC–APCI–MS for the determination of vitamin K1 in human plasma: Method and clinical application by Qinxin Song; Aidong Wen; Li Ding; Li Dai; Lin Yang; Xiemin Qi (541-545).
A sensitive HPLC–APCI–MS method for the determination of vitamin K1 (VK-1) in human plasma was established. Target ions at [M+H]+ m/z 451.5 for VK-1 and [M+H]+ m/z 331.4 for the I.S. (teprenone). Calibration curve was linear over the range of 0.3–1000 ng/ml. The lower limit of quantification was 0.3 ng/ml. The intra- and inter-batch variability values were less than 8% and 15%, respectively. The C max was 210.1 ± 86.7 ng/ml while the elimination half-life (t 1/2) was 8.8 ± 1.7 h and time to the C max was 5.5 ± 0.8 h after administration of soft capsule containing 10 mg VK-1.
Keywords: Vitamin K1; HPLC–APCI–MS; Teprenone; Pharmacokinetics;

Validated high performance liquid chromatography–UV detection method for the determination of daptomycin in human plasma by Jens Martens-Lobenhoffer; Jan T. Kielstein; Catrin Oye; Stefanie M. Bode-Böger (546-550).
Daptomycin is the first approved member of the new class cyclic lipopeptide antibiotic drugs, effective against a broad spectrum of Gram-positive bacteria. Here we present an HPLC method with UV detection capable to obtain pharmacokinetic data of daptomycin in human plasma, exemplarily shown in a critically ill patient with acute renal failure undergoing extended daily dialysis. Sample preparation consists only of protein precipitation with methanol. Chromatographic separation was achieved on a Zorbax Eclipse XDB-C8 column and daptomycin was detected at 224 nm. The calibration function was linear over the range from 3.5 to 350 μg/ml. The relative standard deviations were < 2% in the intra-day and < 6% in the inter-day measurements. The accuracy was always better than 7%. Daptomycin was stable in aqueous solutions for 2 months frozen at–20 °C. However, in plasma frozen at −20 °C a loss of 25% in 1 month was observed.
Keywords: Daptomycin; HPLC; UV; Stability;

Simultaneous determination of three carbapenem antibiotics in plasma by HPLC with ultraviolet detection by Tiphaine Legrand; Stéphanie Chhun; Elisabeth Rey; Benoît Blanchet; Jean-Ralph Zahar; Fanny Lanternier; Gérard Pons; Vincent Jullien (551-556).
A simple, precise and accurate high-performance liquid chromatography (HPLC) method using ultraviolet (UV) detection has been developed for simultaneous determination of carbapenem antibiotics: imipenem, meropenem and ertapenem in human plasma. Samples were spiked with ceftazidime as internal standard and proteins were precipitated by acetonitrile. Separation was achieved on a C8 column with a mobile phase composed of phosphate buffer 0.1 M (pH 6.8) and methanol in gradient elution mode. Detection was performed at 298 nm. Calibration curves were linear from 0.5 to 80 mg/L for each compound, with correlation coefficients over 0.997. Intra- and inter-day validation studies showed accuracy between −4.5 and 8.1% and precision below 10.4%. Mean recoveries were 82.2, 90.8 and 87.7% for imipenem, meropenem and ertapenem, respectively. This method provides a useful tool for the therapeutic drug monitoring of carbapenems.
Keywords: Imipenem; Meropenem; Ertapenem; Carbapenem; HPLC; Ultraviolet detection;

A rapid, robust and selective on-line solid-phase extraction–liquid chromatographic method with ultra-violet detection (on-line SPE-LC-UV) for microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitor screening was developed and validated. Disrupted A549 cells were used as mPGES-1 source and the formation of prostaglandin E2 (PGE2) out of the substrate prostaglandin H2 (PGH2) was determined at 195 nm. Direct on-line sample clean up was achieved by automated column switch (C18 trap column) prior isocratic separation using a C18 analytical column. The on-line SPE-LC-UV method was accurate, precise and reproducible in the range of 71–1763 ng/ml for PGE2 and met the generally accepted criteria for bioanalytical methods. The method was successfully applied to determine the IC50 value of the known mPGES-1 inhibitor NS-398.
Keywords: mPGES-1; On-line SPE-LC-UV; PGE2;

Quantitative determination of 5-aminolaevulinic acid and its esters in cell lysates by HPLC-fluorescence by Francesca Giuntini; Ludovic Bourré; Alexander J. MacRobert; Michael Wilson; Ian M. Eggleston (562-566).
The development of a reliable sensitive method for the HPLC determination of 5-aminolaevulinic acid (ALA) and ALA esters in cell lysates is described. The method relies on the quantification of a fluorescent derivative of ALA following its derivatisation with acetylacetone and formaldehyde. Following this procedure it is possible to quantify ALA in cell lysates with no need for pre-purification of the sample. The method has been validated in two ranges of concentration (0.6–65 μM, 0.1–10 μg/mL, and 30–600 μM, 5–100 μg/mL), and has also been extended and validated for the determination of ALA released from ALA prodrugs after acidic hydrolysis.
Keywords: 5-Aminolaevulinic acid; Photodynamic therapy; HPLC; Fluorescence; Porphyria;

Searching biomarker candidates in serum using multidimensional native chromatography by Stefan Kreusch; Margarete Schulze; Gerhard A. Cumme; Günter Ditze; Thomas Moore; Heidrun Rhode (567-572).
The microplate-based method developed by our group for non-denaturing multidimensional proteome separation was improved on using improved column arrays and a newly developed robot. Currently size exclusion, anion exchange and lectin affinity chromatography are combined orthogonally. Different samples run simultaneously to enhance reliability of intercomparison. LC–ESI (electro-spray ionization) MS/MS analysis of selected fractions identified 32,288 peptides matching 2669 serum proteins. The present contribution (I) shows the characteristics of the method, whereas “prove of principle” by applying it to search for biomarker candidates with model diseases is reported in an accompanying paper (II).
Keywords: Human serum proteome; Multidimensional chromatography; Microplate;

Determination of metaldehyde in human serum by headspace solid-phase microextraction and gas chromatography–mass spectrometry by Takeshi Saito; Seiji Morita; Megumi Motojyuku; Kazuki Akieda; Hiroyuki Otsuka; Isotoshi Yamamoto; Sadaki Inokuchi (573-576).
A rapid headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS–SPME–GC–MS) method has been developed for the determination of metaldehyde in human serum samples. Metaldehyde is extensively used as a molluscicide for the control of slugs and snails, and cases of metaldehyde poisoning have been reported. Metaldehyde was headspace-extracted on a polydimethylsiloxane (PDMS) fiber at 70 °C for 25 min, desorbed, and analyzed rapidly by GC–MS. The method was validated for limit of detection (LOD), linearity, precision, and recovery. Although the recovery of the sample was very low, the method itself was rapid with a low detection limit of 0.25 μg/ml, R.S.D. value 12.6%, and linearity range 0.5–25.0 μg/ml (r 2  = 0.999). The results demonstrated that the SPME–GC–MS method for the analysis of metaldehyde is simple, rapid, solvent-free, and does not require any pre-analysis conversions.
Keywords: Headspace solid-phase microextraction; GC–MS; Metaldehyde; Serum;

Determination of urinary triclosan by stir bar sorptive extraction and thermal desorption–gas chromatography–mass spectrometry by Migaku Kawaguchi; Rie Ito; Hidehiro Honda; Naoyuki Endo; Noriya Okanouchi; Koichi Saito; Yasuo Seto; Hiroyuki Nakazawa (577-580).
We have developed an analytical method for the determination of urinary 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), which utilizes stir bar sorptive extraction (SBSE) and thermal desorption (TD)–gas chromatography–mass spectrometry (GC–MS). Human urine sample is de-conjugated by treatment with β-glucuronidase and sulfatase. A stir bar coated with polydimethylsiloxane (PDMS) is added to the urine sample in a vial and the sample is stirred for 60 min at room temperature (25 °C). Then, the PDMS stir bar is subjected to TD–GC–MS. The detection limit of triclosan is 0.05 ng mL−1. The method shows linearity over the calibration range (0.1–10 ng mL−1) and the correlation coefficient (r) is higher than 0.993 for triclosan standard solution. The average recoveries of triclosan in human urine sample are 102.8–113.1% (RSD: 2.4–6.7%). This simple, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in human urine samples.
Keywords: Triclosan; Urine sample; Stir bar sorptive extraction (SBSE); Thermal desorption (TD); Gas chromatography–mass spectrometry (GC–MS);