Journal of Chromatography B (v.872, #1-2)

Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry by Irene van den Broek; Rolf W. Sparidans; Jan H.M. Schellens; Jos H. Beijnen (1-22).
With the growing interest for peptides and proteins in different kinds of fields, e.g. pharmacy, clinical diagnostics or food industry, the quantification of these compounds is becoming more and more important. Quantitative analysis of these analytes in biological matrices, however, remains a challenging task, due to the complexity of both the matrix and the analytical characteristics of these large bio-molecules. Liquid chromatography coupled to (tandem) mass spectrometry (LC–MS or LC–MS/MS) is the preferred analytical technique for peptide analysis as it allows very selective and sensitive measurements. This article summarizes the numerous published LC–MS applications for the quantification of peptides in biological matrices and discusses all different issues herewith concerned. This includes chromatographic aspects as the selection and effects of mobile and stationary phase, flow rate and temperature, as well as mass spectrometric characteristics such as ionization and detection modes, collision-induced dissociation of peptides and factors influencing the mass spectrometric response. For both techniques the main properties of all described methods have been listed, creating a comprehensive overview with the peptide analytes divided into different classes. Likewise, all other issues concerned with quantitative bioanalysis have been evaluated in detail, including extensive consideration of several different applied sample pre-treatment techniques and reflection of subjects as the choice for an internal standard and assay validation. Furthermore, several issues which are of particular interest for the quantitative bioanalysis of peptide compounds like peptide adsorption and degradation have been regarded.
Keywords: Bioanalysis; Peptides; Quantification; Liquid chromatography–mass spectrometry; Sample pre-treatment;

The way forward, enhanced characterization of therapeutic antibody glycosylation: Comparison of three level mass spectrometry-based strategies by Elsa Wagner-Rousset; Audrey Bednarczyk; Marie-Claire Bussat; Olivier Colas; Nathalie Corvaïa; Christine Schaeffer; Alain Van Dorsselaer; Alain Beck (23-37).
Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs. First a top-down approach was used on intact and reduced MAbs by liquid chromatography coupled to an electrospray ionization-time of flight mass spectrometer (LC–ESI-TOF), which provided fast and accurate profiles of MAbs glycosylation patterns for routine controls. Secondly, after digestion of the antibody with the peptide N-glycosidase F (PNGase F) enzyme, released N-linked glycans were directly analyzed by electrospray ionization–tandem mass spectrometry (ESI–MS/MS) without any prior derivatization, which gave precise details on the structure of the most abundant glycoforms. Finally, a bottom-up approach on tryptic glycopeptides using a nanoLC-Chip–MS/MS ion trap (IT) system equipped with a graphitized carbon column was investigated. Data were compared to those obtained with a more classical C18 reversed phase column showing that this last method is well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.
Keywords: Therapeutic monoclonal antibodies; NS0 and CHO cell lines; Glycosylation; Oligosaccharides; N-glycans; Glycopeptides; Mass spectrometry; Tandem mass spectrometry; LC–ESI-TOF; RP-HPLC; LC–MS; NanoLC-Chip; Graphitized carbon;

A new straightforward method based on cloud-point extraction (CPE) has been developed, optimized and validated for the determination of venlafaxine in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection. The non-ionic surfactant Triton X-114 (polyethylene glycol tert-octylphenyl ether) was chosen as the extract solvent. Separation was obtained using a reversed-phase Diamonsil column (C18, 250 mm × 4.6 mm I.D., 5 μm) and a mobile phase composed of acetonitrile–phosphate buffer solution (pH 3.0)–triethylamine (33.5:66.5:0.4). Fluorescence detection was used (λ ex 276 nm, λ em 598 nm). Maprotiline was used as the internal standard. Under the optimum conditions, the linear range of venlafaxine in human plasma was 10–800 ng mL−1 (r 2  = 0.9995). The limit of detection (LOD) was less than 2 ng mL−1 (S/N = 3) and the limit of quantification (LOQ) was less than 10 ng mL−1 (S/N = 10). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsules in nine healthy volunteers.
Keywords: Venlafaxine; Cloud-point extraction; High-performance liquid chromatography; Triton X-114; Pharmacokinetics;

A high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method allowing the sensitive and specific quantification of rivaroxaban (BAY 59-7939), a Factor Xa inhibitor in advanced development for the prevention and treatment of thromboembolic disorders, in human plasma is described. After precipitation of plasma proteins with methanol containing the internal standard followed by centrifugation, the plasma supernatant was injected directly onto the HPLC–MS/MS system. Concentrations could be determined between 0.50 and 500 μg/L. Inter-assay precision was ≤7.4% and inter-assay accuracy was between 96.3 and 102.9% throughout the entire working range. The method was applied successfully in several clinical studies, which allowed an accurate determination of rivaroxaban pharmacokinetics in human plasma.
Keywords: Rivaroxaban; Factor Xa inhibitor;

Perphenazine is a phenothiazine-type antipsychotic that is a potential candidate for sublingual administration due to its extensive first-pass metabolism. In this study, a gas chromatographic–mass spectrometric method was developed for quantification of perphenazine in rabbit plasma after sublingual administration. The plasma samples were purified by mixed-mode solid phase extraction with good recovery (>83%). The method was linear (r 2  > 0.99) over a range of 2–64 ng/ml, with a lower limit of quantification of 2 ng/ml. The accuracy was 100 ± 4%, and the within-day and between-day precisions were <6.8% R.S.D. and <14% R.S.D., respectively. Perphenazine was stable in stock solutions and plasma. The method was successfully applied for analysing perphenazine in plasma after sublingual administration to rabbits.
Keywords: Perphenazine; GC–MS; Rabbit plasma; Sublingual administration;

Simultaneous determination of triazolam and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry by Ryh-Nan Pan; Chang-Ching Lin; Pei-Wei Huang; Cheng-Huei Hsiong; Li-Heng Pao (58-62).
A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of triazolam and its metabolites, alpha-hydroxytriazolam (α-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), was developed and validated. Triazolam-D4 was used as the internal standard (IS). This analysis was carried out on a Thermo® C18 column and the mobile phase was composed of acetonitrile:H2O:formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) and quantification was performed by multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 343.1 → 308.3, 359.0 → 308.3, 359.0 → 111.2 and 347.0 → 312.0 for triazolam, α-OHTRZ, 4-OHTRZ and triazolam-D4, respectively. LLOQ of the analytical method was 0.05 ng/mL for triazolam and 0.1 ng/mL for α-OHTRZ and 4-OHTRZ. The within- and between-run precisions were less than 15.26% and accuracy was −8.08% to 13.33%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of triazolam in healthy Chinese volunteers.
Keywords: Triazolam; α-OHTRZ; 4-OHTRZ; LC–MS/MS; Pharmacokinetic;

Hollow-fiber-supported liquid phase microextraction with in situ derivatization and gas chromatography–mass spectrometry for determination of chlorophenols in human urine samples by Rie Ito; Migaku Kawaguchi; Hidehiro Honda; Youji Koganei; Noriya Okanouchi; Norihiro Sakui; Koichi Saito; Hiroyuki Nakazawa (63-67).
A simple and highly sensitive method that involves hollow-fiber-supported liquid phase microextraction (HF-LPME) with in situ derivatization and gas chromatography–mass spectrometry (GC–MS) was developed for the determination of chlorophenols (CPs) such as 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TrCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) in human urine samples. Human urine samples were enzymatically de-conjugated with β-glucuronidase and sulfatase. After de-conjugation, HF-LPME with in situ derivatization was performed. After extraction, 2 μl of extract was carefully withdrawn into a syringe and injected into the GC–MS system. The limits of detection (S/N = 3) and quantification (S/N > 10) of CPs in the human urine samples are 0.1–0.2 ng ml−1 and 0.5–1 ng ml−1, respectively. The calibration curve for CPs is linear with a correlation coefficient of >0.99 in the range of 0.5–500 ng ml−1 for DCP and TrCP, and of 1–500 ng ml−1 for TeCP and PCP, respectively. The average recoveries of CPs (n  = 6) in human urine samples are 81.0–104.0% (R.S.D.: 1.9–6.6%) with correction using added surrogate standards. When the proposed method was applied to human urine samples, CPs were detected at sub-ng ml−1 level.
Keywords: Chlorophenols (CPs); Hollow-fiber (HF); Liquid phase microextraction (LPME); Gas chromatography–mass spectrometry (GC–MS); In situ derivatization;

We present herein an ultra-fast quantitative assay for the quantitation of saquinavir in human plasma, without prior chromatographic separation, with matrix-assisted laser desorption/ionization using the selected reaction monitoring quantitation mode (MALDI-SRM/MS). The method was found to be linear from 5 to 10,000 ng/ml using pentadeuterated saquinavir (SQV-d5) as an internal standard, and from 5 to 1000 ng/ml using reserpine as internal standard (IS). Accuracy and precision were in the range of 101–108%, 3.9–11% with SQV-d5 and in the range 93–108%, 3.5–15% with reserpine. Plasma samples (250 μl) were extracted with a mixture of ethyl acetate/hexane. MALDI spotting of the extract was automated using electrodeposition and the dried droplet method using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. A 96 spots MALDI plate was prepared within 20 min in a fully unattended manner. Each sample was spotted four times and quantitation was based on the average of their analyte/IS area ratio. Samples were analyzed on a triple quadrupole linear ion trap (QqQLIT) equipped with a high repetition laser source (1000 Hz). The analysis time of one sample was approximately 6 s, therefore 96 samples could be analyzed in less than 10 min. With liquid–liquid extraction sample preparation no significant matrix effects were observed. Moreover, the assay showed sufficient selectivity for samples to be analyzed at the lower limit of quantification (LLOQ) in the presence of other antiretroviral drugs, without prior chromatographic steps. In parallel, to assess the selectivity of the assay with real samples, a liquid chromatography (LC)–SRM/MS method was developed and a cross validation with clinical samples was successfully performed.
Keywords: MALDI; Saquinavir; Quantitative analysis; Mass spectrometry; Human plasma; High-throughput;

Liquid chromatography–tandem mass spectrometric assay for diclofenac and three primary metabolites in mouse plasma by Rolf W. Sparidans; Jurjen S. Lagas; Afred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (77-82).
The first liquid chromatography–tandem mass spectrometric assay for the simultaneous determination of diclofenac, 4′-hydroxy-diclofenac, 5-hydroxy-diclofenac and diclofenac-acyl-glucuronide in mouse plasma, using a simple sample pre-treatment procedure, was developed and validated. Analytes in plasma were stabilized using acetic acid and ascorbic acid. After addition of the internal standard D4-diclofenac to a 10 μl sample volume and protein precipitation with acetonitrile, the supernatant was supplemented with an equal volume of water and injected into the chromatographic system. A polar embedded reversed-phase column with gradient elution using formic acid and ammonium acetate in water–methanol were used. The eluate was totally transfered into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the ranges 10–5000 ng/ml for 4′-hydroxy-diclofenac and 20–10,000 ng/ml for the other analytes, the lowest levels of these ranges (10 or 20 ng/ml) being the lower limits of quantification. Within day precisions were ≤10%, between day precisions ≤13% and accuracies were between 90 and 108%. The analytes were chemically stable under all relevant conditions. The assay was successfully applied in a pharmacokinetic study with diclofenac in mice.
Keywords: Diclofenac; 4′-Hydroxy-diclofenac; 5-Hydroxy-diclofenac; Diclofenac-acyl-glucuronide; LC/MS/MS; Mouse plasma;

The bacterium Brenneria salicis is the causal agent of watermark disease in willow. This work shows the importance of in situ studies and high-resolution separation of biological samples with ultrahigh performance liquid chromatography combined with ion trap mass spectrometry to unambiguously identify molecular compounds associated with this disease. Approximately 40 oligolignols accumulated in wood sap of watermark diseased willow, and are indicative for degradation of the xylem cell wall, of which 15 were structurally assigned based on an earlier study. Many bacteria are known to produce and release quorum sensing signal molecules that switch on the expression of specific, sometimes pathogenic functions. Two quorum sensing signal molecules, N-(3-oxohexanoyl)-l-homoserine lactone and N-(hexanoyl)-l-homoserine lactone, were present in 4/1 ratios in diseased wood and in high-density in vitro cultures of B. salicis at 0.13–1.2 μM concentrations, and absent in healthy wood and in low-density in vitro cultures of B. salicis. Although it is not a proof, it can be an indication for involvement of quorum sensing in B. salicis pathogenesis. Cyclic dipeptides were present at high concentrations in high-density in vitro cultures of B. salicis, but not in situ, and were found not to be involved in quorum sensing signaling, therefore, the attribution of quorum signal properties to cyclic dipeptides isolated from in vitro cultures of pathogenic bacteria should be reconsidered.
Keywords: Watermark disease; Brenneria salicis; Quorum sensing; Willow; Oligolignols;

Hydroperoxides of 18:2n-6 and 20:4n-6 were obtained by autoxidation and photooxidation. The enantiomers were separated as free acids (Reprosil Chiral-NR column, eluted with hexane containing 1–1.2% alcoholic modifier) and analyzed by on line UV detection (234 nm) and liquid chromatography-MS/MS/MS of carboxylate anions (A  → (A-18) → full scan) in an ion trap. The combination of UV and MS/MS/MS analysis facilitated identification of hydroperoxides even in complex mixtures of autoxidized or photooxidized fatty acids. The signal intensities increased about two orders of magnitude by raising the isolation width of A from 1.5 amu to 5 or 10 amu for cis-trans conjugated hydroperoxy fatty acids, and one order of magnitude or more for non-conjugated hydroperoxy fatty acids. The S enantiomer of 8-, 9-, 10-, and 13-hydroperoxyoctadecadienoic acids and the S enantiomer of cis-trans conjugated hydroperoxyeicosatetraenoic acids eluted before the corresponding R enantiomer with two exceptions (11-hydroperoxylinoleic acid and 8-hydroperoxyeicosa-5Z,9E,11Z,14Z-tetraenoic acid). The separation of enantiomers or regioisomers could be improved by the choice of either isopropanol or methanol as alcoholic modifier.
Keywords: Autoxidation; Hydroperoxy eicosanoids; Hydroperoxy linoleic acids; MS/MS fragmentation; Normal phase HPLC; Photooxidation; Reprosil Chiral-NR;

A new HPLC method for the determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) in human plasma was developed and validated. Plasma samples were analyzed after protein precipitation with methanol and the two analytes were separated using a C18 column with a mobile phase composed of 17.5 mmol/L acetic acid (pH 3.3):acetonitrile = 85:15 (v/v) at 0.2 mL/min flow rate. 4-ASA and N-Ac-4-ASA were used as internal standards. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in negative ionization mode and in multiple reaction monitoring acquisition (m/z 152 → 108 for 5-ASA; m/z 194 → 150 and 194 → 107 for N-Ac-5-ASA). The limit of quantification (LOQ) was 50 ng/mL for both analytes (0.2 ng injected) and matrix-matched standard curves showed linearity up to 4000 ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively ≤6.3% and ≤11% for 5-ASA and ≤8.0% and ≤10% for N-Ac-5-ASA. For both analytes the within- and between-batch accuracy (bias%) values ranged respectively from −8.4% to 7.9% and from −7.9% to 8.0%. The overall recoveries (n  = 6) at three tested concentration levels (i.e. 100, 1000 and 4000 ng/mL) were respectively >90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.% ≤ 10%). The method was applied to evaluate the pharmacokinetic of 5-ASA after a single oral dose administration of this compound (1200 mg) to 24 healthy volunteers. The mean maximum concentration levels were 680 ng/mL for 5-ASA and 1240 ng/mL for N-Ac-5-ASA and the kinetic profiles were in agreement with previous studies.
Keywords: Mesalazine; HPLC-ESI-MS/MS; Method validation; Pharmacokinetic; IBD;

Quantitative analysis of backbone-cyclised peptides in plants by Ivana Saska; Michelle L. Colgrave; Alun Jones; Marilyn A. Anderson; David J. Craik (107-114).
Progress in understanding the biosynthetic pathway of the cyclotides has been hampered as this unique family of cyclic plant peptides are notoriously difficult to analyse by standard proteomic approaches such as gel electrophoresis. We have developed a simple, rapid and robust strategy for the quantification of cyclotides in crude plant extracts using MALDI-TOF MS making use of generic peptides similar in mass to the analyte as internal standards for calibration. Linearity (r 2  > 0.99) over two orders of magnitude (down to femtomole levels) was achieved in plant extracts, allowing quantitative analysis of transgenic and endogenous peptide expression.
Keywords: Cyclotides; MALDI-TOF MS; Plant peptides; Quantification;

Determination of rimonabant in human plasma and hair by liquid chromatography–mass spectrometry by Stanislas Grassin Delyle; Emuri Abe; Philippe Devillier; Jean Claude Alvarez (115-120).
Rimonabant is the first therapeutically relevant cannabinoid antagonist, licensed in Europe for treatment of obesity when a risk factor is associated. The objective of this study was to develop and validate a method for measurement of rimonabant in human plasma and hair using liquid chromatography coupled to mass spectrometry (LC–MS/MS). Rimonabant and AM-251 (internal standard) were extracted from 50 μL of plasma or 10 mg of hair using diethylether. Chromatography was performed on a 150 mm × 2.1 mm C18 column using a mobile phase constituted of formate buffer/acetonitrile. Rimonabant was ionized by electrospray in positive mode, followed by detection with mass spectrometry. Data were collected either in full-scan MS or in full-scan MS/MS mode, selecting the ion m/z 463.1 for rimonabant and m/z 555.1 for IS. The most intense product ion of rimonabant (m/z 380.9) and IS (m/z 472.8) were used for quantification. Calibration curves covered a range from 2.5 (lower limit of quantification) to 1000.0 ng/mL (upper limit of quantification) in plasma and from 2.5 to 1000.0 pg/mg in hair. Validation results demonstrated that rimonabant could be accurately and precisely quantified in both matrixes: accuracy and precision were within 85–115% and within 15% of standard deviation, respectively. Stability studies in plasma showed that rimonabant was stable during the assay procedure, but a 30% decrease was observed for one concentration after 3 weeks at −20 °C. This simple and robust LC–MS/MS method can be used for measuring rimonabant concentrations in human plasma and hair either in clinical or in forensic toxicology.
Keywords: Rimonabant; Liquid chromatography; Mass spectrometry; Human plasma; Human hair; Cannabinoid antagonist;

A fast, sensitive, and enantioselective LC–MS/MS bioanalytical method was developed and validated for the direct determination of individual alprenolol enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP) along with supported liquid extraction (SLE) procedures. Complete baseline separation of enantiomeric alprenolol was achieved within 2 min in reversed phase chromatography at 0.9 ml/min. SLE in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.500–500 ng/ml for each alprenolol enantiomer using 0.0500 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤7.3% relative standard deviation (RSD) and −6.2 to 8.0% relative error (RE) for both alprenolol enantiomers.
Keywords: Chiral separation; Alprenolol; LC–MS/MS; Cellobiohydrolase (CBH); Supported liquid extraction (SLE);

HPLC–MS/MS determination of a hardly soluble drug in human urine through drug–albumin binding assisted dissolution by Ramona Rodila; Grace E. Kim; Leimin Fan; Min S. Chang; Jun Zhang; Huaiqin Wu; Tawakol A. El-Shourbagy (128-132).
ABT-263 is under development for treatment of cancer. In order to support clinical trials, an analytical method for ABT-263 quantification in human urine became necessary. Due to the extremely poor solubility of ABT-263 in aqueous and most common organic solvents, a critical step was to dissolve the drug into urine matrix. Although other potential approaches could be used, addition of powder albumin was found to be the most advantageous. Albumin powder does not significantly alter urine sample volume (≤2.8%) and a range of albumin to urine sample volume ratios can be allowed for full recovery of drug and thus accurate quantification. The procedure is fairly simple and can potentially be a universal approach for compounds with low solubility in urine, but strong protein binding. The method has been validated to support clinical trials.
Keywords: ABT-263; Hardly soluble drug; Hydrophobic drug; Urine; Albumin; Determination; Liquid chromatography; Tandem mass spectrometry; Plasma protein binding;

Quantification of urinary F2-isoprostanes with 4(RS)-F4t-neuroprostane as an internal standard using gas chromatography–mass spectrometry by Emilie Mas; Françoise Michel; Alexandre Guy; Valérie Bultel; Yoan Falquet; Patrick Chardon; Jean-Claude Rossi; Jean Paul Cristol; Thierry Durand (133-140).
Isoprostanes are a family of prostaglandin isomers produced from oxidation of polyunsaturated fatty acids through a non-enzymatic free radical-catalyzed mechanism. Quantification of F2-isoprostanes (F2-IsoPs) provides a good index of oxidative stress and allows non-invasive assessment of lipid peroxidation in vivo. Since “interferences peaks” at m/z 573 co-elute with d 4-15-F2t-IsoP preferentially used, we propose a new GC–NICI-MS approach to quantify urinary F2-IsoPs by using 4(RS)-F4t-neuroprostane as the internal standard. This method was applied to quantify urinary F2-IsoPs excretion in healthy volunteers and polytraumatized patients. Our results showed a significant increase (p  < 0.0001) in urinary F2-IsoPs in polytraumatized patients compared with healthy volunteers (4.73 ± 2.75 ng/mg vs. 0.811 ± 0.359 ng/mg creatinine).
Keywords: Isoprostanes; GC–MS; 15-F2t-IsoP; 4(RS)-F4t-NeuroP; Polytraumatized patients;

ABT-888, a poly(ADP-ribose) polymerase (PARP) -inhibitor in clinical trials, potentiates DNA-damaging agents. We developed and validated, according to FDA guidelines, an LC–MS assay for sensitive, accurate and precise quantitation of ABT-888 and its metabolite M8 in 0.2 mL human plasma. After ethyl acetate extraction, separation is achieved with a hydro-Synergi column and a 0.1% formic acid in acetonitrile/water-gradient. Detection uses electrospray, positive-mode ionization mass spectrometry. Between 10 (LOQ) and 1000 ng/mL, accuracy was 95.5–98.5% for ABT-888 and 91.4–100.9% for M8, and precision was 0.1–4.9% for ABT-888 and 0–13.7% for M8. The assay is being applied to samples generated in several clinical trials.
Keywords: ABT-888; Mass spectrometry; PARP-inhibitor;

A sensitive and specific LC–MS/MS method employing positive electrospray ionization for the determination of ARQ 501 (β-lapachone) in (nu/nu) mouse plasma and tumor tissue is described. Samples were processed using protein precipitation with acetonitrile. A d6 analog of ARQ 501 was used as the internal standard (IS). The analytes were separated using a Zorbax SB8 column (30 mm × 2.1 mm i.d. 5 μm particle size) and analyzed in the multiple reaction monitoring (MRM) mode using mass transitions of 243 > 159 and 249 > 159  m/z for ARQ 501 and d6-ARQ 501, respectively. The lower limit of quantitation (LLOQ) for ARQ 501 was 3.0 ng/mL. The calibration curve was linear in the range of 3.0–2000 ng/mL with a correlation coefficient better than 0.99. Intra- and inter-batch precisions were within 8.4% for plasma and 11.8% for tumor samples. Accuracy expressed as percentage relative error (%R.E.) ranged from −9.0 to 7.7 for both plasma and tumor samples. Recovery was between 106 and 113% for both ARQ 501 and its d6 analog. Plasma pharmacokinetic data of ARQ 501 in mouse from intraperitoneal (IP) dosing at 60 mg/kg obtained using this validated method is presented along with tumor concentration data. The C max, AUC(0−∞), t 1/2, Cl/F, and V d/F were determined to be 4016 ng/mL, 4392 h ng/mL, 3.9 h, 13.7 L/h/kg, and 76.5 L/kg, respectively. Tumor tissue concentrations were in the range 1–2 μM for approximately 2 h post-dose.
Keywords: β-Lapachone; LC–MS/MS; Electrospray ionization; HPLC; Pharmacokinetics; Mouse plasma; Mouse tumor;

Simultaneous quantitation of seven endogenous C-21 adrenal steroids by liquid chromatography tandem mass spectrometry in human serum by Valdemir Melechco Carvalho; Odete Hirata Nakamura; José Gilberto Henriques Vieira (154-161).
Quantitation of endogenous steroids is important in the diagnosis of several endocrine disorders. In this study we present a new method for simultaneous quantitation of cortisol, cortisone, 11-desoxycortisol, 21-desoxycortisol, corticosterone, 17-hydroxyprogesterone and 11-desoxycorticosterone in human serum by on-line extraction and LC–MS/MS. Analytes extraction was performed on-line using a 2-position and 6-port valve equipped with a monolithic silica cartridge. After chromatographic separation of all analytes, detection was performed in the multiple reaction monitoring mode using positive atmospheric pressure chemical ionization mode. Total imprecision of the assay ranged from 5.5 to 15.5%. Comparison with immunoassays yielded coefficients of 0.893 for cortisol, 0.848 for 11-desoxycortisol and 0.924 for 17-hydroxyprogesterone. The sensitivity of this method provides meaningful data for patients within normal and elevated range and it may be useful for the diagnosis of a variety of adrenal dysfunctions.
Keywords: 11-Desoxycortisol; 21-Desoxycortisol; Corticosterone; 11-Desoxycorticosterone; 17-Hydroxyprogesterone; LC–MS/MS;

The quality coefficient (Q) has frequently been used to select weighting formulae in calibration, and especially so in bioanalytical work, where there has been increasing awareness of the importance of data heteroscedasticity in recent years. However, this quantity is statistically flawed and should not be used for this purpose. The quality coefficient is computed from the differences between the apparent and true concentrations of the calibration samples as obtained from the least-squares calibration fit. Q is defined as the sum of either the squares or the absolute values of these differences, taken directly or as percentage (relative) deviations. It is calculated for several different trial weighting formulae, and the lowest Q value is then deemed to identify the best weighting choice. However, these Qs are predisposed to favor data consistent with their definitions—homoscedastic data for tests employing absolute differences, and data having proportional error (constant coefficient of variance) for tests using relative differences—because the Q in each case closely resembles the quantity actually minimized by the least-squares fit of the calibration data. The problem is examined and illustrated through Monte Carlo computations on data having either constant or proportional uncertainty and subjected to both tests. A modified Q based on the results of both the absolute and relative tests is much more reliable than either test alone but is still not recommended as a solution to the weighting problem, as other, statistically sound approaches are available and readily used.
Keywords: Calibration; Weighted least squares; Quality coefficient; Monte Carlo; Heteroscedasticity;

Automated liquid–liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) for the quantitation of methoxsalen in human plasma by Manish Yadav; Pritesh Contractor; Vivek Upadhyay; Ajay Gupta; Swati Guttikar; Puran Singhal; Sailendra Goswami; Pranav S. Shrivastav (167-171).
A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid–liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2 mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10 mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5 mL/min. The linear dynamic range was established over the concentration range 1.1–213.1 ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5 min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10 mg extended release methoxsalen formulation under fasting condition.
Keywords: Methoxsalen; 96-Well plate liquid–liquid extraction; UPLC–MS/MS; High throughput; Human plasma;

This paper described a method for quantification of ulifloxacin, the active metabolite of prulifloxacin in human plasma by capillary zone electrophoresis using lomefloxacin as the internal standard. The separation was carried out at 25 °C in a 60.2 cm × 75 μm fused-silica capillary with an applied voltage of 20 kV using 200 mM borate buffer (pH 10.5). The detection wavelength was 275 nm. Clean-up and preconcentration of the samples were developed by 96-well format solid-phase extraction. 0.25 ml of plasma sample and 0.25 ml of IS were loaded onto the preconditioned wells, and the wells were washed using 1 ml of 20% methanol in acid water (1% phosphoric acid), and the analytes were eluted using 1 ml of 95/5 methanol/ammonia water. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and robustness. The calibration graph was linear for ulifloxacin from 0.02 to 2 μg/ml. The lower limit of quantification was 0.02 μg/ml. The intra- and inter-day precisions were within 4.0 and 8.2%, respectively. The method developed was successfully applied to the evaluation of clinical pharmacokinetic study of prulifloxacin formulation product after oral administration to healthy volunteers.
Keywords: Prulifloxacin; Ulifloxacin; 96-Well format SPE; CZE;

Sample preparation method for plasma membrane proteome analysis by Seung-Ah Park; Mi-Ryung Kim; Pan-Kyeom Kim; Hyung-Don Cho; Gi-Yeon Han; Chan-Wha Kim (177-180).
The preparation of plasma membrane (PM) proteome samples is seriously difficult and time-consuming, owing to their profound hydrophobicity and low abundance. We have developed an efficient PM sample preparation method using Ultracentrifugation with Percoll and an aqueous two-phase extraction. The developed method was rapid (3 h) and provided high purities (26-fold of cell lysate) with a high yield (2.6% of whole cell lysate proteins). This method is especially useful for PM proteome studies using 2D gel electrophoresis.
Keywords: hMSCs; Percoll; Plasma membrane; Two-phase extraction; Two-dimensional electrophoresis; Ultracentrifugation;

Preparative high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) was used to isolate and separate bioactive constituents from the roots of Aconitum coreanum. Two new diterpenoid alkaloid isomers were successfully separated for the first time by HSCCC with an optimized two-phase solvent system composed of ethyl acetate–n-butanol–methanol–2% acetic acid (3.5:1.5:2:4.5, v/v/v/v), 25.4 mg of GFT (1) and 18.3 mg of GFU (2) were isolated form 1 g crude extract in one step HSCCC experiment. The purities of the two new compounds were all over 95% as analyzed by HPLC and their structures were identified by ESI–MS, 1H NMR, 13C NMR, and 2D NMR analysis.
Keywords: Counter-current chromatography; Aconitum coreanum; Preparative separation and purification; Isomeric diterpenoid alkaloids;

Simultaneous determination of dibucaine and naphazoline in human serum by monolithic silica spin column extraction and liquid chromatography–mass spectrometry by Takeshi Saito; Seiji Morita; Izumi Kishiyama; Shota Miyazaki; Akihiro Nakamoto; Manami Nishida; Akira Namera; Masataka Nagao; Sadaki Inokuchi (186-190).
A simple, sensitive, and specific liquid chromatography–mass spectrometry (LC–MS) method for simultaneous determination of dibucaine and naphazoline from serum was developed and validated. The extraction procedure was performed using a monolithic silica spin column. Chromatographic separation of dibucaine and naphazoline was achieved on a C18 reverse phase column with a mobile phase gradient (mobile phase A: 10 mM ammonium formate and mobile phase B: acetonitrile) at a flow rate of 0.2 mL/min. LC–MS was operated under the selective ion monitoring mode using the electrospray ionization technique in the positive mode. The retention times for naphazoline, dibucaine, and the internal standard (IS) were 6.7, 7.8, and 8.0 min, respectively. A linear graph was obtained for dibucaine and naphazoline with correlation coefficients >0.998 for all analytes by this method. The limit of quantification of dibucaine and naphazoline was 10 and 25 ng/mL, respectively. The mean recoveries were greater than 70%. Both compounds were stable under conditions of short-term storage, long-term storage as well as after freeze–thaw cycles. Monolithic spin column extraction and LC–MS analysis enabled the separation of dibucaine and naphazoline within 20 min.
Keywords: Dibucaine; Naphazoline; LC–MS; Monolithic silica spin column; Serum;