Journal of Chromatography B (v.877, #18-19)

A rapid and selective method for simultaneous determination of cyclophosphamide and its metabolite carboxyethylphosphoramide mustard (CEPM) was developed using online sample preparation and separation with tandem mass spectrometric detection. Diluted plasma was injected onto an extraction column (Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed with an aqueous solution, and retained analytes were transferred to an analytical column (Gemini 3 μm C18 110A, 100 mm × 2.0 mm) using a gradient mobile phase prior to detection by MS/MS. Analytes were detected in an API-3000 LC-MS/MS system using positive multiple-reaction monitoring mode (m/z 261/140 and 293/221 for CTX and CEPM, respectively). Online extraction recoveries were 76% and 72% for cyclophosphamide and CEPM. Within-day and between-day variabilities were <3.0%, and accuracies were between −6.9% and 5.2%. This method has been used to measure plasma cyclophosphamide and CEPM concentrations in an ongoing Phase II study in children with newly diagnosed medulloblastoma.
Keywords: Cyclophosphamide; Carboxyethylphosphoramide mustard; High turbulence liquid chromatography and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS);

Determination of metoclopramide in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry by Hye Won Lee; Hye Young Ji; Hoe Yoon Kim; Eun-Seok Park; Kang Choon Lee; Hye Suk Lee (1716-1720).
For the rapid, selective and sensitive analysis of metoclopramide in human plasma, hydrophilic interaction chromatography with electrospray ionization tandem mass spectrometric (HILIC/MS/MS) method was developed. This method involved liquid–liquid extraction with dichloromethane followed by separation on an Atlantis HILIC silica column using the mobile phase of acetonitrile–ammonium formate (100 mM, pH 6.5) (85:15, v/v). Analytes were quantified using electrospray ionization mass spectrometry in the selected reaction monitoring mode. The standard curve was linear (r 2  = 0.998) over the concentration range of 2.00–150 ng/mL using 50 μL of plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.8–7.7% and −7.5 to 3.6%, respectively. The matrix effect for metoclopramide and levosulpiride (internal standard) was practically absent. The present method was successfully applied to the pharmacokinetic study of metoclopramide after oral dose of metoclopramide hydrochloride (10 mg) to male healthy volunteers.
Keywords: Metoclopramide; HILIC/MS/MS; Human plasma;

New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma by Silvia De Francia; Antonio D’Avolio; Francesca De Martino; Elisa Pirro; Lorena Baietto; Marco Siccardi; Marco Simiele; Silvia Racca; Giuseppe Saglio; Francesco Di Carlo; Giovanni Di Perri (1721-1726).
A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.
Keywords: Imatinib; Dasatinib; Nilotinib; HPLC–MS; Quantification;

Dronedarone, a noniodinated benzofuran derivative of amiodarone, is believed to have a better side effect profile, and is currently undergoing phase III clinical trials. A novel method was developed for the determination of dronedarone and its principal metabolite debutyldronedarone in both plasma and myocardial tissue by high-performance liquid chromatography (HPLC) coupled with UV-detection. The assay was also validated for determination of amiodarone and desethylamiodarone. Samples were obtained from healthy humans (plasma) and goats (plasma and myocardium). Sample preparation included deproteinization with acetonitrile and extraction with a mixture of heptane and dichloromethane (50/50, v/v). Chromatographic separation was performed on a Pathfinder PS polymeric C18 column (50 mm × 4.6 mm, 2.5 μm) with a mobile phase of acetonitrile, isopropanol, water and ammonia (80/10/10/0.025, v/v/v/v) at a flow-rate of 1 ml/min. Calibration curves of all analytes were linear in the range of 0.01–5 μg/ml for plasma samples, with a lower limit of quantification (LLOQ) of 0.04 μg/ml. For myocardial tissue samples, linear curves of all analytes were observed in the range of 0.02–500 μg/g, with a LLOQ of 0.08 μg/g. Within- and between-day precision was <18%, and within- and between-day accuracy ranged from 97.5 to 109.7%, with a recovery of 67.6–79.9%. The present method enables sensitive and specific detection of dronedarone, amiodarone and principal metabolites in plasma as well as myocardial tissue.
Keywords: Dronedarone; Debutyldronedarone; Amiodarone; Desethylamiodarone; High-performance liquid chromatography;

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids, directly from a crude extract of Ipomoea muricata. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)–acetonitrile–water (4:1:5, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and trifluoroacetic acid (10 mM) to the aqueous mobile phase as an eluter. From 4 g of crude extract, 210 mg lysergol and 182 mg chanoclavine were obtained in 97% and 79.6% purities. Total yield recovery was >95%. Isolated alkaloids were characterized on the basis of their 1H, 13C NMR and ESI-MS data.
Keywords: Ipomoea muricata; pH-zone-refining centrifugal partition chromatography; Preparative chromatography; Lysergol; Chanoclavine;

The analysis of pegylated-interferon-α2a in patient serum samples is of high interest for clinic research trials, as this therapeutic protein has become an important antiviral treatment. In this study, an LC–MS/MS method for the absolute quantification of pegylated-interferon-α2a in human serum was developed. The assay achieved a lower limit of quantification of 3.6 ng/mL (60 pM) with the use of a monolithic C18 solid phase extraction to enrich the target protein. The linear range of the assay was defined up to 54 ng/mL to measure the typical clinical pegylated-interferon-α2a levels, and within this range, the precision and accuracy were found to be within ±20%. The method was applied to a clinical study and found suitable for high-throughput analysis of pegylated-interferon-α2a in human serum. In addition, further investigations suggested the enrichment step may have general application to the sensitive analysis of other low molecular weight proteins.
Keywords: LC–MS/MS; Therapeutic protein; Quantification; Pegylated-interferon; PEG-IFN; Human serum; Monolithic SPE;

A rapid and cleanup-free microwave-assisted extraction (MAE) method is proposed for the simultaneous extraction of six illegal drugs of abuse – cocaine, benzoylecgonine (BZE), cocaethylene (CCE), morphine, 6-monoacethylmorphine (6AM) and codeine – from human hair samples. The analytes were determined using high performance liquid chromatography (HPLC) with photodiode array UV detection. The influence of several variables on the efficiency of the MAE procedure was investigated in detail by a multi-objective optimization approach based on a hybrid experimental design (17 experiments) and desirability functions. Six drugs were successfully extracted from human hair with recoveries close to 100% and good reproducibility (<3.6% RSD) under the optimal MAE conditions: 11 mL dichloromethane (DCM) extraction solvent, 60 °C extraction temperature, 9 min extraction time and 0.5 mL of methanol (MeOH) added to 50 mg of the hair sample in the extraction vessels. Limits of quantification of 0.2 ng mg−1 were found for the studied compounds. A comparison of sample preparation procedures, including MAE, enzymatic digestion and digestion by aqueous acids, was also conducted. The results indicated that the global behaviour of sample procedures provided similar satisfactory recoveries ranging from 86 to 100%. Indeed, the MAE procedure resulted in a reduction of extraction time by 100-fold and the elimination of cleanup steps. Slightly higher recoveries of morphine, 6AM, BZE and CCE, at 1 ng mg−1 concentration level and cocaine at 40 ng mg−1 concentration level, were achieved using MAE. Lastly, the proposed MAE method was applied to several human hair samples from multidrug abusers.
Keywords: Human hair; Drug of abuse; Microwave-assisted extraction; Experimental design;

Organic solvent extraction and metabonomic profiling of the metabolites in erythrocytes by Zhang Ying; A Jiye; Guangji Wang; Huang Qing; Yan Bei; Zha Weibin; Gu Shenghua; Liu Linsheng; Ren Hongcan; Ren Meiting; Sheng Longsheng (1751-1757).
We used erythrocytes as the model tissue to evaluate an optimal solution for the extraction of intracellular metabolites and time-dependent variation of the metabolome in living cells. Projection to latent structure (PLS) of the GC/MS and LC/MS data suggested that the most efficient solution for the extraction of metabolites from wet erythrocytes (50 mg) could be a methanol–chloroform–water mixture (950 μL, 700:200:50, v/v/v). PLS-discriminant analysis (DA) clearly profiled a time-dependent alternation of metabolic phenotype of erythrocytes. Identification of the metabolites showed that the process was characterized by accumulating of metabolic products and depleting of nutritious substances in erythrocytes during incubation.
Keywords: Metabonomics; Erythrocytes; Intracellular metabolites; Principal component analysis; GC/MS; LC/MS;

In the present work, a simple and high sensitive method based on hollow fiber liquid phase microextraction (HF-LPME) was developed followed by high performance liquid chromatography (HPLC) for determination of ultra-trace amounts of Se(IV) after derivatization in biological and natural water samples. Se(IV) was complexed with o-phenylenediamine to form piazselenol. The formed piazselenol was extracted into 20 μL of 1-octanol located in the lumen of a hollow fiber and the solution was injected into HPLC-UV for analysis. Using the Taguchi method, an orthogonal array design (OAD), OA16 (45) was employed to optimize the HF-LPME of piazselenol. The effect of five experimental factors (each factor at four levels) including the volume of the organic phase, extraction time, pH of the solution, stirring rate and ionic strength on the extraction efficiency of piazselenol was studied and optimized. The maximum extraction efficiency of piazselenol was obtained at 20 μL of 1-octanol as the extracting solvent, 30 min extraction time, pH 2, stirring rate of 500 rpm and 30% (w/v) NaCl. Under the optimum conditions, preconcentration factors up to 130 were achieved and the relative standard deviation (%RSD) of the method was <3.7% for different concentrations of Se(IV). The calibration curves were obtained in the ranges of 0.2–100 and 0.05–10 μg L−1 for the 11 and 50 mL of the sample volumes with reasonable linearity, respectively (r 2  > 0.995). The limits of detection (LOD) were 0.1 and 0.02 μg L−1 for the 11 and 50 mL sample volumes, respectively (S/N  = 3). Finally, the applicability of the proposed method was evaluated by the extraction and determination of Se(IV) in the plasma, urine and water samples.
Keywords: Se(IV); Hollow fiber liquid phase microextraction; Orthogonal array design; Piazselenol; Plasma; Urine;

Validation and application of an LC–ESI-MS method for simultaneous determination of astilbin and its major metabolite 3′-O-methylastilbin in rat plasma by Yan Liang; Qiang Xu; An Kang; Yuan Xie; Tong Xie; Li Liu; Haiping Hao; Lin Xie; Guang-ji Wang (1765-1770).
We herein describe the development of an LC–MS method for simultaneous determination of astilbin and 3′-O-methylastilbin in rat plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts on to a Shim-pack C18 column (150 mm × 2.0 mm I.D., 5 μm) with gradient elution and detection in negative ionization mode. Initially, the method was validated regarding linearity, accuracy and precision. The correlation coefficients of all the calibration curves showed good linearity (r  > 0.999) within test ranges, and the relative deviation was less than 10% for intra- and inter-day assays. Besides, this method was also validated for its stability, extraction efficiency, matrix effect and so on. Finally, this proposed method was successfully applied to rat pharmacokinetic study and yielded the most comprehensive data on systemic exposure of them to date.
Keywords: Astilbin; 3′-O-methylastilbin; LC–ESI-MS; Metabolite;

Simultaneous determination of clarithromycin, rifampicin and their main metabolites in human plasma by liquid chromatography–tandem mass spectrometry by Femke de Velde; Jan-Willem C. Alffenaar; A. Mireille A. Wessels; Ben Greijdanus; Donald R.A. Uges (1771-1777).
The drug combination rifampicin and clarithromycin is used in regimens for infections caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the “Guidance for Industry – Bioanalytical Method Validation” of the FDA. The calibration curves were linear in the range of 0.10–10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20–5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to −10%. The components in human plasma are stable after freeze–thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days; rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma.
Keywords: LC/MS/MS; Clarithromycin; Rifampicin; TDM; Pharmacokinetics;

A highly sensitive and robust UPLC–MS with electrospray ionization method for quantitation of taxifolin in rat plasma by Xiaodan Wang; Hongjun Xia; Feng Xing; Guifeng Deng; Qi Shen; Su Zeng (1778-1786).
A sensitive ultra performance liquid chromatography–mass spectrometry method has been developed and validated for the quantification of taxifolin in rat plasma. Following liquid/liquid extraction by ethyl acetate, the analytes were separated on a Sunfire™ (2.1 mm × 50 mm, 3.5 μm) column and analyzed in the selected ion recording with a negative electrospray ionization mode. The method was linear over the concentration range of 6–6750 ng/mL. Intra- and inter-day precisions were all within 8% and accuracy ranged from 92.9% to 105.1%. The lower limit of quantification was 6 ng/mL. The present method was successfully applied to the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration to rats. The absolute bioavailability of taxifolin was 0.17% in rat.
Keywords: Taxifolin; Rat plasma; Method validation; UPLC–MS; Pharmacokinetics;

A HPLC method to determine and quantify sulphachloropyrazine residues from broilers and turkeys is reported. This procedure permitted sulphachloropyrazine to be separated from muscle tissue, liver, kidneys and fat with skin after extraction with dichloromethane under slightly acidic conditions. The analytical methodology showed a high specificity and sensitivity and an adequate precision and accuracy with a limit of quantification of 56 ng mL−1. The peak area showed a linear relationship with a concentration over the range 50–750 ng mL−1 for sulphachloropyrazine standard solutions. Recovery dates were also satisfactory with values between 69.7 and 77.5%.
Keywords: Sulphachloropyrazine; Broilers and turkeys; HPLC determination; Method validation;