Journal of Chromatography B (v.931, #C)

Determination of deltonin in rat plasma by using HPLC–MS/MS and the application of this method in pharmacokinetic studies by Dan Du; Bo Gao; Guang Xin; Aimin Sun; Baozhan Huang; Rui Zhang; Zhihua Xing; Qianming Chen; Yang He; Wen Huang (1-5).
Deltonin is a naturally occurring spirostanol glycoside from Dioscorea zingiberensis C.H. Wright, which is used in traditional Chinese medicine. It exerts strong cytotoxic effect on C26 cells, inhibits C26 derived-tumor growth, and prolongs the survival of tumor-bearing mice after its oral administration, indicating its potential for use as an anti-tumor drug. To investigate the pharmacokinetic profiles of deltonin, a rapid, sensitive, and simplified high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay was developed and validated for the determination of deltonin in rat plasma. After acetonitrile-mediated plasma protein precipitation, chromatographic separation of deltonin was achieved using a reversed phase Hypersil Gold column (150 mm × 2.1 mm, 5 μm), with gradient elution using 0.1% formic acid and acetonitrile. Thereafter, deltonin was quantified using MS/MS with electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode. The flow rate of the mobile phase was 200 μL/min, and the retention time was 9.03 min for deltonin and 6.31 min for the internal standard (IS: 20(S)-ginsenoside Rb1). The linear range of the calibration curve was 2–5000 ng/mL (r 2  > 0.99), and the limit of detection (LOD) was 0.46 ng/mL. The intra- and inter-day accuracies ranged from −2.8% to 11.1% and precisions (RSD) were within 13.1%. Deltonin was found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after 3 freeze–thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of deltonin (50 and 100 mg/kg). The pharmacokinetics is characterized by high apparent clearance (CL/F) and apparent volume of distribution (Vd/F).
Keywords: Deltonin; HPLC–MS/MS; Rat plasma; Pharmacokinetics;

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 10 cephalosporins and desacetylcefapirin in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS). Samples were directly purified through HLB cartridge after dilution with 50 mM phosphate buffer solution (pH 8.5). Then the eluate was dried under nitrogen and the residue was redissolved in mobile phase. Samples were analyzed by LC–MS/MS on an Acquity UPLC BEH Shield RP18 column with gradient elution. The samples were quantified using ceftiofur-D3 as internal standard. The proposed method was validated according to the European Commission Decision 2002/657/EC. The CCα values were 111, 0.04, 140, 55, 55, 67, 23, 23, 68, 0.10 and 113 μg/kg for cefalexin, cefradine, cefacetrile, cefazolin, cefoperazone, cefapirin, cefalonium, cefquinome, desacetylcefapirin, cefotaxime and ceftiofur, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CVr), and reproducibility (CVR) varied from 94.6% to 117.1%, from 5.6% to 13.6% (CVr), and from 5.9% to 27.9% (CVR), respectively. The method is demonstrated to be suitable for the determination of 10 cephalosporins and desacetylcefapirin in bovine milk. The total time required for the analysis of one sample, including sample preparation, was about 40 min.
Keywords: Cephalosporins; Milk; LC–MS/MS; Solid phase extraction; Validation;

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the simultaneous quantitation of five major active ingredients of Ixeris sonchifolia (Bge.) Hance in rat plasma has been developed and validated. After liquid–liquid extraction of 50 μL plasma with ethyl acetate, analytes and internal standard (I.S.), astilbin, were chromatographed on a Zorbax SB-C18 column (150 mm × 4.6 mm, 5 μm) using acetonitrile – 10 mM ammonium acetate (60:40, v/v, pH 5.6) as mobile phase. The five analytes: chicoric acid, luteolin 7-O-β-d-glucuronide, luteolin 7-O-β-d-glucopyranoside, luteolin 7-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside, apigenin 7-O-β-d-glucuronide and I.S., were detected by negative ion electrospray ionization followed by multiple reaction monitoring of the ions with m/z 473.0 → 311.0, 461.0 → 285.0, 447.0 → 285.0, 609.1 → 285.0, 445.1 → 269.0 and 449.1 → 150.9, respectively. The method was linear for all analytes in the concentration range 10–3000 ng/mL with intra- and inter-day precision (as relative standard deviation) ≤8.99% and accuracy (as relative error) ≤4.00%. The limits of detection (LOD) were 5, 1, 5, 5, 2 ng/mL for chicoric acid, luteolin 7-O-β-d-glucuronide, luteolin 7-O-β-d-glucopyranoside, luteolin 7-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside, apigenin 7-O-β-d-glucuronide, respectively. The method was successfully applied to a pharmacokinetic study of the five analytes in rat after a single intravenous dose of Kudiezi Injection.
Keywords: LC–MS/MS; Ixeris sonchifolia (Bge.) Hance; Pharmacokinetics; Rat;

As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (P F) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%.
Keywords: Purification; Organic solvent; Aqueous organic phase system; Pectinase; Mango peel;

Retinol, tocopherols, coenzyme Q10, carotenoids, and vitamin D are lipophilic compounds shown to function as important health-protective agents by mitigating the damaging effects of oxidative and other injury. Scientific interest in evaluating these compounds has resurfaced in recent years, particularly in the nutritional, clinical and epidemiologic fields, and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC-based assays since 2007 for the simultaneous determination of these lipid-phase compounds utilizing exclusively serum or plasma as these matrices are mostly used in clinical and epidemiological investigations. We also provide an overview of blood measurements for selected carotenoids, tocopherols, coenzyme Q10 and retinol from the last 15 years of healthy umbilical cord blood, children, and adults.
Keywords: Plasma; Serum; Micronutrients; HPLC; Lipid-phase antioxidants;

A selective method analyzing separately o-, m- and p-methylhippuric acid isomers in workers’ urine samples has been developed using ultra performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation has been optimized to resolve the three isomers at baseline. Combined with this optimal separation, the mass spectrometer allowed rapid switching from MRM scan to full scan and product ion scan within the chromatographic peak. This feature allowed the retention of analyte chemical structure information for the three methylhippuric acid isomers in parallel with the simultaneous acquisition of quantitative data. Such an approach is unequaled for the reliability of the data generated and it can be applied to each isomer separately. The method was adjusted to a dynamic range between 0.2 mM and 8.12 mM for o-methylhippuric acid and p-methylhippuric acid, and between 0.41 mM and 16.23 mM for m-methylhippuric acid in order to cover the biological exposure index. A negligible matrix effect was observed with the conditions used. Also, intra-day and inter-day precisions were both <6% for all the concentration levels tested and the accuracy was evaluated at 97 ± 4%. The inclusion of simultaneous full scan acquisitions did not prevent the robustness of the quantitative data. The method applied to the determination of inter-laboratory proficient test samples led to results in the tolerated range. Moreover, urine samples from workers were robustly quantified and the MHA levels were all below the biological exposure index reference value.
Keywords: Methylhippuric acids; Biological monitoring; Tandem mass spectrometry; Ultra performance liquid chromatography; Selectivity;

Selective extraction of lamivudine in human serum and urine using molecularly imprinted polymer technique by Maryam Shekarchi; Mojgan Pourfarzib; Behrouz Akbari-Adergani; Ali Mehramizi; Mehran Javanbakht; Rassoul Dinarvand (50-55).
In this work, a novel technique is described for determination of lamivudine in biological fluids by molecularly imprinted polymers (MIPs) as the sample clean-up method joint with high performance liquid chromatography (HPLC). MIPs were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker, acetonitrile and tetrahydrofuran as porogen and lamivudine as the template molecule. The new imprinted polymer was used as a molecular sorbent for the separation of lamivudine from human serum and urine. Molecular recognition properties, binding capacity and selectivity of the MIPs were evaluated and the results showed that the obtained MIPs have a high affinity for lamivudine in aqueous medium. HPLC analyses showed that the extraction of lamivudine from serum and urine by MIPs had a linear calibration curve in the range of 60–700 μg/L with excellent precisions of 2.73% for serum and 2.60% for urine. The limit of detection and quantization of lamivudine was 19.34 and 58.6 μg/L in serum and 7.95 and 24.05 μg/L in urine respectively. MIP extraction provided about 10 fold LOQ improvement in serum and 5 fold LOQ improvement in urine samples. The recoveries of lamivudine in serum and urine samples were found to be 84.2–93.5% and 82.5–90.8% respectively. Due to the high precision and accuracy, this method may be the UV-HPLC choice with MIP extraction for bioequivalence analysis of lamivudine in serum and urine.
Keywords: Molecularly imprinted polymer; Lamivudine; Drug analysis; Human serum; Urine; HPLC;

A novel method for the analysis of 3-mercaptopyruvate using high-performance liquid chromatography with fluorescence detection by Yuki Ogasawara; Tomoaki Hirokawa; Kaori Matsushima; Shin Koike; Norihiro Shibuya; Shinzo Tanabe; Kazuyuki Ishii (56-60).
3-Mercaptopyruvate (3-MP) is a metabolite of cysteine present in mammalian tissues and is known to be a substrate of 3-mercaptopyruvate sulfurtransferase (3MST, EC.3.4.1.2). The physiological relevance of the 3-MP pathway has not been fully recognized because the metabolic behavior of 3-MP remains unclear. Here, we describe a novel method using high-performance liquid chromatography with fluorescence detection to measure 3-MP formation from cysteine. To demonstrate the practical value of the present method, we applied it to analyze the 3-MP produced in biological samples from mouse tissue.
Keywords: 3-Mercaptopyruvate; Cysteine metabolism; Sulfurtransferase; Bound sulfur; Hydrogen sulfide;

Application of a liquid chromatography–tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of brazilin in rats by Yanyan Jia; Hujun Wang; Ying Song; Kai Liu; Fang Dou; Chengtao Lu; Jie Ge; Ningjuan Chi; Yi Ding; Wenli Hai; Aidong Wen (61-67).
Brazilin is an important constituent of Caesalpinia sappan L., and has several bioactivities. In this study, a rapid and sensitive analytical method based on high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) has been developed for the determination of brazilin in rat plasma, urine, feces and tissues (brain, heart, liver, lung and kidney and spleen). Biological samples were processed with ethyl acetate containing 5% formic acid extraction, and salicylic acid (SA) was chosen as the internal standard (IS). The separation of brazilin was achieved on an Inspire C18 column (4.6 mm × 150 mm, 5 μm) with a mobile phase consisting of methanol/5 mM ammonium acetate (80:20, v/v). The MS/MS detection was carried out by monitoring the fragmentation of m/z 285.1 → 163.0 for brazilin and m/z 137.1 → 93.1 for SA on a triple quadrupole mass spectrometer. The total run time was only 5.0 min. The analyte showed good linearity over a wide concentration range (R 2  > 0.995) and its lower limit of quantification was 2 ng/mL. The accuracy and precision ranged from 97.1 to 103.3% and 1.7 to 9.1%, respectively. Recoveries (78.9–93.8%) and matrix effects (81.0–97.8%) were satisfactory in all the biological matrices examined. Stability studies (86.4–99.8%) showed that brazilin was stable during the assay procedure and long-term storage. The assay was successfully applied to plasma pharmacokinetics, tissue distribution and excretion study of rats. The pharmacokinetic parameters, such as half-life, mean residence time, maximum concentration were determined. These preclinical data of brazilin would be useful for the clinical reference.
Keywords: Brazilin; LC–MS/MS; Pharmacokinetics; Tissue distribution; Excretion;

Pinosylvin (trans-3,5-dihydroxystilbene), a naturally occurring analogue of resveratrol (trans-3,5,4′-trihydoxystilbene), exhibited various beneficial pharmacological activities in pre-clinical studies. To further probe its potential medicinal application, a sensitive liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the quantification of pinosylvin in rat plasma. A simple protein precipitation procedure was used for plasma cleanup before analysis by LC–MS/MS with electrospray ionisation and multiple reaction monitoring in its negative ion mode. This LC–MS/MS method demonstrated good selectivity, accuracy (intra- and inter-day analytical recovery within 100 ± 7.7%), precision (intra- and inter-day coefficient of variation < 12.0%) and sensitivity (lower limit of detection = 1.0 ng/mL), with excellent linearity (R 2  > 0.99) over the range of 1–1000 ng/mL. The pharmacokinetic profiles of pinosylvin were subsequently assessed in Sprague–Dawley rats. Following intravenous administration (5 or 10 mg/kg), plasma levels of pinosylvin declined rapidly with a short half-life (t 1/2  < 10 min). Upon oral administration at 15 mg/kg, pinosylvin could not be quantified in plasma (<1 ng/mL) while dose-escalation to 50 mg/kg led to a low and erratic plasma exposure with very poor estimated oral bioavailability (F  < 1%). The short half-life and limited systemic exposure of pinosylvin prompt caution in its therapeutic application and it warrants exploration in developing pinosylvin pro-drug.
Keywords: Pinosylvin; Resveratrol; LC–ESI-MS/MS; Pharmacokinetics; Oral bioavailability;

Since 1992, formoterol is included in the prohibited list of doping substances and methods, presently reviewed and updated by the World Anti-Doping Agency. Recently a threshold value of 40 ng/mL has been established to differentiate between the prohibited (oral) and the permitted (inhalatory) administration of formoterol to athletes. This paper considers the urinary excretion profile of formoterol and its main metabolites after inhalation of different doses of two of the most used medicaments, available in Italy, containing formoterol fumarate bihydrate (12 and 36 μg twice a day of Foradil® or 9 and 27 μg twice a day of Symbicort®), focusing also on the effects, on the measured levels of formoterol, of potential alteration processes (thermal and/or microbiological) that may take place after the collection of the urine samples. Urine sample preparation included an enzymatic hydrolysis and a dilution step. Detection of analytes was performed by a newly developed and validated direct LC–ESI-MS/MS procedure, using a triple quadrupole mass spectrometer under positive ion electro-spray ionization conditions and selected reaction monitoring acquisition mode. The results showed the capability and suitability of the direct LC–ESI-MS/MS analysis for the quantitative confirmation analysis of formoterol in urine samples. The data from the analysis of the urine samples obtained in the excretion studies showed that formoterol is excreted mainly as unmodified drug and to a lesser degree as O-demethylated metabolite. The urinary levels of formoterol (40–60%) and its metabolites (O-demethylated metabolite 5–25%; glucuronide metabolites 25–40%) vary significantly depending both on the administered drug formulation and the subject tested. The maximum urinary concentration reached in this study was 15 ng/mL (free + glucuronide), that is significantly lower than the threshold value fixed to report an adverse analytical finding. Finally, our results also showed that formoterol is stable for at least 4 weeks in urine samples correctly collected and stored.
Keywords: Anti-doping analysis; Formoterol; LC–MS/MS; Urinary metabolites;

Detection of ligand–receptor binding using microfluidic frontal affinity chromatography on proteoliposomes derived directly from native cell membranes by Kenneth Olesen; Roger Karlsson; Ulrika Lind; Max Davidson; Anders Blomberg; Anders Karlsson (84-89).
A method for characterization of ligand binding to membrane receptors in their native cell membrane is presented. The methodology is based on microfluidic frontal affinity chromatography coupled to mass spectrometry (FAC-MS). Proteoliposomes with receptor of interest are prepared directly from cell membranes and serve as a stationary phase in a microfluidic flow cell for frontal analysis. The G-Protein-Coupled Receptor (GPCR) Ste2 involved in the pheromone-induced yeast mating pathway is used as a model receptor for proof of principle characterization. The ligand affinity of the natural pheromone peptide, the α-factor, is compared to a set of pheromone analogs having different receptor affinities. With short preparation time, preserved lipid composition and the ability to immobilize proteoliposomes from any cell membrane, we propose that our methodology with immobilized proteoliposomes together with microfluidics FAC-MS can be an important improvement for ligand-receptor studies in native membranes.
Keywords: Microfluidics; Frontal affinity chromatography; Ligand–receptor binding; GPCR; Mass spectrometry;

Volatile biomarkers from human melanoma cells by Jae Kwak; Michelle Gallagher; Mehmet Hakan Ozdener; Charles J. Wysocki; Brett R. Goldsmith; Amaka Isamah; Adam Faranda; Steven S. Fakharzadeh; Meenhard Herlyn; A.T. Charlie Johnson; George Preti (90-96).
Dogs can identify, by olfaction, melanoma on the skin of patients or melanoma samples hidden on healthy subjects, suggesting that volatile organic compounds (VOCs) from melanoma differ from those of normal skin. Studies employing gas chromatography–mass spectrometry (GC–MS) and gas sensors reported that melanoma-related VOCs differed from VOCs from normal skin sources. However, the identities of the VOCs that discriminate melanoma from normal skin were either unknown or likely derived from exogenous sources. We employed solid-phase micro-extraction, GC–MS and single-stranded DNA-coated nanotube (DNACNT) sensors to examine VOCs from melanoma and normal melanocytes. GC–MS revealed dozens of VOCs, but further analyses focused on compounds most likely of endogenous origin. Several compounds differed between cancer and normal cells, e.g., isoamyl alcohol was higher in melanoma cells than in normal melanocytes but isovaleric acid was lower in melanoma cells. These two compounds share the same precursor, viz., leucine. Melanoma cells produce dimethyldi- and trisulfide, compounds not detected in VOCs from normal melanocytes. Furthermore, analyses of the total volatile metabolome from both melanoma cells and normal melanocytes by DNACNT sensors, coupled with the GC–MS results, demonstrate clear differences between these cell systems. Consequently, monitoring of melanoma VOCs has potential as a useful screening methodology.
Keywords: Volatile organic compounds; Gas chromatography–mass spectrometry; Melanoma; Carbon nanotubes; Volatile metabolome;

An alternative derivatization method for the analysis of amino acids in cerebrospinal fluid by gas chromatography–mass spectrometry by Maria José Nunes de Paiva; Helvécio Costa Menezes; Paulo Pereira Christo; Rodrigo Ribeiro Resende; Zenilda de Lourdes Cardeal (97-102).
The determination of the concentrations of l-amino acids in cerebrospinal fluid (CSF) has been used to gain biochemical insight into central nervous system disorders. This paper describes a microwave-assisted derivatization (MAD) method using N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) as a derivatizing agent for determining the concentrations of l-amino acids in human CSF by gas chromatography with mass spectrometry (GC/MS). The experimental design used to optimize the conditions showed that the optimal derivatization time was 3 min with a microwave power of 210 W. The method showed good performance for the validation parameters. The sensitivity was very good, with limits of detection (LODs) ranging from 0.01 μmol L−1 to 4.24 μmol L−1 and limits of quantification (LOQs) ranging from 0.02 to 7.07 μmol L−1. The precision, measured using the relative standard deviation (RSD), ranged from 4.12 to 15.59% for intra-day analyses and from 6.36 to 18.71% for inter-day analyses. The coefficients of determination (R 2) were above 0.990 for all amino acids. The optimized and validated method was applied to the determination of amino acid concentrations in human CSF.
Keywords: l-Amino acids; Microwave-assisted derivatization; Gas chromatography; Mass spectrometry;

An improved quantitative assay was developed and validated for fludarabine in human plasma. Fludarabine and its internal standard, cladribine, were separated on a C18 analytical column after sample purification by strong anion-exchange solid-phase extraction. Quantitation was performed by electrospray triple-quadrupole mass spectrometry in positive ionization mode using multiple-reaction monitoring. This assay had excellent inter- and intra-assay precisions within 8%, and accuracies ranging from 100 to 116%. The method was linear within the concentration range of 0.2–250 ng/mL using 100 μL of plasma with mean R 2  = 0.9999. The extraction recoveries were 85% for fludarabine and 95% for the internal standard, which represent a significant improvement over the previously published methods. We utilized this method for pharmacokinetic (PK) investigations in 215 patients. Interference peaks constantly observed in each blank plasma sample were well resolved from fludarabine using our optimized LC–MS/MS conditions, demonstrating the reliability of this improved assay. The validated method will be further applied to PK studies within our bone marrow transplant program, which will allow for optimal dose and scheduling of fludarabine in these patients.
Keywords: Fludarabine; Bone marrow transplant; LC–MS/MS;

A novel method which involved dispersive liquid–liquid microextraction (DLLME)-back extraction based on ionic liquid (IL) was developed for the determination of three phosphodiesterase-5 (PDE-5) inhibitors, sildenafil (SD), vardenafil (VD) and aildenafil (AD), in human plasma. DLLME based on IL as the extractant solvent and methanol as the dispersive solvent was the first step to extract PDE-5 inhibitors from sample solution; the other step of back extraction was followed by transferring target analytes from the IL to acidified aqueous solution. This two-step extraction ensured the compatibility of the final extractant phase, acidified aqueous solution herein, with the reversed phase high performance liquid chromatography-UV detection, and afforded clean extractant phase. The optimal extraction condition was obtained after systematical optimization. The sample solution (960 μL) was extracted by 20 μL of 1-octyl-3-methylimidazolium hexafluorophosphate in the presence of 20 μL methanol and 300 mg mL−1 NaCl with the assistance of vortex; IL phase enriched with the target analytes was then extracted by 10% acetic acid aqueous solution. Good linearity ranges (SD 1–500 ng mL−1, VD 2–2000 ng mL−1 and AD 2–2000 ng mL−1) with suitable r 2 (=0.9999) were achieved. Limits of detection (LODs) in pure water were 0.15 ng mL−1, 0.30 ng mL−1 and 0.43 ng mL−1 for VD, SD and AD, respectively. Intra-day and inter-day relative standard deviations were below 6.38%. Finally, this method was applied for the determination of PDE-5 inhibitors in human plasma with satisfactory LODs of 0.92 ng mL−1, 1.19 ng mL−1 and 2.69 ng mL−1 for VD, SD and AD, respectively. Acceptable absolute recoveries were obtained from 100.4% to 103.9%. The developed method afforded a convenient, fast and cost-saving operation with high extraction efficiency for the test analytes. It has potential to be applicable to biological samples.
Keywords: Phosphodiesterase-5 inhibitors; Ionic liquid; Dispersive liquid–liquid microextraction; Back extraction; Human plasma; Sildenafil; Vardenafil; Aildenafil;

High throughput LC–MS/MS method for simultaneous determination of tenofovir, lamivudine and nevirapine in human plasma by Rajani Kumar Valluru; Phani Bhushana Reddy B; Kalyan Sumanth S; Praveen Kumar V; Naveen Babu Kilaru (117-126).
A selective and high throughput liquid chromatography–mass spectrometry method has been developed and validated for the simultaneous quantification of tenofovir (TFV), lamivudine (3TC) and nevirapine (NVP) in human plasma using emtricitabine (FTC) as internal standard (ISTD). Following solid phase extraction (SPE), the analytes and ISTD were run on Prontosil C18AQ column (100 mm × 4.6 mm, 3 μm) using an isocratic mobile phase consisting of 1 mM ammonium acetate in water (pH 6.5 ± 0.3):acetonitrile (50:50, v/v). The precursor and product ions of the drugs were monitored on a triple quadrupole instrument operated in the negative ionization mode. The method was validated over a concentration range of 2–500 ng/mL for TFV and over a concentration range of 10–4000 ng/mL for 3TC and NVP with relative recoveries ranging from 61 to 85%. The intra and inter batch precision (%CV) across four validation runs was less than 12.2%. The accuracy determined at four QC levels (LLOQ, LQC, MQC and HQC) was within ±8.5%, in terms of relative error.
Keywords: Tenofovir (TFV); Lamivudine (3TC); Nevirapine (NVP); Solid phase extraction (SPE); Internal standard (ISTD);

Gegen is one of the most commonly used traditional Chinese medicines for promoting blood circulation and removing blood stasis. Puerarin and daidzin are the main active constituents of Gegen. Puerarin is mainly metabolized in rats by glucuronidation and the major metabolite from rat urine has been identified as puerarin-7-O-glucuronide through semi-preparative HPLC isolation and then spectroscopic analysis. The study investigated the pharmacokinetic behavior of puerarin-7-O-glucuronide (without enzymatic hydrolysis), puerarin and daidzin when total flavonoid from Gegen was administered in normal and blood stasis animals or in blood stasis animals alone or in combination with Sanqi. The plasma samples were processed by protein precipitation with methanol, and chromatographed on a Thermo Syncronis C18 column (10 cm × 2.1 mm, 1.7 μm) by gradient elution at a flow rate of 0.25 mL/min, and detected with a triple quadrupole tandem mass spectrometer by selected reaction monitoring via electrospray ionization source with positive ionization mode. An unpaired Student’s t-test was used for the statistical comparison of the main pharmacokinetic parameters. There were statistically significant differences (P  < 0.05) in the pharmacokinetic parameters of puerarin-7-O-glucuronide, puerarin and daidzin involving the AUC, CL and Vd not only between normal rats and blood stasis rats after administration of total flavonoid from Gegen, but also between administration of total flavonoid from Gegen alone and in combination with total saponin from Sanqi in blood stasis rats. The results obtained suggest that the pharmacokinetic behavior of puerarin-7-O-glucuronide, puerarin and daidzin are changed when total flavonoid from Gegen was administered in blood stasis animals or in combination with total saponin from Sanqi.
Keywords: Total flavonoid from Gegen; Glucuronide metabolite; Pharmacokinetics; Rat plasma; Blood stasis syndrome; Ultra-HPLC–MS/MS;

A method based on liquid chromatography coupled to tandem mass spectrometry was developed for quantitative determination of varenicline in human plasma. Varenicline and the internal standard (25.0 ng/mL of Clarithromycin) were extracted from human plasma by liquid–liquid extraction, using methyl tertiary butyl ether as the organic solvent. The chromatographic separation was achieved using C8 column with isocratic elution using a mixture of acetonitrile:0.001 M ammonium acetate (adjusted to pH 4.0) (70:30%, v/v). The method was validated over the concentration range of 0.1–10.0 ng/mL by investigating specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to United State Food and Drug Administration guideline. The validated bioanalytical method was successfully applied to evaluate bioequivalence of two commercial products of 1 mg varenicline single dose.
Keywords: Varenicline; LC–MS/MS; Champix; Clinical study; Human plasma;

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry by S. Al-Shehri; M. Henman; B.G. Charles; D. Cowley; P.N. Shaw; H. Liley; A. Tomarchio; C. Punyadeera; J.A. Duley (140-147).
Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0 μM. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7–111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3 ± 25.4; 30.9 ± 19.5 μM respectively) compared to adults (4.3 ± 3.3; 4.6 ± 4.5 μM); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photodiode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults.
Keywords: Saliva; Neonates; Nucleotide metabolites; Purines; HPLC; Mass spectrometry;

In the current study, a fast and simple preconcentration and sample clean up procedure was developed based on carrier mediated three phase solvent bar liquid phase microextraction (TPSB-LPME) method prior to high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) absorbance detector for simultaneous extraction and determination of trace amounts of dexamethasone sodium phosphate (DSP) in human plasma, human urine and bovine milk. According to this procedure, dexamethasone sodium phosphate was extracted from an acidic aqueous sample (SP, 7.5 mL with pH = 6) into the organic solvent 1-octanol (containing 5%, w/v of Aliquat-336 as carrier) residing in the pores of a hollow fiber and then back extracted into an alkali receiving phase (RP, 5 μL of 0.65 M NaClO4 with pH = 10) was located inside the lumen of the fiber. After the extraction period, the receiving phase was directly injected into HPLC. The effect of different extraction conditions (i.e., pH of source and receiving phases, ionic strength, stirring rate, counter-ion concentration and extraction time) on the extraction efficiency of DSP was investigated and optimized using central composite design (CCD) as a powerful tool. Under the optimal conditions, preconcentration factor of 320, extraction recovery of 23%, dynamic linear range of 1–1000 ng mL−1 (r 2  = 0.997) and limit of detection of 0.1 ng mL−1 were obtained. Eventually, applicability of the proposed method was successfully confirmed by extraction and determination of drug in plasma and urine samples and bovine milk with R.S.D.s < 8%. Comparing to the traditional methods, the proposed method exhibits high sensitivity and high preconcentration factors as well as good precision. The extraction setup is simple and due to active transport of analytes, high cleanup effect and good selectivity are obtained in the extraction process. This extraction technique is also the most economical sample preparation and preconcentration technique as compared to traditional extraction techniques.
Keywords: Dexamethasone sodium phosphate; Solvent bar liquid phase microextraction; Response surface methodology; High performance liquid chromatography; Human plasma and urine; Bovine milk;

Identification of honokiol metabolites in rats by the method of stable isotope cluster technique and ultra-high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry by Huijun Lai; Minghai Tang; Juan Liu; Yinfeng Dong; Neng Qiu; Shucai Li; Liang Ma; Jianhong Yang; Hang Song; Yongkui Zhang; Aihua Peng; Lijuan Chen (157-163).
Honokiol, a natural molecule isolated from Magnolia officinalis Rehd. et Wils., is widely known as an antitumor agent. In present work, an analysis of in vivo biotransformation and metabolites of honokiol has been performed by a combined method based on stable isotope cluster technique with honokiol-[13C6]-labeled and ultra-high performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UHPLC/Q-TOF-MS). The metabolites could be easily identified by the determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to that between isotope-labeled and non-isotope-labeled honokiol. A total of eighteen metabolites were detected and tentatively identified, fourteen of which were reported for the first time. The results indicated that the main metabolic pathways of honokiol in rats were hydroxylation, methylation, sulfation and glucuronidation. This study provided the first essential information on biotransformation and metabolites of honokiol in rats, which was very useful for further pharmacological and clinical studies of honokiol as a potent drug candidate.
Keywords: Honokiol; Metabolite; Rats’ plasma; UHPLC/Q-TOF-MS; Stable isotope cluster technique;

Design and implementation of an imprinted material for the extraction of the endocrine disruptor bisphenol A from milk by John O’Mahony; Mary Moloney; Martin McCormack; Ian A. Nicholls; Boris Mizaikoff; Martin Danaher (164-169).
This paper describes the determination of bisphenol A (BPA) in milk samples, using a novel molecularly imprinted polymer. The imprinted polymer was developed using a rational design approach, and pre-polymerization interactions were investigated using molecular dynamics simulations and X-ray crystallography. A hydroquinone-imprinted polymer was used for solid phase extraction (SPE) clean-up of samples. BPA was quantified by high performance liquid chromatography (HPLC) and fluorescence (FLD) detection. Following validation, the method described was capable of determining bisphenol A in milk down to a limit of detection of 1.32 μg kg−1. The method was applied to a survey (n  = 27) of commercial milk products; BPA was detected in one of the samples, at a level of 176 μg kg−1. Test results were confirmed by a parallel UHPLC–MS/MS analytical method. This demonstrates the utility of the hydroquinone-imprinted polymer for application to selective sample clean-up and analysis of bisphenol A in milk, avoiding possible detrimental affects associated with template bleeding and without the need for expensive or difficult-to-obtain template.
Keywords: MIP; Bisphenol A; SPE; Milk; UHPLC–MS/MS; HPLC-FLD;

Determination of leelamine in mouse plasma by LC–MS/MS and its pharmacokinetics by Min Song; Doohyun Lee; Taeho Lee; Sangkyu Lee (170-173).
Leelamine may be applicable to treat diabetes and is known to inhibit pyruvate dehydrogenase kinase 4. In this study, we developed and validated a quantification method using liquid chromatography (LC) coupled with tandem mass spectrometry analysis, which was applied to a pharmacokinetic investigation in mouse plasma. Leelamine transition ions in multiple reaction-monitoring modes using positive ionization were observed at m/z 286.4 to m/z 173.2. LC was performed using an ACE 5 C18 column, and a mixture of acetonitrile and water containing 0.1% formic acid was used as the mobile phase at a flow rate of 0.22 mL/min. Leelamine and the internal standard (reserpine) had retention times of 4.1 and 3.9 min, respectively. Acceptable linearity (r 2  = 0.995) was observed over the concentration range of 10–3000 ng/mL, with a lower limit of quantification of 10 ng/mL in mouse plasma. The intra-day and inter-day accuracy and precision were less than 15%, which was sufficient for quality-control purposes. This method was used to determine leelamine concentrations in mouse plasma and showed that the oral bioavailability of leelamine was 7.6%.
Keywords: Leelamine; LC–MS/MS; Pharmacokinetic; Mouse; Bioavailability;

A novel, simple and eco-friendly ionic liquid based dispersive liquid–liquid microextraction followed by HPLC determination of anti-hypertensive drugs viz., eprosartan, valasartan, irbesartan, losartan and telmisartan in rat serum has been developed and validated. Experimental parameters influencing the extraction efficiency, nature and volume of the ionic liquid, dispenser solvent, extraction time and effect of salt were optimized. Under the optimum conditions, the extraction recoveries were between 92.85 and 98.50%. The relative standard deviations of intra-and inter-day accuracy varied between 1.9 and 9.1% (n  = 3). The linearity of the proposed method was 0.1–20 μg/mL with coefficients of determination varying between 0.9979 and 0.9992.
Keywords: Ionic liquids; Anti-hypertensives; Microextraction; Rat serum; Liquid chromatography;