Journal of Chromatography B (v.878, #26)

A porous polymethacrylate ester-based monolithic column for capillary electrochromatography (CEC) was designed by mean of in situ co-polymerizing lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent including cyclohexanol, 1,4-butanediol and water. After investigating the influence factors of the CEC monolithic columns, four flavonoids (i.e., Rutin, Quercetin, Kaempferol, and Quercitrin) were separated and assayed to evaluate this monolithic column with CEC method. Under optimum conditions, the CEC method exhibited high separation efficiency, with rapid separation time of 3–4 min, for the four flavonoid samples using 10 mM phosphate buffer containing 70% acetonitrile (pH 9.0). Importantly, the proposed method could provide a promising approach for rapid separation and detection in biomedicine.
Keywords: Capillary electrochromatography; Flavonoids; Monolithic columns;

Development of an LC–MS/MS assay to determine plasma pharmacokinetics of the radioprotectant octadecyl thiophosphate (OTP) in monkeys by Hari Kosanam; Fei Ma; Hui He; Suma Ramagiri; Veeresa Gududuru; Gabor J. Tigyi; Koen Van Rompay; Duane D. Miller; Charles R. Yates (2379-2383).
Octadecenyl thiophosphate (OTP), a synthetic analogue of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD80/30 radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid–liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10 mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1 → 95.0 and 403.1 → 95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3 mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.
Keywords: Octadecenyl thiophosphate (OTP); Lysophosphatidic acid (LPA); Liquid chromatography/tandem mass spectrometry (LC–MS/MS); Pharmacokinetics;

A new simple, sensitive and precise liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of valacyclovir-HCl and acyclovir in tsetse flies (Glossina pallipides). Tsetse flies were extracted by ultrasonication with acidified methanol/acetonitrile, centrifuged and cleaned up by solid phase dispersion using MgSO4 and MSPD C18 material. Samples were analysed using a Waters Alliance 2695 series HPLC with a C18 Gemini analytical column (150 mm × 4.6 mm × 5 μm) and a guard cartridge column connected to a Waters Quattro-Micro triple-quadrupole mass spectrometer. The isocratic mobile phase consisted of methanol:acetonitrile:water (60:30:10, v/v/v) plus formic acid (0.1%) at a flow rate of 0.25 ml/min. The precursor > product ion transition for valacyclovir (m/z 325.1 > 152) and acyclovir (m/z 226.1 > 151.9) were monitored in positive electrospray multiple reaction monitoring mode. The method was validated at fortification levels of 0.5, 1 and 2 μg/g. The range of calibration for both drugs was 0.45–4.5 μg/g. The overall accuracy of the method was 92% for valacyclovir and 95% for acyclovir with corresponding within-laboratory reproducibilities of 4.4 and 3.4%, respectively. Mean recoveries were above 80% for both drugs and repeatability ranged from 0.7 to 6.1%. For both drugs the limits of detection and quantification were 0.0625 and 0.2 μg/g, respectively. The method was applied in experiments on the mass rearing of tsetse flies for sterile insect technique (SIT) applications, in which the flies were fed with blood meals containing acyclovir or valcyclovir-HCl prior to analysis to assess effects on Glossina pallidipes Salivary Gland Hypertrophy syndrome.
Keywords: LC–MSMS; MRM; Valacyclovir; Acyclovir; Tsetse; Sterile insect technique (SIT);

The aim of this study was to develop an analytical method to monitor the saliva matrix for ototoxic solvents absorption: the method is based on headspace gas chromatography/mass spectrometry and represents an alternative biological monitoring for investigating low exposure to hazardous ototoxic solvents. Simultaneous determination of toluene, ethylbenzene, xylenes and styrene has been carried out and the method has been optimized for both instrumental parameters and samples treatment. Chromatographic conditions have been set in order to obtain a good separation of xylene isomers due to the interest in p-xylene as ototoxic one. Method validation has been performed on standards spiked in blank saliva by using two internal standards (2-fluorotoluene and deuterated styrene-d8). This method showed the possibility to detect the target compounds with a linear dynamic range of at least a 2 orders of magnitude characterized by a linear determination coefficient (r 2) greater than 0.999. The limit of detection (LOD) ranged between 0.19 ng/mL (styrene) and 0.54 ng/mL (m-xylene) and the lower limit of quantification (LLOQ) ranged between 0.64 ng/mL (styrene) and 1.8 ng/mL (m-xylene). The method achieved good accuracy (from 99 to 105%) and precision for both intra- and inter-assay (relative standard deviation ranging from 1.7 to 13.8%) for all six compounds concerned. The repeatability was improved by adding sodium sulphate to the matrix. Saliva samples resulted stable for at least 7 days after collection, if stored in headspace vials, at the temperature of 4 °C. An evaluation of the main sources of uncertainty of the method is also included: expanded uncertainties ranges between 10 and 16% for all of the target compounds. In summary, the headspace gas chromatography/mass spectrometry method is a highly sensitive, versatile and flexible technique for the biological monitoring of exposure to ototoxic solvents by saliva analysis.
Keywords: Ototoxic solvents; Saliva matrix; Static headspace analysis; Biological monitoring;

Rapid and sensitive determination of nicotine in formulations and biological fluid using micellar liquid chromatography with electrochemical detection by Mei-Liang Chin-Chen; Maria Rambla-Alegre; Abhilasha Durgavanshi; Devasish Bose; Josep Esteve-Romero (2397-2402).
Nicotine can be determined in pharmaceuticals and biological fluids by micellar liquid chromatography (MLC) using a C18 column, a mobile phase containing sodium dodecyl sulphate 0.15 M–6% (v/v) pentanol–NaH2PO4 0.01 M (pH 6)–KCl 0.001 M, with electrochemical detection at 0.8 V. In the optimization step, the influence of the modifiers propanol, butanol and pentanol, and the voltage has been studied. With the proposed method the analysis time is below than 8 min, linearity better than 0.999, limits of detection and quantification (ng/ml) was 4 and 12 respectively, repeatability and intermediate precision below 1.8%, and all these parameters are adequate for the quantification of nicotine in chewing gum, dermal patches, tobacco and serum samples either by a pharmacologist, pathologist or toxicologist.
Keywords: Nicotine; Electrochemical detector; Micellar liquid chromatography; Direct injection;

A simple and sensitive HPLC method was established and validated for the determination of docetaxel (DTX) in rabbit plasma. Biosamples were spiked with paclitaxel (PCX) as an internal standard (I.S.) and pre-treated by solid-phase extraction (SPE). The SPE procedure followed a simple protein digestion was based on nylon6 electrospun nanofibers mats as sorbents. Under optimized conditions, target analytes in 500 μL of plasma sample can be completely extracted by only 2.5 mg nylon6 nanofibers mat and eluted by 100 μL solvent. The HPLC separation was obtained on C18 column and UV detector was used to quantify the target analytes. The extraction recovery was more than 85%; the standard curve was linear over the validated concentrations range of 10–5000 ng/mL and the limit of detection was 2 ng/mL. The inter-day coefficient of variation (CV%) of the calibration standards was below 5.0% and the mean accuracy was in the range of 92.8–113.4%. Moreover, analysing quality control plasma samples in 3 days, the results showed that the method was precise and accurate, for the intra- and inter-day CV% within 10% and the accuracy from 96.0% to 114.0%. The developed and validated method was successfully applied to relative bioavailability study for the preclinical evaluation of a new injectable DTX–sulfobutyl ether beta-cyclodextrin (DTX–SBE-β-CD) inclusion complex freeze-dried powder (test preparation), compared with the reference preparation (DTX injection, Taxotere®) in healthy rabbits. On the basis of the mean AUC(0–t) and AUC(0–infinity), the relative bioavailability of the test preparation was found to be 113.1%.
Keywords: Docetaxel; DTX–SBE-β-CD inclusion compound freeze-dried powder; Electrospun nylon6 nanofibers mat; Solid-phase extraction; Relative bioavailability;

A sensitive and specific liquid chromatography–tandem mass spectrometric method for determination of belinostat in plasma from liver cancer patients by Ling-Zhi Wang; Daniel Chan; Winnie Yeo; Seow-Ching Wan; Stephen Chan; Anthony Chan; Soo-Chin Lee; How-Sung Lee; Boon-Cher Goh (2409-2414).
A novel, sensitive and reliable liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of belinostat (PXD101) in human plasma. Oxamflatin was used as the internal standard. Liquid–liquid extraction of the plasma sample was performed using tert-butyl methyl ether as the organic solvent. Chromatographic separation was achieved on a BDS Hypersil C18 column (2.1 mm × 100 mm, 5 μm) using gradient elution mode using 0.05% formic acid in water and 0.05% formic acid in acetonitrile as solvents A and B, respectively, 60/40. The run time was 6 min. The mass spectrometer was operated under a positive electrospray ionization condition and a multiple reaction monitoring mode. An excellent linear calibration was achieved in the range of 0.5–1000 ng/mL. An average recovery of belinostat for four quality controls was 72.6% and the recovery of the internal standard at 1000 ng/mL was 67.8%. The intra-day and inter-day precisions for belinostat were ≤8.0 and ≤10.3%, respectively, and their accuracy ranged from 100.2 to 106.7%. No significant matrix effect was identified. In analysis of patient samples, belinostat glucuronide was identified and baseline separated from belinostat. This well-validated assay has been applied for quantification of belinostat in plasma samples within 24 h after the start of infusion for Asian hepatocellular carcinoma patients in a dose escalation study.
Keywords: Belinostat; Belinostat glucuronide; LC–MS/MS; Human plasma;

An enantioselective and sensitive method was developed and validated for determination of doxazosin enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. The enantiomers of doxazosin were extracted from plasma using ethyl ether/dichloromethane (3/2, v/v) under alkaline conditions. Baseline chiral separation was obtained within 9 min on an ovomucoid column using an isocratic mobile phase of methanol/5 mM ammonium acetate/formic acid (20/80/0.016, v/v/v) at a flow rate of 0.60 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 452 → 344 for doxazosin enantiomers, and m/z 384 → 247 for prazosin (internal standard). The method was linear in the concentration range of 0.100–50.0 ng/mL for each enantiomer using 200 μL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.100 ng/mL. The intra- and inter-assay precision was 5.0–11.1% and 5.7–7.6% for R-(−)-doxazosin and S-(+)-doxazosin, respectively. The accuracy was 97.4–99.5% for R-(−)-doxazosin and 96.8–102.8% for S-(+)-doxazosin. No chiral inversion was observed during the plasma storage, preparation and analysis. The method proved adequate for enantioselective pharmacokinetic studies of doxazosin after oral administration of therapeutic doses of racemic doxazosin.
Keywords: Doxazosin; Enantiomers; Enantioselective LC–MS/MS; Stereoselective pharmacokinetics;

An adaptable HPLC method for the analysis of frequently used antibiotics in ocular samples by Lindsay T. Davis; Neeru Kumar; Lisa M. Nijm; Lawrence J. Ulanski; Elmer Y. Tu; Richard G. Fiscella; Randal J. Peterson; Randolph D. Glickman (2421-2426).
Four different antibiotics, delivered individually to rabbit eyes via hydrophilic intraocular lenses soaked in the drug solution prior to implantation, were measured in aqueous and vitreous humor samples from the eyes. To meet this analytical need, we developed a sensitive, high performance liquid chromatographic (HPLC) method for measuring the concentrations of moxifloxacin, gatifloxacin, linezolid, and cefuroxime in the ocular tissue. Separations were carried out on a LichroSpher RP-18 column, maintained at room temperature. The fluoroquinolones were eluted with a mobile phase consisting of 20% acetonitrile, in 0.1% trifluoroacetic acid (pH 3.0) with 30 mM tetrabutylammonium chloride. Linezolid and cefuroxime were eluted with 25% acetonitrile in 25 mM Na acetate buffer, pH 5.0. All elutions were isocratic. With ultraviolet detection, the lower limit of quantitation (LLOQ) for these compounds approached 1 ng (on-column injection). By using fluorescence detection, the LLOQ for the fluoroquinolones improved to 200 pg. The overall accuracy of the method was ≥90%. With minor modifications, the method was optimized for each of the agents, and the resulting analytical sensitivity made the method suitable for clinical investigations of the ocular penetration of these drugs.
Keywords: Antibiotics; β-Lactams; Fluoroquinolones; HPLC; Ocular tissue; Oxazolidinones;

A novel assay of cellular stearoyl-CoA desaturase activity of primary rat hepatocytes by HPLC by Chao Su; Hjalmar Gullberg; Hanna Simko; Marguerite Luthman; Per-Olof Edlund; Thomas Lundbäck (2427-2432).
The stearoyl-CoA desaturase (SCD) activity is involved in regulation of metabolism, energy storage, and membrane fluidity. However, only few cellular assays have been developed. We describe a simple and robust method to quantitate SCD activity and its inhibition in primary rat hepatocytes. Hepatocytes assimilate stearic acid, with or without modification by SCD, into its lipid pool. To measure the extent of this conversion primary rat hepatocytes were cultivated 4 h or overnight with [1-14C]18:0 and extracellular fatty acids were washed out. Total cell lipids were then hydrolyzed and extracted. Recoveries of 18:0 were secured with a modified Folch method by addition of 0.1% Triton X-114 to the samples. The extracted fatty acids were dissolved in 85% ethanol and separated by reverse phase HPLC, which took 10 min including column recovery time. [1-14C]18:0 and [1-14C]18:1(n9) were detected and quantified by on-line flow scintillation analysis. Incubation of the cells with SCD inhibitors resulted in decreased ratios of 18:1/18:0 in dose-dependent manners. The improvements enabled us to establish a novel robust assay based solely on HPLC analysis of cellular SCD activity, which was developed in 12-well format.
Keywords: HPLC; Stearoyl-CoA desaturase; Primary rat hepatocyte; Desaturation index; Stearic acid; Folch method;

LC–ESI-Q-TOF-MS for faster and accurate determination of microcystins and nodularins in serum by Milla-Riina Neffling; Lisa Spoof; Michael Quilliam; Jussi Meriluoto (2433-2441).
Microcystins (MC) and nodularins (Nod) are cyclic peptide hepatotoxins and tumour promoters produced by cyanobacteria. This study deals with liquid chromatography–mass spectrometry (LC–MS) analyses of 9 major cyanobacterial peptide toxins, starting with a comparison of six small particle size reversed-phase HPLC columns, from which one, Phenomenex Synergi Hydro-RP, was chosen for further chromatography with accurate mass MS studies in a complex biological fluid, serum. The instrumentation used for the serum sample analysis included a Bruker micrO-TOF-Q-MS coupled to an Agilent 1200RR LC system. Total analysis run time per sample was 8.5 min. The Q-TOF-MS instrument was operated on auto MS–MS mode to obtain fragment ions (such as the characteristic fragment m/z 135 from Adda amino acid residue) for toxin identification purposes. Detected mass errors in serum samples were in the range of from 0.3 mDa to 9.1 mDa. The narrow mass window (±20 mDa) for mass chromatograms used in quantitation gave benefits by background noise reduction. We conclude that a LC–ESI-Q-TOF-MS instrumentation is a powerful tool for identification and quantitation of cyanobacterial peptide toxins in a biological matrix.
Keywords: LC–ESI-Q-TOF-MS; Sub-3 micron particle columns; Cyclic peptides; Microcystins; Serum;

A robust and validated high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for simultaneous determination of F351 (5-methyl-1-(4-hydroxylphenyl)-2-(1H)-pyridone) and three major metabolites in human urine sample. This assay method has also been validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. Chromatography was carried out on an XTerra RP 18 column and mass spectrometric analysis was performed using an API 4000 mass spectrometer coupled with electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 202 → 109, 232 → 93, 282 → 202 and 378 → 202 were used to quantify F351 and three metabolites, respectively. Retention times for F351 and three metabolites were 2.54, 1.38, 1.53 and 1.34 min, respectively. The assay was validated from 20 to 4000 ng/mL for F351 and M1, from 80 to16,000 ng/mL for M2 and M3. Intra- and inter-day precision for all analytes was <6.3%, method accuracy was between −11.2 and 0.3%. This assay was used to support a clinical study where multiple oral doses were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of F351.
Keywords: F351; Metabolite; Selectivity; LC–MS/MS; Human urine;

Determination of esmolol and metabolite enantiomers within human plasma using chiral column chromatography by Lei Fang; Crystal Bykowski-Jurkiewicz; Jeffrey G. Sarver; Paul W. Erhardt (2449-2452).
A high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous determination of each of esmolol's enantiomers at the 25–1000 ng/ml concentrations observed in human plasma upon intravenous administration of this rapidly metabolized beta-adrenergic receptor blocking agent. Alternatively, a high performance liquid chromatography (HPLC) UV detection method has been developed for the simultaneous determination of each of the enantiomers for esmolol's metabolite which, in turn, achieve 2.5–50 μg/ml concentrations in human plasma. Utilizing chiral columns, these methods do not require a precolumn asymmetric derivatization step. Linearity in all cases was >0.99. Precision and accuracy at all but the lowest concentrations were within ±6% for the esmolol enantiomers and within ±2.5% for the esmolol metabolite enantiomers. These values should be suitable for performing thorough pharmacokinetic studies for all of the stereoisomers of this prototypical soft drug and its corresponding metabolite.
Keywords: Esmolol; Esmolol metabolite; Enantiomers; Chiral column chromatography; LC–MS/MS; HPLC;

Ion exchange chromatographic conditions for obtaining individual subunits of soybean β-conglycinin by Miryam Amigo-Benavent; Vasileios I. Athanasopoulos; M. Dolores del Castillo (2453-2456).
Soybean β-conglycinin is a complex protein possessing health-promoting properties. β-Conglycinin is a trimeric glycoprotein. Little information related to methods for separation of the individual chains forming β-conglycinin has been so far published and it is of great interest. As a consequence, less data on the bioactivities of α, α′ and β subunits of this glycoprotein have been published. The present research aimed to find out new alternative chromatographic conditions to obtain β-conglycinin subunits that are free of contaminating proteins. In the present short communication, we propose the use of a two-step ion exchange chromatographic protocol to achieve this goal. Firstly, β subunit was separated by means of anionic exchange fast protein liquid chromatography. Secondly, α and α′ chains were separated from each other by cationic exchange. Our data indicated the feasibility of proposed fractionation protocol to separate soybean β-conglycinin α and α′ subunits from other contaminating proteins and to obtain enough amounts of the three individual chains forming this glycoprotein for further characterization and application. The procedure may be easily up-scaled.
Keywords: Soybean β-conglycinin subunits; Isolation; FPLC (fast protein liquid chromatography); Ion exchange chromatography;

Measurement of menadione in urine by HPLC by Ala Al Rajabi; James Peterson; Sang-Woon Choi; John Suttie; Susan Barakat; Sarah L Booth (2457-2460).
Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C30 column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H2O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R 2) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.
Keywords: Menadione; HPLC; Vitamin K; Phylloquinone; Menaquinone-4;

A novel β-cyclodextrin (β-CD) functionalized organic polymer monolith was prepared by covalently bonding ethylenediamine-β-CD (EDA-β-CD) to poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monolith via ring opening reaction of epoxy groups. SEM characterization was performed to confirm the homogeneity of the monolithic polymer. The resulting monolith was then characterized by DSC and XPS elemental analysis to study the thermal stability of the monolith, and to prove the successful immobilization of β-CD on the polymer substrate. The β-CD ligand density of 0.68 mmol g−1 was obtained for the modified monolith, indicating the high reactivity and efficiency of the EDA-β-CD modifier. The ethylenediamine-β-CD functionalized monoliths were used for the chiral separation of ibuprofen racemic mixture and showed promising results.
Keywords: Ethylenediamine-β-CD; Poly(GMA-co-EGDMA); Chiral separation; Ibuprofen;