Journal of Chromatography B (v.909, #C)

► Interaction of l-methionine-agarose with nucleotides. ► The techniques used are STD-NMR and chromatographic experiments. ► 5′-TMP has the highest retention time on support while 5′-CMP has the lowest. ► The polynucleotide retention time increases with the salt concentration. ► 5′-TMP promoted more STD contacts with the support mainly through the base.The interaction of l-methionine-agarose with 5′-mononucleotide was investigated by saturation transfer difference (STD)-nuclear magnetic resonance (NMR) spectroscopy. Chromatographic experiments were also performed using homo-oligonucleotides of distinct molecular masses (1–30 nucleotides) to explore the effect of base hydrophobicity, temperature, pH and salt concentration on the retention of homo-oligonucleotides to l-methionine-agarose support. With STD-NMR, the results reveal that hydrophobic residues, such as the CH3 of thymine and adenine, can preferentially recognise the l-methionine side chain of the support. Also, 5′-TMP led to more contacts with the support, while 5′-UMP presented fewer STD contacts. For 5′-UMP, 5′-CMP and 5′-GMP, the main interaction with the support was through the sugar-phosphate backbone. Similar binding profiles were obtained using chromatographic experiments. Indeed, 5′-TMP had the highest retention time, followed by 5′-GMP, 3′-AMP, 5′-UMP and 5′-CMP. In general, the retention factor of homo-oligonucleotides was higher for ammonium sulphate concentration 1.5 M. For the polyT3–polyT30 series, the retention time increased by about three-fold, indicating that larger homo-oligonucleotides have more hydrophobic bases, thus enhancing contact with the l-methionine support. The temperature (5, 20 and 35 °C) did not influence homo-oligonucleotide retention. However, the retention time slightly increased when the pH was lower than 9. The STD-NMR technique combined with chromatographic experiments was thus successfully used to screen amino acid–nucleotide interactions.
Keywords: Nuclear magnetic resonance; Chromatographic experiments; Homo-oligonucleotides; l-Methionine support; Molecular interactions;

► A multi target analytical approach for clinical drugs of abuse screening was explored. ► The analysis time was short. ► The selectivity was based on high efficiency chromatography and high resolution MS. ► A method comparison was made with LC–tandem MS and immunochemistry. ► A large number (800) authentic urine specimens were used for evaluation.In this study a rapid liquid chromatography–time-of-flight mass spectrometry method was developed, validated and applied in order to evaluate the potential of this technique for routine urine drug testing. Approximately 800 authentic patient samples were analyzed for amphetamines (amphetamine and methamphetamine), opiates (morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine and codeine-6-glucuronide) and buprenorphines (buprenorphine and buprenorphine-glucuronide) using immunochemical screening assays and mass spectrometry confirmation methods for comparison. The chromatographic application utilized a rapid gradient with high flow and a reversed phase column with 1.8 μm particles. Total analysis time was 4 min. The mass spectrometer operated with an electrospray interface in positive mode with a resolution power of >10,000 at m/z 956. The applied reporting limits were 100 ng/mL for amphetamines and opiates, and 5 ng/mL for buprenorphines, with lower limits of quantification were 2.8–41 ng/mL. Calibration curves showed a linear response with coefficients of correlation of 0.97–0.99. The intra- and interday imprecision in quantification at the reporting limits were <10% for all analytes but for buprenorphines <20%. Method validation data met performance criteria for a qualitative and quantitative method. The liquid chromatography–time-of-flight mass spectrometry method was found to be more selective than the immunochemical method by producing lower rates of false positives (0% for amphetamines and opiates; 3.2% for buprenorphines) and negatives (1.8% for amphetamines; 0.6% for opiates; 0% for buprenorphines). The overall agreement between the two screening methods was between 94.2 and 97.4%. Comparison of data with the confirmation (LC–MS) results for all individual 9 analytes showed that most deviating results were produced in samples with low levels of analytes. False negatives were mainly related to failure of detected peak to meet mass accuracy criteria (±20 mDa). False positives was related to presence of interfering peaks meeting mass accuracy and retention time criteria and occurred mainly at low levels. It is concluded that liquid chromatography–time-of-flight mass spectrometry has potential both as a complement and as replacement of immunochemical screening assays.
Keywords: Screening; Drugs of abuse; Urine; Time-of-flight mass spectrometry; Liquid chromatography;

► HILIC SPE was developed for cGMP and cAMP analysis in biological samples. ► Low ng/mL quantification limits were obtained by LC–MS/MS. ► Plasma volumes as low as 200 μL were thereby used. ► 2′,3′ and 3′,5′ isomers could simultaneously be monitored. ► The four isomers were monitored in tissue samples.3′,5′-Cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are essential second messenger molecules. They are involved in signal transduction within cells, in physiological functions such as neurotransmission and in the modulation of cell growth and differentiation of organisms, respectively. A quantitative solid phase extraction method (SPE) based on hydrophilic interaction on silica was developed and applied to both plasma and tissue samples. The stable isotope-labeled internal standards 2D1,15N3-3′,5′-cGMP and 13C10,15N5-3′,5′-cAMP were added prior to the sample preparation to ensure high precision and accuracy. The samples were analyzed by reversed-phase liquid chromatography (RP-LC). Negative electrospray (ESI)-MS/MS was used to selectively monitor several transitions of each metabolite. The method for the analysis of 3′,5′-cAMP and 3′,5′-cGMP in plasma was validated in the range of 0.15–20 ng/mL (R 2  = 0.9996 and 0.9994 for 3′,5′-cAMP and 3′,5′-cGMP, respectively). Basal plasma concentrations for fifteen healthy human patients determined with this method varied between 4.66–9.20 ng/mL for 3′,5′-cAMP and between 0.30–1.20 ng/mL for 3′,5′-cGMP, with precisions better than 9.1%. 3′,5′-cGMP and 3′,5′-cAMP together with their 2′,3′-isomers were also determined in a semi quantitative way in animal tissues. The structures of the isomers were confirmed by analysis with LC-high resolution time-of-flight MS and subsequently by comparison of retention times with standards.
Keywords: Liquid chromatography; Triple quadrupole mass spectrometry; Cyclic nucleotides; Sample preparation; Human plasma; Animal tissues;

► On-line combination of BS and PFSG methods with CE. ► Enhance sensitivity and decrease analysis time without any modification. ► 10-fold higher detection sensitivity and ∼2-fold faster analysis of FP virus. ► Rapid and highly sensitive analysis technique for disease-related specific DNA.A rapid on-line capillary electrophoresis (CE) method for highly sensitive detection of DNA molecules with specific lengths was developed based on the combination of base stacking (BS) and programmed field strength gradients (PFSG). The BS method has been performed for on-column concentration to improve detection sensitivity without any modification of the CE system. PFSG increased the electrophoretic velocity of DNA molecules, which effectively decreased analysis time. Using the BS and PFSG combination, the amplified PCR product (340-bp DNA) of cats infected with feline panleukopenia virus was detected within 6.5 min. Detection sensitivity (∼10-fold) was enhanced compared to conventional CE analysis. The combined on-line CE/BS-PFSG methodology could be an effectively rapid analysis technique for the highly sensitive detection of disease-related specific DNA molecules.
Keywords: Feline panleukopenia virus; Capillary electrophoresis; Enhanced detection sensitivity; Rapid detection; On-line combination;

Analysis of new potential anticonvulsant compounds in mice brain tissue by SPE/HPLC/DAD by Jolanta Flieger; Magdalena Pizoń; Tomasz Plech; Jarogniew J. Łuszczki (26-33).
► Separation method in sample together with standard antiepileptic drugs taking into account observed their synergic action. ► Quantitative determination of new ant-epileptic compounds in mouse brain tissue by HPLC–DAD after sample clean-up step by the use of SPE procedure. ► Explanation of the different anti-epileptic activity.This paper describes a novel reversed-phase high performance liquid chromatography (RP-HPLC) with photo-diode array detection (DAD) method for the determination of three new derivatives of 4-alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with different antiepileptic activity in the brains of mice treated with the doses of 300 mg kg−1 of body weight. Samples were prepared by solid-phase extraction (SPE) method using BAKERBOND™ spe Octadecyl (C18) and analyzed by the use of an isocratic elution mode over an Zorbax Extend-C18 column (150 mm × 4.6 mm I.D., 5-μm, Agilent Technologies). The mobile phase consisted of 80% methanol (for compound TP-315) and 85% acetonitrile (for compound TP-321) for 80% 2-propanol (for TP-323) at a flow rate of 1.0 mL min−1 and 0.5 mL min−1 in the last case. Gradient elution mode was also proposed for all examined analytes in mixture with common antiepileptic drugs: carbamazepine, phenobarbital and phenytoin in view of possible synergistic activity. Photodiode-array investigations of the peaks after degradation studies indicate the stability of the compounds under conditions proposed for sample preparation procedure. Linear coefficients of correlation (r 2) were >0.995 for all analytes. The proposed strategy gives extraction yields higher than 95% with the intra- and inter-day relative standard deviation lower than 3% and 5%, respectively. This method was applied to the analysis of brain tissue of mice treated with investigated compounds. Obtained results enable to explain the differences in their pharmacological activity.
Keywords: Anti-epileptic agents; Mouse brain tissue; HPLC; SPE; DAD;

► HPLC detection system was established to quantify chelators specific to soft-metal ions. ► The system employed dequenching of Cu(I)–BCS complexes as a detection system. ► GSH and PC2–PC4 were successfully determined. ► Methanol was superior over acetonitrile as an eluent.HPLC eluent systems employing acetonitrile and methanol were evaluated for the quantitation of glutathione (GSH) and phytochelatin (PC n ), a family of peptides implicated in heavy-metal detoxification in higher plants. The detection system is based on the dequenching of copper(I)–bathocuproine disulfonate and is specific for soft-metal chelators. Although both elution systems yielded comparable analytical performance for each PC n , the acetonitrile system had a lower sensitivity for GSH and a steadily increasing baseline. The inferior properties of the acetonitrile system may be due to complex formation between acetonitrile and Cu(I) ions. Both methods were applied to measure peptide levels in the primitive red alga Cyanidioschyzon merolae. Coefficients of variation (CVs) were less than 5%, except for GSH and PC4 determinations in the acetonitrile system, in cases when CV values were found to be 8.8% and 6.3%, respectively. Recoveries were greater than 96%, except for GSH determination in the acetonitrile system, with a recovery of 84.4%; however, the concentration measured in the acetonitrile system did not differ from that measured in the methanol system at a significance level of 0.05.
Keywords: Phytochelatin; Glutathione; Bathocuproine disulfonate; Dequenching;

► HF-LPME was used to quantitate the oestrogens from individual zebrafish liver. ► Injection port derivatization of the oestrogens was performed. ► Bioaccumulation of zebrafish liver was investigated using tank experiments.The applicability of hollow fibre protected liquid phase microextraction (HF-LPME) for the determination of three oestrogens, namely estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) from individual zebrafish liver samples, in a bioaccumulation study on these organisms, is reported. The oestrogens were extracted from single, mechanically crushed and minced livers from fish that were heaved in tubes containing water spiked at low concentration of the analytes. Extraction was performed with ∼3 μL of toluene contained in the hollow fibre. In order to achieve high extraction efficiency, the parameters that could affect the effectiveness of HF-LPME were optimized, i.e. the extracting organic solvent, extraction time, stirring speed and pH of the aqueous phase. For gas chromatography/mass spectrometry (GC/MS) analysis, injection port derivatization of the oestrogens with bis(trimethylsilyl)trifluoroacetamide was conducted. Under the most favourable extraction and derivatization conditions, enrichment factors of 158–279 were obtained. Linearity of the HF-LPME–GC/MS method was evaluated from 1 to 50 μg/L and the coefficient of determination (r 2 ) ranged from 0.9687 to 0.9926. The LODs were between 0.017 and 0.033 μg/L (at a signal to noise ratio of 3) with relative standard deviations (RSDs, analytes spiked at 5 μg/L) of between 15 and 17% (n  = 3).
Keywords: Oestrogens; Bioaccumulation; Microextraction; Single zebrafish liver; Injection port derivatization;

► Analysis of four urinary metabolites of JWH-018 and JWH-073 was streamlined. ► β-Glucuronide hydrolysis step was reduced to only 10 min at room temperature. ► A salting-out assisted liquid–liquid extraction reduced processing time and waste. ► The analysis run time was shortened to 3.2 min by utilizing UPLC.Herbal smoking mixtures which are sold as incense or potpourri and often referred to as ‘Spice’ are actually inactive plant matter adulterated with alkylamino indole based synthetic cannabinoids such as JWH-018 and JWH-073. Due to the inclusion of five synthetic cannabinoids, including JWH-018 and JWH-073, as Schedule I drugs by the Drug Enforcement Agency (DEA) in March 2011, it has become necessary for forensic laboratories to develop analytical methods to test for the presence of metabolites of synthetic cannabinoids. When a new analyte of interest emerges, most laboratories strive to develop a sample preparation procedure and validate an analytical method as quickly as possible and therefore, rely on effective but time consuming traditional protocols such as solid phase and liquid–liquid extraction. This research focuses on the examination of all aspects of sample preparation and analytical method development to streamline the analysis of four urinary metabolites of JWH-018 and JWH-073. A detailed evaluation of the β-glucuronide hydrolysis step lead to the reduction of time required for hydrolysis from 1 h at 50 °C to only 10 min at room temperature. By utilizing a salting-out assisted liquid–liquid extraction (SALLE) in place of traditional liquid–liquid extraction with a volatile solvent, processing time was saved and waste was reduced. The analysis run time was also shortened to one-third of a typical published run time by utilizing UPLC with isocratic conditions in place of conventional HPLC running a gradient method.
Keywords: Salting-out assisted liquid–liquid extraction; UPLC; Synthetic cannabinoids; Spice; Enzymatic hydrolysis; Urine;

► Aminophenyl boronic acid functionalized macroporous particles was synthesized. ► Selectivity of particles to cis-diol-containing flavonoids was presented. ► The adsorption behavior of particles was tested with Hypericum perforatum extracts. ► Increment of antioxidant activity in desorption solution was observed. ► The highest radical scavenging capacity was obtained with post-adsorption solution.Aminophenyl boronic acid (APBA) carrying uniform-macroporous poly(chloromethylstyrene-co-divinylbenzene), poly(CMS-co-DVB) particles were synthesized for selective separation of cis-diol-containing flavonoids from plant extracts. For this purpose, 2.5 μm polystyrene seed particles were first swelled by a mixture of dibutyl phthalate (DBP), toluene and dodecanol, then by a monomer mixture including CMS and DVB. The repolymerization of the monomer phase in the swollen seed particles provided macroporous and uniform particles, approximately 7 μm in size. Chlorine atoms on the surface of these particles were derivatized with APBA to gain affinity properties for flavonoids containing vicinal hydroxyl groups. Model adsorption studies showed that these particles selectively adsorbed quercetin and rutin containing cis-diol groups, but did not adsorb apigenin similar to quercetin and not carrying cis-diol groups. These particles were also tested in adsorption/desorption studies for ethanol and ethyl acetate extracts of the Hypericum perforatum (HP) stems to obtain high antioxidant mixtures. With ethanol extract, the antioxidant activity of the desorption solution was a bit higher than that of the post-adsorption solutions. However, the DPPH radical scavenging activity of the desorption solution decreased with respect to the original extract and post-adsorption solutions. A similar result was obtained for the antioxidant activity of the desorption solution using ethyl acetate extract. An interesting result was obtained that DPPH radical scavenging activity of the post-adsorption solution was higher than that of the original ethyl acetate extract and desorption solutions. These results were attributed to selective adsorption of antioxidant characterized cis-diol-containing apolar molecules much more rather than that radical scavenger characterized polar molecules.
Keywords: Uniform latex particles; Activated swelling method; Chromatographic packing material; Adsorption–desorption; Antioxidant activity; Hypericum perforatum;

► MIPs for sulfadimethoxine were prepared non-covalently. ► Imprinting factors for four template:monomer:cross-linker ratios were evaluated. ► Monomer ratio should at least equal the hydrogen bonding sites on the template. ► Excess monomer or cross-linker can decrease the imprinting factor. ► Retention of SDM on MIP by HPLC increases with higher water in mobile phase.Four non-covalently prepared molecularly imprinted polymers (MIPs) for sulfadimethoxine (SDM) were prepared using different ratios of SDM template, methacrylic acid monomer, and ethylene glycol dimethacrylate cross-linker. The imprinting factor (IF) was calculated by comparing the retention of SDM on the imprinted polymer with a comparable non-imprinted polymer. The template:monomer:cross-linker ratio of 1:6:20 resulted in an IF of 3.94 which is higher than found in previous studies. A significant decrease in IF to 0.89 when template:cross-linker ratio was 1:40 contradicts most literature where higher cross-linker concentration improves selectivity. IF was 4.36 when 20% water was added to the acetonitrile HPLC mobile phase during evaluation. Retention of SDM increased as water concentration changed as: 20, 40, 0, 60 and 70%, indicating a combination of shape recognition, hydrogen bonding and hydrophobic interactions contributing to retention of analyte. The MIP has the potential for use in SPE for purification and concentration of SDM and with further optimization, possibly direct HPLC analysis.
Keywords: Molecularly imprinted polymer; Sulfadimethoxine;

Determination of tamsulosin in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study by Chang-Ik Choi; Hye-In Lee; Jung-Woo Bae; Yun-Jeong Lee; Ji-Yeong Byeon; Choon-Gon Jang; Seok-Yong Lee (65-69).
► The more rapid, sensitive and simplified LC–MS/MS method for human plasma tamsulosin. ► A liquid–liquid sample extraction procedure without a sample-alkalinizing step. ► The lower limit of quantification using 500 μL of human plasma was 0.01 ng/mL.Tamsulosin, a selective α1-adrenoceptor antagonist, is used for the treatment of benign prostatic hyperplasia (BPH). We developed and validated a rapid, sensitive, and simplified liquid chromatography analytical method utilizing tandem mass spectrometry (LC–MS/MS) for the determination of tamsulosin in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tamsulosin was achieved using a reversed-phase Luna C18 column (2.0 mm × 50 mm, 5 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5)–methanol (25:75, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of tamsulosin and the internal standard (IS, diphenhydramine) were 0.8 and 0.9 min, respectively. The calibration curves were linear over a range of 0.01–20 ng/mL (r  > 0.999). The lower limit of quantification using 500 μL of human plasma was 0.01 ng/mL. The mean accuracy and precision for intra- and inter-day validation of tamsulosin were both within acceptable limits. The present LC–MS/MS method showed improved sensitivity for quantification of tamsulosin in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.
Keywords: Tamsulosin; LC–MS/MS; Human plasma; Pharmacokinetics;

Molecularly imprinted solid phase extraction of urinary diethyl thiophosphate and diethyl dithiophosphate and their analysis by gas chromatography–mass spectrometry by Mariane Gonçalves Santos; Ricardo Vilela Vitor; Felipe Lopes Andrade; Isarita Martins; Eduardo Costa Figueiredo (70-76).
► A MIP was developed for selective extraction of dialkyl phosphates in urine samples. ► The eluate was efficiently analyzed by gas chromatography–mass spectrometry. ► Good figures of merit were attained. ► The method is an interesting alternative to analyze dialkyl phosphates in urine.An analytical method involving molecularly imprinted solid phase extraction (MISPE) and gas chromatography–mass spectrometry (GC–MS) was developed for the analysis of organophosphates metabolites (diethyl thiophosphate – DETP and diethyl dithiophosphate – DEDTP) in human urine samples. A DETP molecularly imprinted polymer (MIP) was synthesized using 4-vinylpiridine as the functional monomer and ethylene glycol dimethacrylate as the cross-linker. The conditioning step of the MISPE was conducted by running 3 mL of acetonitrile, 3 mL of 0.1 mol L−1 dibasic phosphate buffer at pH 11 and 2 mL of water through the molecularly imprinted polymer (MIP) cartridge. The extraction step was executed using 1.0 mL of a urine sample, with the pH previously adjusted to 3.0. Finally, the analytes were eluted with 3 mL of acetonitrile and derivatized with 3% 2,3,4,5,6-pentafluorobenzyl bromide solution at room temperature for 1 h. The sample was analyzed by GC–MS in the SIM (selected ion monitoring) mode. Analytical calibration curves for DETP and DEDTP were constructed using a pool of urine samples and six levels of concentration. The method was found to be linear from 10 to 500 μg L−1 (r  > 0.99) with limits of quantification of 10 μg L−1 for both analytes. The within-day and between-day precisions were evaluated (as %RSD) and all the results were <15% for both analytes. The method was accurate (relative error < ±15%), with good robustness.
Keywords: MIP; Dialkyl phosphates; SPE; Pentafluorobenzyl bromide; Gas chromatography–mass spectrometry; Organophosphates;