Optimizing a High-Throughput Solid-Phase Microextraction System to Determine the Plasma Protein Binding of Drugs in Human Plasma.

Plasma protein binding refers to the binding of a drug to plasma proteins after entering the body. The measurement of plasma protein binding is essential during drug development and in clinical practice, as it provides a more detailed understanding of the available free concentration of a drug in the blood, which is in turn critical for pharmacokinetics and pharmacodynamics studies. In addition, the accurate determination of the free concentration of a drug in the blood is also highly important for therapeutic drug monitoring and in personalized medicine. The present study uses C18-coated solid-phase microextraction 96-pin devices to determine the free concentrations of a set of drugs in plasma, as well as the plasma protein binding of drugs with a wide range of physicochemical properties. It should be noted that the extracted amounts used to calculate the binding constants and plasma protein bindings should be measured at respective equilibrium for plasma and phosphate buffer. Therefore, special attention is placed on properly determining the equilibration times required to correctly estimate the free concentrations of drugs in the investigated systems. The plasma protein binding values obtained with the 96-pin devices are consistent with those reported in the literature. The 96-pin device used in this research can be easily coupled with a Concept96 or other automated robotic systems to create an automated plasma protein binding determination protocol that is both more time and labor efficient compared to conventional equilibrium dialysis and ultrafiltration methods.

Modification of polyacrylate sorbent coatings with carbodiimide crosslinker chemistry for sequence-selective DNA extraction using solid-phase microextraction.

Selective DNA extraction is immensely useful for the isolation and detection of low-abundance sequences. Oligonucleotide-modified substrates are often used to capture sequences of interest for downstream analysis. In this study, we explore the chemical modification of commercial-available polyacrylate solid-phase microextraction fibers for selective DNA analysis using carbodiimide crosslinker chemistry. Reproducible modification conditions are found and the fibers were subsequently applied for selective DNA analysis. Several experimental parameters such as stir-rate, desorption time, and buffer-type are optimized. The developed method was able to selectively extract the target DNA sequence (260 bp) in the presence of 100-fold excess interfering salmon testes DNA.

Analysis of polynuclear aromatic hydrocarbons in olive oil after solid-phase extraction using a dual-layer sorbent cartridge followed by high-performance liquid chromatography with fluorescence detection.

A simple and easy direct solid-phase extraction (SPE) method was developed for the analysis of polynuclear aromatic hydrocarbons (PAHs) in olive oil using a dual-layer cartridge containing activated Florisil and a mixture of octadecyl (C18)-bonded and zirconia-coated silicas. Undiluted olive oil was applied directly to the SPE cartridge, and the sample was eluted with acetonitrile solvent. Background in the extract was found to be low enough for either gas chromatography-mass spectrometry (GC-MS) or high-performance liquid chromatography with fluorescence detection (HPLC-FLD) analysis. Average recoveries for 16 different PAHs from spiked olive oil replicates were >75%, with intraday precisions of <20% relative standard deviation (% RSD). Detection limits ranged from 0.2 to 1.0 µg/kg and, specifically for the PAHs listed in EC Regulation 835/2011, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, and benzo(a)pyrene, were from 0.3 to 0.7 µg/kg. The method was then applied to determine the PAH content present in commercial samples of refined versus extra-virgin olive oils.

Nontargeted analysis of DNA adducts by mass-tag MS: reaction of p-benzoquinone with DNA.

Using a method in which DNA adducts are discovered based on their conversion in a nucleotide form to phosphorimidazolides with isotopologue benzoylhistamines (or p-bromobenzoylhistamine) prior to detection by MALDI-TOF-MS, we have profiled the adducts that form when calf thymus DNA is reacted in vitro with p-benzoquinone (BQ). We find, as relative values normalized to 100% of adducts observed, 79% BQ-dCMP, 21% BQ-methyl-dCMP (a new DNA adduct), and trace amounts of BQ-dAMP and BQ-dGMP. Because mC is 5% of C in this DNA, the reaction of BQ with DNA in vitro is about five times faster at methyl-C than C. When equal amounts of dCMP and methyl-dCMP are reacted with BQ, equal amounts of the corresponding adducts are observed. Thus, the microenvironment of methyl-C in DNA enhances its reactivity relative to C with BQ. In a prior, similar study, but based on analysis by (32)P-postlabeling, the second most abundant adduct was assigned to BQ-A, apparently because of comigration of the BQ-A and BQ-methyl-C adducts (as bisphosphates) in the chromatographic step. Because the calf thymus DNA (used as received) was contaminated with RNA, we also detected the ribonucleotide adduct, BQ-CMP.

Novel pre-fractionation method of trans fatty acids by gas chromatography with silver-ion cartridge column.

We developed a novel pre-separation method of trans fatty acids (TFAs) using a silver-ion cartridge column and GC. As a preliminary study, a mixture of fatty acid methyl esters consisting of saturated, cis-unsaturated, and trans-unsaturated fatty acids was dissolved in dichloromethane and loaded onto a Bond Elut SCX ion-exchange cartridge column that was converted to the silver-ion form. The column was then eluted with dichloromethane to obtain the saturated fatty acids, dichloromethane/ethyl acetate (90/10) for the trans mono-ene, dichloromethane/ethyl acetate (65/35) for the cis mono-ene, dichloromethane/acetone (60/40) for the trans di-ene, and acetone/acetonitrile (80/20) for the others. Satisfactory separation of the cis/trans isomers was confirmed by GC analysis. To generalize this technique, the elution conditions of the ready-to-use Discovery Ag-ION SPE cartridge column were also optimized. Both cartridge columns had good separation, recovery, and repeatability. Peer laboratory verification was carried out between two laboratories using different production lots of the ready-to-use cartridge column, and the robustness of the product and reproducibility of the method were found to be satisfactory. This technique is therefore a powerful tool not only for routine analyses of TFAs in oils, fats, and foods but also for detailed analyses of TFAs in various research fields.

Determination of malachite green residues in fish using molecularly imprinted solid-phase extraction followed by liquid chromatography-linear ion trap mass spectrometry.

This paper describes the development of an analytical procedure to determine malachite green (MG) residues in salmon samples using molecularly imprinted polymers (MIPs) as the extraction and clean-up material, followed by liquid chromatography-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS). MG and two structurally related compounds, crystal violet (CV) and brilliant green (BG) were employed for the selectivity test. The imprinted polymers exhibited high binding affinity for MG, while CV and BG showed less binding capacity: 47% and 34%, respectively. The recovery values of MG in salmon samples fortified with leucomalachite green (LMG) were determined by measuring the amount of MG in the sample, after carrying out the oxidation reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which converts the LMG back into chromic-form. The average recovery of MG in spiked salmon muscle over the concentration range 1-100 ng g(-1) was 98% with a relative standard deviation value (R.S.D.) below 12%. The method detection limits (MDLs) obtained for MG, CV, BG and their leuco-metabolites were in the range of 3-20 ng kg(-1) (ppt).

Development of an improved method for trace analysis of chloramphenicol using molecularly imprinted polymers.

A confirmatory method is described for the determination of the illegal antibiotic chloramphenicol using a specifically developed molecularly imprinted polymer (MIP) as the sample clean-up technique. The newly developed MIP was produced using an analogue to chloramphenicol as the template molecule. Using an analogue of the analyte as the template avoids a major traditional drawback associated with MIPs of residual template leeching or bleeding. The MIP described was used as a solid-phase extraction phase for the extraction of chloramphenicol from various sample matrices including honey, urine, milk and plasma. A full analytical method with quantification by LC-MS/MS is described. The method was fully validated according to the European Union (EU) criteria for the analysis of veterinary drug residues.

Evaluation of a solid-phase extraction dual-layer carbon/primary secondary amine for clean-up of fatty acid matrix components from food extracts in multiresidue pesticide analysis.

The use of dual-layer solid-phase extraction (SPE), a primary-secondary amine (PSA) in combination with graphitized carbon black (GCB), was evaluated for sample clean-up during multiresidue pesticide screening of agricultural and food products. The retention of fatty acids by the PSA sorbent was quantified and the effect of the elution solvent on the retention of fatty acid on the SPE cartridge was evaluated. The use of stronger elution solvents to elute certain pesticides from graphitized carbon was shown to interfere with the capacity of PSA to bind fatty acids. A suitable protocol was tested using GCB/PSA dual-layer SPE to clean-up several food matrices and to simultaneously screen multiple fortified pesticides with a wide range of physico-chemical properties. With a few exceptions, pesticide recoveries were between 85% and 110%, and sample-to-sample differences of less than 5% were achieved, demonstrating the versatile suitability of the dual-layer SPE to sample clean-up.

Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation.

We have developed a practical method for quantifying DNA. The method is practical in two ways. First, a single enzyme is used to digest the DNA to nucleotides that are then quantified by HPLC under ordinary conditions. Second, the method quantifies DNA even when it is impure. In our method, "nuclease P1/HPLC," the DNA is hydrolyzed by nuclease P1 and the resulting 2'-deoxynucleoside 5'-monophosphates are quantified by HPLC with UV detection. This method was applied to several kinds of genomic DNA in terms of origin and method by which it had been purified. Calf thymus DNA (purified by salt precipitation by the supplier), pig liver DNA (purified by phenolic extraction or by anion-exchange chromatography using a Genomic Tip from Qiagen) and mouse skin DNA (similarly purified) were tested. In some cases a given sample was purified by two of these methods. The values for the amount of DNA by our method were compared with those by three other methods: acid hydrolysis/HPLC (selected as a reference procedure), UV absorbance, and dye binding. Agreement for all DNA samples between the values by our method versus those provided by acid hydrolysis/HPLC was within 10% for amounts of DNA in the 19-54 microg range. In contrast, UV absorbance and the dye-binding assay gave differences up to 30-40% relative to the consistent values furnished by acid hydrolysis and our method. Overall, normalizing the concentrations of the DNA (thymus, liver, skin) by acid hydrolysis/HPLC in 10 samples to values of 1.0 gave the following, relative values and standard deviations: 1.01+/-.07 (nuclease P1/HPLC), 0.8+/-0.17 (dye binding), and 1.1+/-0.1 (UV). Since one cannot assume that any sample of DNA is pure, and determining purity of DNA is difficult, then nuclease P1/HPLC or acid hydrolysis/HPLC is recommended rather than the UV absorbance or dye binding for quantifying DNA whenever an accurate value is important.

Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides.

Towards a goal of detecting scaled-up DNA adducts as altered deoxynucleotides by mass spectrometry, we have set up a practical and general method for isolating DNA-derived deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides starting with a 1 g sample of mammalian tissue. The method is practical because costs have been minimized, and it is general because it can be applied to a more difficult sample such as mouse skin or non-fresh calf liver. The procedure, consisting of a series of steps that were largely gleaned and tuned from prior literature, proceeds as follows: (1) homogenize the tissue in sodium dodecyl sulfate; (2) digest with ribonuclease A, ribonuclease TI, alpha-amylase and proteinase K; (3) partition between water and phenol; (4) precipitate the DNA with ethanol followed by redissolving and dialysis; and (5) digest with nuclease P1 and phosphodiesterase I followed by ultrafiltration and boric acid gel chromatography. The yellow to brown color of DNA from difficult tissues only persisted up to the ultrafiltration step. Apparently this DNA was contaminated with iron-containing proteins. Residual ribonucleotides were not observable (<0.1%) by HPLC in the final sample. Without boric acid gel chromatography, residual contamination by ribonucleotides was about 1% even when the DNA was purified before digestion by phenol partitioning followed by use of a Genomic Tip kit from Qiagen.

Handling and detection of 25 amol of near-infrared dye deoxynucleotide conjugates by capillary electrophoresis with laser-induced fluorescence detection.

Near-infrared dyes are attractive as labeling reagents to enhance sensitivity in trace analysis largely because background fluorescence is low in this spectral region. Here we demonstrate, towards a goal of detecting DNA adducts in small biological samples, that some near-infrared (IR) dye-labeled deoxynucleotides can be separated and detected with high sensitivity by capillary electrophoresis (CE)-laser-induced fluorescence detection (LIF) in a realistic way (handling detection limit of 25 amol) for near-IR dye-labeled deoxynucleotides. This detection limit is achieved by polarity-switching injection of 2.0 microl from a volume of 5.0 microl, in which the compounds are 5 x 10(-12) mol/l in 50% aqueous methanol. Although the adenine and cytosine-containing conjugates co-migrated, the other three (guanine, N2-ethylguanine and thymine) were resolved.

Handling and detection of 0.8 amol of a near-infrared cyanine dye by capillary electrophoresis with laser-induced fluorescence detection.

We are interested in the detection of DNA adducts and other trace analytes by labeling them with a fluorescent tag followed by use of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for high resolution and sensitivity. Towards this goal, here we report the following: (1) synthesis and handling properties of a near-IR, carboxyl-substituted heptamethine cyanine dye; (2) modification of an existing ball lens LIF detector to provide near-LIF detection with excitation at 785 nm for CE; and (3) corresponding handling and detection of as little as 0.8 amol of the dye by enrich-injection of 4.7 microl of 1 x 10(-13) mol/l dye in methanol from an 8-microl volume into a corresponding CE-LIF system. The electrolyte for the separation was methanol-40 mmol/l aqueous sodium borate (98:2, v/v). This finding encourages further exploration of the dye by functionalization of its carboxyl group for chemical labeling purposes.

Monensin mediates a rapid and selective transport of Pb(2+). Possible application of monensin for the treatment of Pb(2+) intoxication.

The carboxylic acid ionophore monensin, known as an electroneutral Na(+) ionophore, an anticoccidial agent, and a growth-promoting feed additive in agriculture, is shown to be highly efficient as an ionophore for Pb(2+) and to be highly selective for Pb(2+) compared with other divalent cations. Monensin transports Pb(2+) by an electroneutral mechanism in which the complex PbMonOH is the transporting species. Electrogenic transport via the species PbMon(+) may also be possible. Monensin catalyzed Pb(2+) transport is little affected by Ca(2+), Mg(2+), or K(+) concentrations that are encountered in living systems. Na(+) is inhibitory, but its effectiveness at 100 mm does not exceed approximately 50%. The poor activity of monensin as an ionophore for divalent cations other than Pb(2+) is consistent with the pattern of complex formation constants observed in the mixed solvent 80% methanol/water. This pattern also explains why Ca(2+), Mg(2+), and K(+) are ineffective as inhibitors of Pb(2+) transport, but it does not fully explain the actions of Na(+), where kinetic features of the transport mechanism may also be important. When given to rats at 100 ppm in feed together with Pb(2+) at 100 ppm in drinking water, monensin reduces Pb accumulation in several organs and tissues. It also accelerates the excretion of Pb that was accumulated previously and produces this effect without depleting the organs of zinc or copper. Monensin, used alone or in combination with other agents, may be useful for the treatment of Pb intoxication.