Determination of pterostilbene in rat plasma by a simple HPLC‐UV method and its application in pre‐clinical pharmacokinetic study

A simple HPLC‐UV method was developed and validated for the quantification of pterostilbene (3,5‐dimethoxy‐4'‐hydroxy‐trans‐stilbene), a pharmacologically active phytoalexin in rat plasma. The assay was carried out by measuring the UV absorbance at 320 nm. Pterostilbene and the internal standard, 3,5,4'‐trimethoxy‐trans‐stilbene eluted at 5.7 and 9.2 min, respectively. The calibration curve (20–2000 ng/mL) was linear (R² > 0.997). The lower limits of detection and of quantification were 6.7 and 20 ng/mL, respectively. The intra‐ and inter‐day precisions in terms of RSD were all lower than 6%. The analytical recovery ranged from 95.5 ± 3.7 to 103.2 ± 0.7% while the absolute recovery ranged from 101.9 ± 1.1 to 104.9 ± 4.4%. This simple HPLC method was subsequently applied in a pharmacokinetic study carried out in Sprague–Dawley rats. The terminal elimination half‐life and clearance of pterostilbene were 96.6 ± 23.7 min and 37.0 ± 2.5 mL/min/kg, respectively, while its absolute oral bioavailability was 12.5 ± 4.7%. Pterostilbene appeared to have better pharmacokinetic characteristics than its natural occurring analog, resveratrol. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all four DNA mono-nucleotides and 5′UMP. The ability of LC-ICP-MS to provide an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The method for quantification was validated by analysis of NIST SRM 2,372; a total speciated DNA recovery of 52.1 ng/μL, compared with an expected value of 53.6 ng/μL, was determined by external calibration. From repeat measurements, a mass balance of 97% ± 0.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 and 1.7 μg L⁻¹ (³¹P) for individual nucleotides by LC-ICP-MS, and 360 ng L⁻¹ for 5′AMP by direct nebulisation.     

A new methodology for simultaneous quantification of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 by column-switching LC/MS/MS

This article details the development of a novel method that overcomes the drawbacks of sandwich ELISA (sELISA) and allows reliable evaluation of simultaneous quantification of the amyloid (Aβ)-peptides, total-Aβ, Aβx-38, Aβx-40, and Aβx-42, in rat brain by optimized sample purification and column-switching liquid chromatographic-tandem mass spectrometry (LC/MS/MS). This method provides accurate analyses of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 with a linear calibration range between 0.05 and 45 ng/mL. Verification for accuracy and precision of biological samples were determined by a standard addition and recovery test, spiked with synthetic Aβ1-38, Aβ1-40, and Aβ1-42 into the rat brain homogenate. This method showed <20% relative error and relative standard deviation, indicating high reproducibility and reliability. The brain concentrations of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 after oral administration of flurbiprofen in rats were measured by this method. Aβx-42 concentrations (4.57 ± 0.69 ng/g) in rats administered flurbiprofen were lower than those in untreated rats (6.48 ± 0.93 ng/g). This was consistent with several reports demonstrating that NSAIDs reduced the generation of Aβ. We report here a method that allows not only the quantification of specific molecular species of Aβ but also simultaneous quantification of total-Aβ, Aβx-38, Aβx-40, and Aβx-42, thus overcoming the drawbacks of sELISA.     

Determination of vancomycin in human plasma using high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm × 4 mm i.d. × 10 μm Waters Associates μBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples. Figure The effect of the deproteinization solvent on the signal of the interference peak at retention time of 15.0 min. The peak which interferes with the peaks of Erythromycin and Vancomycin has been disappeared by using 2 mL acetonitrile as the deproteinization solvent.     

Dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry for the rapid and sensitive determination of UV filters in environmental water samples

The performance of the dispersive liquid–liquid microextraction (DLLME) technique for the determination of eight UV filters and a structurally related personal care species, benzyl salicylate (BzS), in environmental water samples is evaluated. After extraction, analytes were determined by gas chromatography combined with mass spectrometry detection (GC-MS). Parameters potentially affecting the performance of the sample preparation method (sample pH, ionic strength, type and volume of dispersant and extractant solvents) were systematically investigated using both multi- and univariant optimization strategies. Under final working conditions, analytes were extracted from 10 mL water samples by addition of 1 mL of acetone (dispersant) containing 60 μL of chlorobenzene (extractant), without modifying either the pH or the ionic strength of the sample. Limits of quantification (LOQs) between 2 and 14 ng L⁻¹, inter-day variability (evaluated with relative standard deviations, RSDs) from 9% to 14% and good linearity up to concentrations of 10,000 ng L⁻¹ were obtained. Moreover, the efficiency of the extraction was scarcely affected by the type of water sample. With the only exception of 2-ethylhexyl-p-dimethylaminobenzoate (EHPABA), compounds were found in environmental water samples at concentrations between 6 ± 1 ng L⁻¹ and 26 ± 2 ng mL⁻¹.     

Size characterization and quantification of silver nanoparticles by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry

A method for determining the size of silver nanoparticles and their quantification by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry (ICP-MS) is proposed and was tested in consumer products. Experimental conditions were studied in detail to avoid aggregation processes or alteration of the original size distributions. Additionally, losses from sorption processes onto the channel membrane were minimized for correct quantification of the nanoparticles. Mobile phase composition, injection/focusing, and fractionation conditions were evaluated in terms of their influence on both separation resolution and recovery. The ionic strength, pH, and the presence of ionic and nonionic surfactants had a strong influence on both separation and recovery of the nanoparticles. In general, better results were obtained under those conditions that favored charge repulsions with the membrane. Recovery values of 83 ± 8% and 93 ± 4% with respect to the content of silver nanoparticles were achieved for the consumer products studied. Silver nanoparticle standards were used for size calibration of the channel. The results were compared with those obtained by photon correlation spectroscopy and images taken by transmission electron microscopy. The quantification of silver nanoparticles was performed by direct injection of ionic silver standard solutions into the ICP-MS system, integration of the corresponding peaks, and interpolation of the fractogram area. A limit of detection of 5.6 μg L^-1 silver, which corresponds to a number concentration of 1×10¹² L^-1 for nanoparticles of 10 nm, was achieved for an injection volume of 20 μL.     

New CZE-DAD method for honeybee venom analysis and standardization of the product

The aim of this study was to develop a new precise and accurate CZE-DAD method for honeybee venom analysis using cytochrome c as an internal standard. The 64.5 cm total length, 56 cm effective length, 75 μm ID, and 360 μm OD uncoated fused-silica capillary was used. The samples were injected into the capillary under a 50-mbar pressure for 7 s. There were 15 kV of electric field across the capillary applied. The current intensity was 26 μA. The separation was carried out at 25 °C. The analysis was run with the normal electrode polarity. The following steps and parameters were taken into account for the validation of the developed method: selectivity, precision, accuracy, linearity, limit of detection and limit of quantitation. All steps of the validation procedure proved that the developed analytical procedure was suitable for its intended purpose. Possibly this was the first study in which several honeybee venom components were separated and five of them were identified by capillary zone electrophoresis. In addition, the developed method was applied for quantitative analysis of 38 honeybee venom samples. The content (relative to the dry venom mass) of analyzed peptides in honeybee venom samples collected in 2002–2007 was as follows: apamine from 0.93% to 4.34% (mean, 2.85 ± 0.79%); mast cell degranulating peptide (MCDP) from 1.46% to 4.37% (mean, 2.82 ± 0.64%); phospholipase A₂ from 7.41% to 20.25% (mean, 12.95 ± 3.09%); melittin from 25.40% to 60.27%, (mean, 45.91 ± 9.78%). The results were compared with the experimental data obtained for the same venom samples analyzed earlier by the HPLC method. It was stated that HPCE and HPLC data did not differ significantly and that the HPCE method was the alternative for the HPLC method. Moreover, using the results obtained principal component analysis (PCA) was applied to clarify the general distribution patterns or similarities of four major honeybee venom constituents collected from two different bee strains in various months and years. PCA has shown that the strain of bee appears to be the only criteria for bee venom sample classification. Strong correlations between apamine, MCDP, phospholipase A₂, and melittin were confirmed. These correlations have to be taken into account in the honeybee venom standardization. The developed method due to its simplicity can be easily automated and incorporated into routine operations both in the bee venom identification, quality control, and standardization of the product.     

HPLC assay method for ceftibuten in plasma and its application to pharmacokinetic study

The purpose of this study was to validate a reliable analytical method for pharmacokinetic study of ceftibuten in human plasma by high performance liquid chromatography (HPLC) system with UV detection. Ceftizoxime was used as the internal standard. After plasma sample was precipitated with acetonitrile and dichloromethane, the supernatant was directly injected into the HPLC system. Separation was performed on a Capcell Pak C₁₈ UG120 column (4.6 mm × 250 mm, 5 μm particles) with a mobile phase of acetonitrile/50 mM ammonium acetate (5: 95, v/v) and UV detection at a wavelength of 262 nm. The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The lower limit of quantification was 0.5 hg/mL of ceftibuten using 0.5 mL of plasma. The calibration curve was linear in concentration range of 0.5–30 μg/mL (r ² = 0.9998). The mean accuracy was 96–102%. The coefficient of variation (precision) in the intra- and inter-day validation was 0.9–3.9 and 0.9–2.4%, respectively. The pharmacokinetics of ceftibuten was evaluated after a single oral administration of 400 mg to healthy volunteers. The AUC_{0–9 h}, c _max, T _max, and T _1/2 were 86.6 ± 12.7 μg h/mL, 18.4 ± 1.5 μg/mL, 2.63 ± 0.83 and 2.65 ± 0.41 h, respectively. The method was demonstrated to be highly reproducible and feasible for pharmacokinetic studies of ceftibuten in eight volunteers after oral administration (400 mg as ceftibuten).     

An improved and fast UHPLC-PDA methodology for determination of <Emphasis Type="Italic">L</Emphasis>-ascorbic and dehydroascorbic acids in fruits and vegetables. Evaluation of degradation rate during storage

This study provides a versatile validated method to determine the total vitamin C content, as the sum of the contents of L-ascorbic acid (L-AA) and dehydroascorbic acid (DHAA), in several fruits and vegetables and its degradability with storage time. Seven horticultural crops from two different origins were analyzed using an ultra-high-performance liquid chromatographic–photodiode array (UHPLC-PDA) system, equipped with a new trifunctional high strength silica (100% silica particle) analytical column (100 mm × 2.1 mm, 1.7 μm particle size) using 0.1% (v/v) formic acid as mobile phase, in isocratic mode. This new stationary phase, specially designed for polar compounds, overcomes the problems normally encountered in HPLC and is suitable for the analysis of large batches of samples without L-AA degradation. In addition, it proves to be an excellent alternative to conventional C18 columns for the determination of L-AA in fruits and vegetables. The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, accuracy, and inter/intra-day precision. Validation experiments revealed very good recovery rate of 96.6 ± 4.4% for L-AA and 103.1 ± 4.8 % for total vitamin C, good linearity with r ² -values >0.999 within the established concentration range, excellent repeatability (0.5%), and reproducibility (1.6%) values. The LOD of the method was 22 ng/mL whereas the LOQ was 67 ng/mL. It was possible to demonstrate that L-AA and DHAA concentrations in the different horticulture products varied oppositely with time of storage not always affecting the total amount of vitamin C during shelf-life. Locally produced fruits have higher concentrations of vitamin C, compared with imported ones, but vegetables showed the opposite trend. Moreover, this UHPLC-PDA methodology proves to be an improved, simple, and fast approach for determining the total content of vitamin C in various food commodities, with high sensitivity, selectivity, and resolving power within 3 min of run analysis.     

Quantification of the six major α-dicarbonyl contaminants in peritoneal dialysis fluids by UHPLC/DAD/MSMS

During heat sterilization of peritoneal dialysis solutions, glucose is partially transformed into glucose degradation products (GDPs), which significantly reduce the biocompatibility of these medicinal products. Targeted α-dicarbonyl screening identified glyoxal, methylglyoxal, 3-deoxyglucosone, 3,4-dideooxyglucosone-3-ene, glucosone, and 3-deoxygalactosone as the major six GDPs with α-dicarbonyl structure. In the present study, an ultra-high-performance liquid chromatography method was developed which allows the separation of all relevant α-dicarbonyl GDPs within a run time of 15 min after derivatization with o-phenylenediamine. Hyphenated diode array detection/tandem mass spectrometry detection provides very robust quantification and, at the same time, unequivocal peak confirmation. Systematic evaluation of the derivatization process resulted in an optimal derivatization period that provided maximal derivatization yield, minimal de novo formation (uncertainty range ±5%), and maximal sample throughput. The limit of detection of the method ranged from 0.13 to 0.19 μM and the limit of quantification from 0.40 to 0.57 μM. Relative standard deviations were below 5%, and recovery rates ranged between 91% and 154%, dependent on the type and concentration of the analyte (in 87 out of 90 samples, recovery rates were 100 ± 15%). The method was then applied for the analysis of commercial peritoneal dialysis fluids (nine different product types, samples from three lots of each).     

"Dilute-and-shoot" triple parallel mass spectrometry method for analysis of vitamin D and triacylglycerols in dietary supplements

A method is demonstrated for analysis of vitamin D fortified dietary supplements that eliminates virtually all chemical pretreatment prior to analysis, which is referred to as a “dilute-and-shoot” method. Three mass spectrometers, in parallel, plus a UV detector, an evaporative light-scattering detector (ELSD), and a corona charged aerosol detector (CAD) were used to allow a comparison of six detectors simultaneously. Ultraviolet data were analyzed using internal standard, external standard, and response factor approaches. The contents of gelcaps that contained 2,000 IU (50 μg) vitamin D₃ in rice bran oil, diluted to 100 mL, were analyzed without the need for lengthy saponification and extraction. Vitamin D₃ was analyzed using UV detection, extracted ion chromatograms, selected ion monitoring (SIM) atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and two transitions of multiple reaction monitoring (MRM) APCI-MS. The internal standard, external standard, and response factor methods gave values of 0.5870 ± 0.0045, 0.5893 ± 0.0041, and 0.5889 ± 0.0045 μg/mL, respectively, by UV detection. The values obtained by MS were 0.6117 ± 0.0140, 0.6018 ± 0.0244, and 0.5848 ± 0.0146 μg/mL by SIM and two transitions of MRM, respectively. The triacylglycerols in the oils were analyzed using full-scan APCI-MS, electrospray ionization (ESI) MS, up to MS⁴, an ELSD, and a CAD. The method proved to be very sensitive for vitamin D₃, as well as triacylglycerols (TAGs), allowing identification of intact TAGs containing fatty acids up to 28 carbons in length. LC-ESI-MS of glycerin polymers is also demonstrated.     

Simultaneous quantification of loureirin A and loureirin B in rat urine, feces, and bile by HPLC-MS/MS method and its application to excretion study

A simple HPLC-MS/MS method for simultaneous determination of loureirin A and loureirin B in rat urine, feces, and bile after oral administration of 10.6 g/kg of longxuejie (one rare traditional Chinese medicinal herb) was developed for the first time. The analytes and buspirone (internal standard) were separated on a C₅ column with acetonitrile–water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.4 min/mL. The detector was a Q-trap™ mass spectrometer with an electrospray ionization interface operating in the multiple reaction monitoring mode. Calibration curves of loureirin A in rat urine, feces, and bile were linear over the concentration range of 1.00–5,000 ng/mL. Loureirin B in rat urine, feces, and bile ranged between 0.08 and 20, 0.20 and 20, and 0.10 and 500 ng/mL, respectively. Validation revealed that the method was specific, accurate, and precise. The fully validated method was applied to the excretion study of loureirin A and loureirin B in rats. After oral administration of 10.6 g/kg longxuejie, cumulative excretion amount of loureirin A and loureirin B in rat urine were 2.94 ± 0.81 and 0.36 ± 0.16 μg at 72 h, respectively. Of the total dose, 5.35% of loureirin A and 5.46% of loureirin B were excreted from feces at 60 h. The cumulative amounts of loureirin A and loureirin B in rat bile reached 4.49 ± 0.98 and 5.11 ± 0.83 μg, respectively, at 36 h after dosing, accounting for 0.054% and 0.056% of the total dose.     

Molecularly imprinted monolith in-tube solid-phase microextraction coupled with HPLC/UV detection for determination of 8-hydroxy-2′-deoxyguanosine in urine

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage. Measurements of 8-OHdG in urinary samples are challenging owing to the low level of 8-OHdG and the complex matrix. In this study, a novel molecularly imprinted polymer (MIP) monolithic column was synthesized with guanosine as a dummy template which was used as the medium for in-tube solid-phase microextraction (SPME). In-tube SPME coupled with HPLC/UV detection for extraction and determination of urinary 8-OHdG was developed. The synthesized MIP monolithic column exhibited high extraction efficiency owing to its greater phase ratio with convective mass transfer and inherent selectivity. The enrichment factor for 8-OHdG was found to be 76 and the limits of detection and quantification of the method for urinary samples were 3.2 nmol/L (signal-to-noise ratio 3) and 11 nmol/L (signal-to-noise ratio 10), respectively. The MIP^’s selectivity also made the sample preparation procedure and chromatographic separation much easier. The linear range of the proposed method was from 0.010 to 5.30 μmol/L (r = 0.9997), with a relative standard deviation of 1.1–6.8%, and the recovery for spiked urine samples was 84 ± 3%. The newly developed method was successfully applied to determine urinary samples of healthy volunteers, coking plant workers, and cancer patients. The 8-OHdG level in cancer patients was significantly higher than that in healthy people.     

Ultrasensitive method for the determination of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts by gas chromatography–high resolution mass spectrometry in mucosal biopsies of the lower esophagus

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3–2.0 g of human target tissues such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of 14.9 fmol HBP and a recovery of 45 ± 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of <3.1%. Method applicability was evaluated with biopsies of esophageal mucosa (N = 14) yielding 5.6 ± 1.9 mg tissue and a mean adduct level of 6.13 ± 9.35 pmol HPB/mg DNA.     

Sensitive quantification of clozapine and its main metabolites norclozapine and clozapine-<Emphasis Type="Italic">N</Emphasis>-oxide in serum and urine using LC-MS/MS after simple liquid–liquid extraction work-up

An LC-MS/MS method for the determination of the atypic neuroleptic clozapine and its two main metabolites norclozapine and clozapine-N-oxide has been developed and validated for serum and urine. After addition of d4-clozapine as deuterated internal standard a fast single-step liquid–liquid extraction under alkaline conditions and with ethyl acetate as organic solvent followed. The analytes were chromatographically separated on a Synergi Polar RP column using gradient elution with 1 mM ammonium formate and methanol. Data acquisition was performed on a QTrap 2000 tandem mass spectrometer in multiple reaction monitoring mode with positive electrospray ionization. Two transitions were monitored for each analyte in order to fulfill the established identification criteria. The validation included the determination of the limits of quantification (1.0 ng/mL for all analytes in serum and 2.0 ng/mL for all analytes in urine), assessment of matrix effects (77% to 92% in serum, 21 to 78% in urine) and the determination of extraction efficiencies (52% to 85% for serum, 59% to 88% for urine) and accuracy data. Imprecision was <10%, only the quantification of norclozapine in urine yielded higher relative standard deviations (11.2% and 15.7%). Bias values were below ±10%. Dilution of samples had no impact on the correctness for clozapine and norclozapine in both matrices and for clozapine-N-oxide in serum. For quantification of clozapine-N-oxide in urine a calibration with diluted calibrators has to be used. Calibration curves were measured from the LOQ up to 2,000 ng/mL and proved to be linear over the whole range with regression coefficients higher than 0.98. The method was finally applied to several clinical serum and urine samples and a cerebro-spinal fluid sample of an intoxicated 13-month-old girl.     

Development and validation of an HPLC-UV detection assay for the determination of rufinamide in human plasma and saliva

The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min^-1, with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [r ² = 0.998 ± 0.002 for plasma (n = 10) and r ² = 0.999 ± 0.001 for saliva (n = 9)] over the range of 0.25–20.0 μg ml^-1, with a limit of quantification at 0.25 μg ml^-1. Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring.     

Rapid quantification of tilidine,nortilidine, and bisnortilidine in urine by automated online SPE-LC-MS/MS

The opioid tilidine is a prodrug which is hepatically metabolized to active nortilidine and bisnortilidine. Due to the increasing abuse of tilidine by drug users and the lack of a specific immunoassay, we developed an analytical method for the quantification of tilidine, nortilidine, and bisnortilidine in urine suitable for screening. In a following step, this method was used to establish data on excretion kinetics of the substances in order to evaluate the time window of detection after a single oral dose of tilidine/naloxone and also was applied to authentic urine samples from correctional facilities. Urine samples were mixed with internal standard solution and extracted on a weak cation exchanger at pH 6 using a Symbiosis Pico system. The chromatographic separation was achieved within a 3.5-min run time on a Phenylhexyl column (50 × 2.0 mm, 5 μm) via gradient elution (methanol and 0.2% formic acid) at a flow rate of 0.50 mL/min. The ESI-MS/MS was performed on a QTrap 3,200 in positive multiple reaction monitoring mode using two mass transitions per analyte. Validating the method resulted in a lower limit of quantification of 1.0 μg/L followed by a linear calibration range to 100 μg/L for each analyte (r ² > 0.99). The analytical method allowed the detection of a single dose of a commercially available tilidine solution up to 7 days after administration. Using this highly sensitive method, 55 of 3,665 urine samples were tested positive.     

Selective determination of ubiquinone in human plasma by HPLC with chemiluminescence reaction based on the redox cycle of quinone

Ubiquinone is an important biologically active compound in the living body. The determination of ubiquinone in human plasma is useful for the investigation of bioavailability of ubiquinone and for early diagnosis of several diseases. Therefore, we developed a high-performance liquid chromatography (HPLC) with chemiluminescence detection method for the analysis of ubiquinone in plasma samples. The method is based on luminol chemiluminescence detection of super oxide anion that is generated by the redox cycle reaction between ubiquinone and dithiothreitol. The HPLC system involved an octyl column with a mobile phase of methanol. Ubiquinone eluted from the column was mixed with dithiothreitol and luminol solutions simultaneously, and generated chemiluminescence was monitored by chemiluminescence detector. The calibration curve for standard ubiquinone solution was linear from 0.09 to 43.2 μg/mL (0.45–216 ng on column) with the correlation coefficient of 0.999, and the detection limit (S/N = 3) was 26 ng/mL (130 pg on column). Using the proposed HPLC method, the peak of ubiquinone in human plasma could be clearly detected on the chromatogram without any interference from plasma components.     

Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine,cotinine, <Emphasis Type="Italic">trans</Emphasis>-3′-hydroxycotinine and norcotinine in human oral fluid

An analytical procedure was developed and validated for the simultaneous identification and quantification of nicotine, cotinine, trans-3′-hydroxycotinine, and norcotinine in 0.5 mL of human oral fluid collected with the Quantisal™ oral fluid collection device. Solid phase extraction and liquid chromatography-tandem mass spectrometry with multiple reaction monitoring were utilized. Endogenous and exogenous interferences were extensively evaluated. Limits of quantification were empirically identified by decreasing analyte concentrations. Linearity was from 1 to 2,000 ng/mL for nicotine and norcotinine, 0.5 to 2,000 ng/mL for trans-3′-hydroxycotinine, and 0.2 to 2,000 ng/mL for cotinine. Correlation coefficients for calibration curves were >0.99 and analytes quantified within ±13% of target at all calibrator concentrations. Suitable analytical recovery (>91%) was achieved with extraction efficiencies >56% and matrix effects <29%. This assay will be applied to the quantification of nicotine and metabolites in oral fluid in a clinical study determining the most appropriate nicotine biomarker concentrations differentiating active, passive, and environmental nicotine exposure.     

Immunochemical analysis of 3-phenoxybenzoic acid,a biomarker of forestry worker exposure to pyrethroid insecticides

Pyrethroid insecticides widely used in forestry, agricultural, industrial, and residential applications have potential for human exposure. Short sample preparation time and sensitive, economical high-throughput assays are needed for biomonitoring studies that analyze a large number of samples. An enzyme-linked immunosorbent assay (ELISA) was used for determining 3-phenoxybenzoic acid (3-PBA), a general urinary biomarker of exposure to some pyrethroid insecticides. A mixed-mode solid-phase extraction reduced interferences from acid hydrolyzed urine and gave 110 ± 6% recoveries from spiked samples. The method limit of quantification was 2 μg/L. Urine samples were collected from forestry workers that harvest pine cone seeds where pyrethroid insecticides were applied at ten different orchards. At least four samples for each worker were collected in a 1-week period. The 3-PBA in workers classified as high, low, or no exposure based on job analysis over all sampling days was 6.40 ± 9.60 (n = 200), 5.27 ± 5.39 (n = 52), and 3.56 ± 2.64 ng/mL (n = 34), respectively. Pair-wise comparison of the differences in least squares means of 3-PBA concentrations among groups only showed a significant difference between high and no exposure. Although this difference was not significant when 3-PBA excretion was normalized by creatinine excretion, the general trend was still apparent. No significant differences were observed among days or orchards. This ELISA method using a 96-well plate was performed as a high-throughput tool for analyzing around 300 urine samples measured in triplicate to provide data for workers exposure assessment.