Validation of a high‐performance liquid chromatography method for the determination of (−)‐α‐bisabolol from particulate systems

A reversed‐phase high performance liquid chromatography method has been developed and validated for determination and quantitation of the natural sesquiterpene (−)‐α‐bisabolol. Furthermore the application of the method was done by characterization of chitosan milispheres and liposomes entrapping Zanthoxylum tingoassuiba essential oil, which contains appreciable amount of (−)‐α‐bisabolol. A reversed‐phase C₁₈ column and gradient elution was used with the mobile phase composed of (A) acetonitrile–water–phosphoric acid (19:80:1) and (B) acetonitrile. The eluent was pumped at a flow rate of 0.8 mL/min with UV detection at 200 nm. In the range 0.02–0.64 mg/mL the assay showed good linearity (R² = 0.9999) and specificity for successful identification and quantitation of (−)‐α‐bisabolol in the essential oil without interfering peaks. The method also showed good reproducibility, demonstrating inter‐day and intra‐day precision based on relative standard deviation values (up to 3.03%), accuracy (mean recovery of 100.69% ± 1.05%) and low values of detection and quantitation limits (0.0005 and 0.0016 mg/mL, respectively). The method was also robust for showing a recovery of 98.81% under a change of solvent in standard solutions. The suitability of the method was demonstrated by the successful determination of association efficiency of the (−)‐α‐bisabolol in chitosan milispheres and liposomes. Copyright © 2009 John Wiley & Sons, Ltd.     

HPLC determination of γ‐aminobutyric acid and its analogs in human serum using precolumn fluorescence labeling with 4‐(carbazole‐9‐yl)‐benzyl chloroformate

In this study, a simple analytical method for the determination of γ‐aminobutyric acid, gabapentin, and baclofen by using high‐performance liquid chromatography with fluorescence detection was developed. An amidogen‐reactive fluorescence labeling reagent, 4‐(carbazole‐9‐yl)‐benzyl chloroformate was first used to sensitively label these analytes. The completed labeling of these analytes can be finished rapidly only within 5 min at the room temperature (25°C) to form 4‐(carbazole‐9‐yl)‐benzyl chloroformate labeled fluorescence derivatives. These labeled derivatives expressed strong fluorescence property with the maximum excitation and emission wavelengths of 280 and 380 nm, respectively. The labeled derivatives were analyzed using a reversed‐phase Eclipse SB‐C18 column within 10 min with satisfactory shapes. Excellent linearity (R² > 0.995) for all analytes was achieved with the limits of detection and the limits of quantitation in the range of 0.25?0.35 and 0.70?1.10 μg/L, respectively. The proposed method was used for the simultaneous determination of γ‐aminobutyric acid and its analogs in human serum with satisfactory recoveries in the range of 94.5–97.5%.     

Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and specific high‐performance liquid chromatography mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of β‐hydroxy‐β‐methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water–acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C₈ (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30–4600 ng/mL (r > 0.998) for HMB. The intra‐ and inter‐day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench‐top, autosampler freeze–thaw cycles and long‐term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Establishment of liquid chromatography/mass spectrometry for determination of bicyclol in rat single‐pass intestinal perfusion

A simple, rapid and sensitive method was developed for determination of bicyclol, a new synthetic anti‐hepatitis drug, in rat plasma from the mesenteric vein using a high‐performance liquid chromatography system coupled to a positive ion electrospray–mass spectrometric analysis. Bicyclol and internal standard (biphenyldicarboxylate, DDB) were isolated from plasma by liquid–liquid extraction, then separated on a Zorbax SB‐C₁₈ column (3.5 µm, 2.1 × 100 mm) with mobile phase of methanol–water (60:40, v/v) at a flow rate of 0.2 mL/min. Detection was performed on a Trap XCT mass spectrometer equipped with an electrospray ionization (ESI) source operated in selected ion monitoring mode. Positive ion ESI was used to form sodium adduct molecular ions at m/z 413 for bicyclol and m/z 441 for DDB, respectively. A linear detection response was obtained for bicyclol ranging from 3.3 to 333.3 ng/mL and the lower limit of quantitation was 3.3 ng/mL. The coefficients of variation for intra‐ and inter‐day precisions were 1.1–7.7 and 2.0–6.6%, respectively. The percentage of absolute recovery of bicyclol was 85.3–94.6%. All analytes proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to determine the plasma concentration of bicyclol in mesenteric vein after intestinal perfusion. Copyright © 2009 John Wiley & Sons, Ltd.     

Switchable‐hydrophilicity solvent liquid‐liquid microextraction versus dispersive liquid‐liquid microextraction prior to HPLC‐UV for the determination and isolation of piperine from Piper nigrum L

Switchable‐hydrophilicity solvent liquid‐liquid microextraction and dispersive liquid‐liquid microextraction were compared for the extraction of piperine from Piper nigrum L. prior to its analysis by using high‐performance liquid chromatography with UV detection. Under optimum conditions, limits of detection and quantitation were found as 0.2–0.6 and 0.7–2.0 μg/mg with the two methods, respectively. Calibration graphs showed good linearity with coefficients of determination (R²) higher than 0.9962 and percentage relative standard deviations lower than 6.8%. Both methods were efficiently used for the extraction of piperine from black and white pepper samples from different origins and percentage relative recoveries ranged between 90.0 and 106.0%. The results showed that switchable‐hydrophilicity solvent liquid‐liquid microextraction is a better alternative to dispersive liquid‐liquid microextraction for the routine analysis of piperine in food samples. A novel scaled‐up dispersive liquid‐liquid microextraction method was also proposed for the isolation of piperine providing a yield of 102.9 ± 4.9% and purity higher than 98.0% as revealed by NMR spectroscopy.     

Sensitive determination of glucose in Dulbecco's modified Eagle medium by high‐performance liquid chromatography with 1‐phenyl‐3‐methyl‐5‐pyrazolone derivatization: application to gluconeogenesis studies

A new pre‐column derivative high‐performance liquid chromatography (HPLC) method for determination of d ‐glucose with 3‐O‐methyl‐d ‐glucose (3‐OMG) as the internal standard was developed and validated in order to study the gluconeogenesis in HepG2 cells. Samples were derivatized with 1‐phenyl‐3‐methy‐5‐pyrazolone at 70°C for 50 min. Glucose and 3‐OMG were extracted by liquid–liquid extraction and separated on a YMC‐Triart C₁₈ column, with a gradient mobile phase composed of acetonitrile and 20 mm ammonium acetate solution containing 0.09% tri‐ethylamine at a flow rate of 1.0 mL/min. The eluate were detected using a UV detector at 250 nm. The assay was linear over the range 0.39–25 μm (R² = 0.9997, n = 5) and the lower limit of quantitation was 0.39 μm (0.070 mg/mL). Intra‐ and inter‐day precision and accuracy were <15% and within ±3%, respectively. After validation, the HPLC method was applied to investigate the gluconeogenesis in Dulbecco's modified Eagle medium (DMEM) cultured HepG2 cells. Glucose concentration was determined to be about 1–2.5 μm in this gluconeogenesis assay. In conclusion, this method has been shown to determine small amounts of glucose in DMEM successfully, with lower limit of quantitation and better sensitivity when compared with common commercial glucose assay kits. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid by derivatization followed by high‐performance liquid chromatography with ultraviolet detection

Methanesulfonic acid is routinely used in pharmaceuticals but can contain potentially genotoxic impurities such as methyl methanesulfonate and ethyl methanesulfonate. The aim of this study was to develop a simple high‐performance liquid chromatography with ultraviolet detection method for determining methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid. Samples (250 mg) in water/acetonitrile (200 μL) were first combined with 10.0 mol/L sodium hydroxide solution (270 μL). Then they were mixed with 2.0 mg/mL N ,N‐diethyldithiocarbamate (500 μL), diluted to 5 mL with N ,N‐dimethylacetamide and allowed to react at 80°C for 1 h. The derivatives were analyzed using gradient high‐performance liquid chromatography with ultraviolet detection (277 nm) and structurally elucidated by liquid chromatography with mass spectrometry. With acetonitrile/5 mmol/L ammonium acetate solution as the eluent and 1 mL/min as the flow rate on a C18 column, the derivatives were eluted at 10.6 and 14.8 min. Good linearity (correlation coefficients > 0.999) and low limits of quantitation (0.6 ppm) were obtained. The recoveries were in the range of 80–115% with relative standard deviation < 5.0%. Finally, the established method was successfully used for the determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid.     

Developing a novel packed in‐tube solid‐phase extraction method for determination ∆9‐tetrahydrocannabinol in biological samples and cannabis leaves

A novel plate‐like nano‐sorbent based on copper/cobalt/chromium layered double hydroxide was synthesized by a simple coprecipitation method. The synthesized nanoparticels were introduced into a stainless steel cartridge using a dry packing method. Then, the packed cartridge was introduced as a novel on‐line “packed in‐tube” configuration and followed by high performance liquid chromatography for the determination of trace amounts of ^?9‐tetrahydrocannabinol from biological samples and cannabis leaves. The as‐prepared sorbent exhibited long lifetime, good chemical stability, and high anion‐exchange capacity. Several important factors affecting the extraction efficiency, such as extraction and desorption times, pH of the sample solution and flow rates of the sample and eluent solutions, were investigated and optimized. Under optimized conditions, this method showed good linearity for ^?9‐tetrahydrocannabinol in the ranges of 0.09–500, 0.3–500, and 0.4–500 µg/L with coefficients of determination of 0.9999, 0.9991, and 0.9994 in water, serum and plasma samples, respectively. The inter‐ and intra‐assay precisions (n = 3) were respectively in the ranges of 1.8–4.6% and 1.9–4.0% at three concentration levels of 10, 50, and 100 µg/L. The limits of detection were also in the range of 0.02–0.1 µg/L.     

Simple and rapid determination of zaltoprofen in human plasma by manual‐shaking‐assisted dispersive liquid–liquid microextraction followed by liquid chromatography with ultraviolet detection

A readily applicable method was developed to determine the concentration level of zaltoprofen, a non‐steroidal antiinflammatory drug from the propionic acid family, in human plasma. This method is based on manual‐shaking‐assisted dispersive liquid–liquid microextraction coupled with liquid chromatography with ultraviolet detection. Factors affecting the extraction efficiency were screened and optimized by experimental design using fractional factorial and central composite designs, respectively. Optimal conditions were: 220 μL of C₂H₄Cl₂ (extraction solvent), 5 mL of 3.75% w/v NaCl aqueous solution at pH 2.0, and manual shaking for 13 s (65 times). The resulting extraction method yielded a reasonable enrichment factor of 18.0 (±0.6, n  = 3) and extraction recovery of 86.0% (±3.3%, n  = 3). The established method was validated for selectivity, linearity, precision, accuracy, matrix effect, recovery, dilution integrity, and stability, and it met the acceptable criteria for all of the tested parameters. Specifically, the method was linear in the range of 0.16–50.0 mg/L, precise (< 8.8% RSD), accurate (–7.5–5.6% deviation), and showed negligible matrix effects (96.1–106.4%) with high absolute recovery (94.5–97.7%). Compared with previous methods involving labor‐intensive liquid–liquid extraction or non‐specific protein precipitation, our method allows the simple, rapid, and efficient determination of zaltoprofen using the most affordable analytical instrument, liquid chromatography with ultraviolet detection.     

Development and validation of an ultra‐performance convergence chromatography method for the quality control of Angelica gigas Nakai

Ultra‐performance convergence chromatography, which integrates the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies, is an environmentally friendly analytical method that uses dramatically reduced amounts of organic solvents. An ultra‐performance convergence chromatography method was developed and validated for the quantification of decursinol angelate and decursin in Angelica gigas using a CSH Fluoro‐Phenyl column (2.1 mm × 150 mm, 1.7 μm) with a run time of 4 min. The method had an improved resolution and a shorter analysis time in comparison to the conventional high‐performance liquid chromatography method. This method was validated in terms of linearity, precision, and accuracy. The limits of detection were 0.005 and 0.004 μg/mL for decursinol angelate and decursin, respectively, while the limits of quantitation were 0.014 and 0.012 μg/mL, respectively. The two components showed good regression (correlation coefficient (r²) > 0.999), excellent precision (RSD < 2.28%), and acceptable recoveries (99.75–102.62%). The proposed method can be used to efficiently separate, characterize, and quantify decursinol angelate and decursin in Angelica gigas and its related medicinal materials or preparations, with the advantages of a shorter analysis time, greater sensitivity, and better environmental compatibility.     

Simultaneous quantification of three tropane alkaloids in goji berries by cleanup of the graphene/hexagonal boron nitride hybrids and ultra‐high‐performance liquid chromatography tandem mass spectrometry

A modified quick, easy, cheap, effective, rugged and safe method was established for simultaneous determination of atropine, anisodamine, and scopolamine in goji berries by using ultra‐high‐performance liquid chromatography with tandem mass spectrometry. The graphene/hexagonal boron nitride hybrids were prepared and first applied as a cleanup adsorbent. Compared to classical cleanup adsorbent (C₁₈), the graphene/hexagonal boron nitride hybrids as adsorbent had better extraction efficiency for the detection of analytes. Under the optimal conditions, the proposed analytical method achieved satisfactory linearity (R² > 0.995), and obtained desirable recoveries ranged from 77.4 to 94.0% with the relative standard deviation of 1.2–6.1% at the concentration levels of 3.2–13.4 µg/kg. The limits of quantitation of atropine, anisodamine, and scopolamine were, respectively, 3.2, 4.6, and 4.5 µg/kg with linearity ranged from 3.2 to 25.4 µg/kg. The modified quick, easy, cheap, effective, rugged, and safe sample preparation with ultra‐high‐performance liquid chromatography and tandem mass spectrometry method was successfully applied to evaluate the safety of goji berries collected from 30 plant areas in China, suggesting its applicability and suitability for the routine analysis of three tropane alkaloids in goji berries.     

Simultaneous determination of trifolirhizin, (–)‐maackiain, (–)‐sophoranone,and 2‐(2,4‐dihydroxyphenyl)‐5,6‐methylenedioxybenzofuran from Sophora tonkinensis in rat plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

A new liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of trifolirhizin, (–)‐maackiain, (–)‐sophoranone, and 2‐(2,4‐dihydroxyphenyl)‐5,6‐methylenedioxybenzofuran from Sophora tonkinensis in rat plasma using chlorpropamide as an internal standard. Plasma samples (50 μL) were prepared using a simple deproteinization procedure with 150 μL of acetonitrile containing 100 ng/mL of chlorpropamide. Chromatographic separation was carried out on an Acclaim RSLC120 C₁₈ column (2.1 × 100 mm, 2.2 μm) using a gradient elution consisting of 7.5 mM ammonium acetate and acetonitrile containing 0.1% formic acid (0.4 mL/min flow rate, 7.0 min total run time). The detection and quantitation of all analytes were performed in selected reaction monitoring mode under both positive and negative electrospray ionization. This assay was linear over concentration ranges of 50–5000 ng/mL (trifolirhizin), 25–2500 ng/mL ((–)‐maackiain), 5–250 ng/mL ((–)‐sophoranone), and 1–250 ng/mL 2‐(2,4‐dihydroxyphenyl)‐5,6‐methylenedioxybenzofuran) with a lower limit of quantification of 50, 25, 5, and 1 ng/mL for trifolirhizin, (–)‐maackiain, (–)‐sophoranone, and 2‐(2,4‐dihydroxyphenyl)‐5,6‐methylenedioxybenzofuran, respectively. All the validation data, including the specificity, precision, accuracy, recovery, and stability conformed to the acceptance requirements. The results indicated that the developed method is sufficiently reliable for the pharmacokinetic study of the analytes following oral administration of Sophora tonkinensis extract in rats.     

Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

The concentrations of l ‐tryptophan (Trp) and the metabolite l ‐kynurenine (KYN) can be used to evaluate the in‐vivo activity of indoleamine 2,3‐dioxygenase (IDO) and tryptophan 2,3‐dioxygenase (TDO). As such, a novel method involving derivatization of l ‐Trp and l ‐KYN with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS) and separation by high‐performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole‐bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH₃CN/10 mm ammonium formate in H₂O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l ‐Trp and l ‐KYN were approximately 50 and 4.0 pm , respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC‐MS/MS method, l ‐Trp and l ‐KYN were successfully determined in 10 μL human serum using 1‐methyl‐l ‐Trp as an internal standard. The precision and recovery of l ‐Trp were in the ranges 2.85–9.29 and 95.8–113%, respectively, while those of l ‐KYN were 2.51–16.0 and 80.8–98.2%, respectively. The proposed LC‐MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.     

A high‐performance liquid chromatographic analysis and preliminary pharmacokinetic characterization of 3′‐hydroxypterostilbene in rats

A high‐performance liquid chromatographic method was developed for the analysis of 3'‐hydroxypterostilbene. This method involves the use of a Luna^® C₁₈ column with ultraviolet detection at 325 nm. The mobile phase consisted of acetonitrile, water and formic acid (50:50:0.01, v/v/v) with a flow rate of 0.8 mL/min. The calibration curves were linear over the range 0.5–100.0 µg/mL. The mean extraction efficiency was between 97.40 and 111.16%. The precision of the assay was 0.196–14.39% (RSD%), and within 15% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <16% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid and specific high‐performance liquid chromatography for the in vitro quantification of d‐Lys6–GnRH in a microemulsion‐type formulation in the presence of peptide oxidation products

A high‐performance liquid chromatography (HPLC) method for assay of d ‐Lys⁶–GnRH contained in a microemulsion‐type formulation is described. The peptide is extracted from the microemulsion matrix and quantified using a two‐step gradient method. Separation from microemulsion compounds and potential peptide oxidation products was achieved on a Jupiter C₁₈ column at 40°C, using a gradient of 10–35% CH₃CN for peptide elution. The correlation of peak intensity measured at 220 nm and peptide concentration was linear over the range 2.5–60 µg/mL with a correlation coefficient of 0.9997 and a y‐intercept not significantly different from zero (p > 0.05). Intraday and interday variability of the assay was less than 5% for multiple injections of samples containing 7.5, 30 and 60 µg/mL. The lower limit of quantitation was calculated to be 0.38 µg/mL, and the lower limit of detection was 0.13 µg/mL. The assay was applied to samples that were stressed under physiological conditions (37°C, pH 7.4) over 4 days. Three degradation peaks were well resolved from the parent peptide, demonstrating the selectivity of the assay. Off‐line MALDI TOF mass spectrometry was applied to identify these degradation species as oxidation products of the peptide. Copyright © 2009 John Wiley & Sons, Ltd.     

Combination of ultrasound‐assisted ethyl chloroformate derivatization and switchable solvent liquid‐phase microextraction for the sensitive determination of l‐methionine in human plasma by GC–MS

A green and fast analytical method for the determination of l ‐methionine in human plasma is presented in this study. Preconcentration of the analyte was carried out by switchable solvent liquid phase microextraction after ethyl chloroformate derivatization reaction. Instrumental detection of the analyte was performed by means of gas chromatography–mass spectrometry. N,N‐Dimethyl benzylamine was used in the synthesis of switchable solvent. Protonated N,N‐dimethyl benzylamine volume, volume/concentration of sodium hydroxide, and vortex period were meticulously fixed to their optimum values. Besides, ethyl chloroformate, pyridine, and ethanol volumes were optimized in order to get high derivatization yield. After the optimization studies, limit of detection and quantitation values were attained as 3.30 and 11.0 ng/g, respectively, by the developed switchable solvent liquid phase microextraction gas chromatography–mass spectrometry method that corresponding to 76.7‐folds enhancement in detection power of the gas chromatography–mass spectrometry system. Applicability and accuracy of the switchable solvent liquid phase microextraction–gas chromatography–mass spectrometry method were also checked by spiking experiments. Percent recovery results were ranged from 97.8 to 100.5% showing that human plasma samples could be analyzed for its l ‐methionine level by the proposed method.     

Simultaneous determination of calycosin‐7‐O‐β‐d‐glucoside,calycosin, formononetin,astragaloside IV and schisandrin in rat plasma by LC‐MS/MS: application to a pharmacokinetic study after oral administration of Shenqi Wuwei chewable tablets

A rapid, sensitive and reliable high‐performance liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for simultaneous quantification of the five main bioactive components, calycosin, calycosin‐7‐O‐β‐d ‐glucoside, formononetin, astragaloside IV and schisandrin in rat plasma after oral administration of Shenqi Wuwei chewable tablets. Plasma samples were extracted using solid‐phase extraction separated on a CEC₁₈ column and detected by MS with an electrospray ionization interface in multiple‐reaction monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r > 0.995. The method had a lower limit of quantitation of 0.1, 0.02, 0.1, 1 and 0.1 ng/mL for calycosin, calycosin‐7‐O‐β‐d ‐glucoside, formononetin, astragaloside IV and schisandrin, respectively. Intra‐ and inter‐day precisions (relative standard deviation) for all analytes ranged from 0.97 to 7.63% and from 3.45 to 10.89%, respectively. This method was successfully applied to the pharmacokinetic study of the five compounds in rats after oral administration of Shenqi Wuwei chewable tablets. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of acyclovir,ganciclovir, and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine in human plasma using high‐performance liquid chromatography

Acyclovir, ganciclovir and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine are active in vitro against the Epstein–Barr virus (EBV) but their in vivo anti‐EBV activity is not well understood. We developed a novel, sensitive high‐performance liquid chromatography assay with ultraviolet detection for measuring acyclovir, ganciclovir and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine in human plasma to identify quantitative relationships between in vitro anti‐EBV activity and therapeutic response. Characteristics of the assay include a low plasma volume (200 µL), perchloric acid protein precipitation, use of penciclovir as the internal standard, run times less than 8 min and a 50 ng/mL lower limit of quantification. The within‐ and between‐assay variability is 0.7–4.8 and 1.0–7.9%, respectively. Accuracy for all three drugs ranges from 89.5 to 106.4% for four quality controls (50, 100, 1000 and 10,000 ng/mL). This assay supports pharmacokinetic and pharmacodynamic studies of candidate anti‐EBV drugs in children and adults with EBV infections. Copyright © 2009 John Wiley & Sons, Ltd.     

Combination of ultracentrifugation and solid‐phase extraction with subsequent chromatographic analysis of α‐tocopherol in erythrocyte membranes

A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid‐phase extraction for the determination of α‐tocopherol in human erythrocyte membranes by high‐performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d ‐mannitol, 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid and calcium chloride. The α‐tocopherol was then extracted from the erythrocyte membranes by solid‐phase extraction with n‐hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP‐18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α‐tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α‐tocopherol in real samples from patients. The combined ultracentrifugation and solid‐phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid–liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.     

3‐(2‐Bromoacetamido)‐N‐(9‐ethyl‐9H)‐carbazol fluorescent probe and its application for the determination of thiophenols in rubber products by HPLC with fluorescence detection and atmospheric chemical ionization mass spectrometry identification

A rapid, sensitive, and selective precolumn derivatization method for the simultaneous determination of eight thiophenols using 3‐(2‐bromoacetamido)‐N‐(9‐ethyl‐9H )‐carbazol as a labeling reagent by high‐performance liquid chromatography with fluorescence detection has been developed. The labeling reagent reacted with thiophenols at 50°C for 50 min in aqueous acetonitrile in the presence of borate buffer (0.10 mol/L, pH 11.2) to give high yields of thiophenol derivatives. The derivatives were identified by online postcolumn mass spectrometry. The collision‐induced dissociation spectra for thiophenol derivatives gave the corresponding specific fragment ions at m/z 251.3, 223.3, 210.9, 195.8, and 181.9. At the same time, derivatives exhibited intense fluorescence with an excitation maximum at λ_ex = 276 nm and an emission maximum at λ_em = 385 nm. Excellent linear responses were observed for all analytes over the range of 0.033–6.66 μmol/L with correlation coefficients of more than 0.9997. Detection limits were in the range of 0.94–5.77 μg/L with relative standard deviations of less than 4.54%. The feasibility of derivatization allowed the development of a rapid and highly sensitive method for the quantitative analysis of trace levels of thiophenols from some rubber products. The average recoveries (n = 3) were in the range of 87.21–101.12%.