A simple assay for the simultaneous determination of rosuvastatin acid,rosuvastatin-5<Emphasis Type="Italic">S</Emphasis>-lactone,and <Emphasis Type="Italic">N</Emphasis>-desmethyl rosuvastatin in human plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS)

A simple and sensitive assay was developed and validated for the simultaneous quantification of rosuvastatin acid (RST), rosuvastatin-5S-lactone (RST-LAC), and N-desmethyl rosuvastatin (DM-RST), in buffered human plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS). All the three analytes and the corresponding deuterium-labeled (d6) internal standards were extracted from 50 μL of buffered human plasma by protein precipitation. The analytes were chromatographically separated using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase comprised of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). The analytes were separated at baseline within 6.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode. The calibration curves for all three analytes were linear (R ≥ 0.9964, n = 3) over the concentration range of 0.1–100 ng/mL for RST and RST-LAC, and 0.5–100 ng/mL for DM-RST. Mean extraction recoveries ranged within 88.0–106%. Intra- and inter-run mean percent accuracy were within 91.8–111% and percent imprecision was ≤15%. Stability studies revealed that all the analytes were stable in matrix during bench-top (6 h on ice–water slurry), at the end of three successive freeze and thaw cycles and at −80°C for 1 month. The method was successfully applied in a clinical study to determine the concentrations of RST and the lactone metabolite over 12-h post-dose in patients who received a single dose of rosuvastatin.     

Development and validation of a sensitive,simple, and rapid method for simultaneous quantitation of atorvastatin and its acid and lactone metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The aim of the proposed work was to develop and validate a simple and sensitive assay for the analysis of atorvastatin (ATV) acid, ortho- and para-hydroxy-ATV, ATV lactone, and ortho- and para-hydroxy-ATV lactone in human plasma using liquid chromatography-tandem mass spectrometry. All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 μL of human plasma by protein precipitation. The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). All analytes including ortho- and para-hydroxy metabolites were baseline-separated within 7.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode, with multiple-reaction monitoring scan. The calibration curves for all analytes were linear (R ² ≥ 0.9975, n = 3) over the concentration range of 0.05–100 ng/mL and with lower limit of quantitation of 0.05 ng/mL. Mean extraction recoveries ranged between 88.6–111%. Intra- and inter-run mean percent accuracy were between 85–115% and percent imprecision was ≤ 15%. Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at −80 °C for 3 months. The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over 12 h post-dose in patients receiving atorvastatin.     

Peroxyoxalate chemiluminescence detection for the highly sensitive determination of fluorescence-labeled chlorpheniramine with Suzuki coupling reaction

A sensitive and selective high performance liquid chromatography-peroxyoxalate chemiluminescence (PO-CL) method has been developed for the simultaneous determination of chlorpheniramine (CPA) and monodesmethyl chlorpheniramine (MDCPA) in human serum. The method combines fluorescent labeling with 4-(4,5-diphenyl-1H-imidazole-2-yl)phenyl boronic acid using Suzuki coupling reaction with PO-CL detection. CPA and MDCPA were extracted from human serum by liquid–liquid extraction with n-hexane. Excess labeling reagent, which interfered with trace level determination of analytes, was removed by solid-phase extraction using a C18 cartridge. Separation of derivatives of both analytes was achieved isocratically on a silica column with a mixture of acetonitrile and 60 mM imidazole-HNO₃ buffer (pH 7.2; 85:15, v/v) containing 0.015% triethylamine. The proposed method exhibited a good linearity with a correlation coefficient of 0.999 for CPA and MDCPA within the concentration range of 0.5–100 ng/mL. The limits of detection (S/N = 3) were 0.14 and 0.16 ng/mL for CPA and MDCPA, respectively. Using the proposed method, CPA could be selectively determined in human serum after oral administration.     

A sensitive liquid chromatographic-mass spectrometric method for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma

A liquid chromatographic–mass spectrometric (LC–MS) method has been developed and validated for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma. After addition of the internal standard, domperidone, plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Apollo C₁₈ column (250 mm × 4.6 mm, 5 μm), with methanol–0.01% formic acid water (60:40, v/v) as mobile phase, within a runtime of 12.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 1.0–500 ng mL⁻¹ for dehydroevodiamine and 2.0–1,000 ng mL⁻¹ for limonin, with lower limits of quantitation of 1.0 and 2.0 ng mL⁻¹, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, respectively, for both analytes, and the accuracy (relative error, RE, %) was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to a comparative pharmacokinetic study of dehydroevodiamine and limonin in rat plasma after oral administration of dehydroevodiamine, limonin, and an aqueous extract of Evodiae fructus. The results indicated there were obvious differences between the pharmacokinetic behavior after oral administration of an aqueous extract of Evodiae fructus compared with single substances.     

Response surface optimization for determination of pesticide residues in grapes using MSPD and GC-MS: assessment of global uncertainty

In this work, a simple and low-cost method based on matrix solid-phase dispersion (MSPD) and gas chromatography to determine eight multi-class pesticides such as vinclozolin, dichlofluanid, penconazol, captan, quinoxyfen, fluquinconazol, boscalid, and pyraclostrobin in grapes is described. Fungicide residues were identified and quantified using gas chromatography–mass spectrometry in selected ion monitoring mode (GC-MS, SIM). The experimental variables that affect the MSPD method, such as the amount of solid phase, solvent nature and elution volume were optimized using an experimental design. The best results were obtained using 0.5 g of grapes, 1.0 g of silica as clean-up sorbent, 1.50 g of C₁₈ as bonded phase and 10 mL of dichloromethane/ethyl acetate (1:1, v/v) as eluting solvent. Significant matrix effects observed for most of the pesticides tested were eliminated using matrix-matched standards. The pesticide recoveries in grapes samples were better than 80% except for captan. Intra-laboratory precision in terms of Horwitz ratio of the pesticides evaluated was below 0.5, suggesting ruggedness of the method. The quantification limits of the pesticides were in the range of 3.4–8.7 μg kg⁻¹, which were lower than the maximum residue limits (MRLs) of the pesticides in grapes samples established by the European legislation. Decision limits (CCα) and detection capability (CCβ) have been calculated. The expanded uncertainties at two levels of concentration were <20% for all analytes.     

Simultaneous detection and quantification of parecoxib and valdecoxib in canine plasma by HPLC with spectrofluorimetric detection: development and validation of a new methodology

Parecoxib is the injectable prodrug of valdecoxib, a cicloxygenase-2 selective drug, currently used in human medicine. Recent studies have suggested both its excellent clinical effectiveness and wide safety profile. The aim of the present study was to develop and validate a new high-performance liquid chromatography (HPLC) with spectrofluorimetric detection method to quantify parecoxib and valdecoxib in canine plasma. Several parameters both in the extraction and the detection method were evaluated. The applicability of the method was determined by administering parecoxib to one dog: the protocol provided the expected pharmacokinetic results. The final mobile phase was acetonitrile: AcONH₄ (10 mM; pH 5.0) 55:45, v/v, with a flow rate of 0.4 mL min⁻¹, and excitation and emission wavelengths of 265 and 375 nm, respectively. The analytical column was a reverse-phase C18 ODS2 3-μm particle size. Protein precipitation in acidic medium followed by two successive liquid–liquid steps was carried out. The best extraction solvent was cyclohexane:Et₂O (3:2, v/v) that gave recoveries ranging from 81.1% to 89.1% and from 94.8% to 103.6% for parecoxib and valdecoxib, respectively. The limits of quantification were 25 and 10 ng mL⁻¹ for parecoxib and valdecoxib, respectively. The chromatographic runs were specific with no interfering peaks at the retention times of the analytes, as confirmed by HPLC–mass spectrometry experiments. The other validation parameters were in agreement with the European Medicines Evaluation Agency and International Conference on Harmonisation guidelines. In conclusion, this method (extraction, separation and applied techniques) is simple and effective. This is the first time that use of a HPLC with spectrofluorimetric detection technique to simultaneously detect parecoxib and valdecoxib in plasma has been reported. This technique may have applications for pharmacokinetic studies.     

A sensitive and selective liquid chromatography−tandem mass spectrometry method for simultaneous determination of five isoquinoline alkaloids from Chelidonium majus L. in rat plasma and its application to a pharmacokinetic study

Chelidonium majus L. is one of the most important medicinal plants of the family Papaveraceae. Its pharmacological effects have been primarily attributed to the presence of a number of alkaloids. In the present study, a sensitive and selective liquid chromatography?tandem mass spectrometry method for simultaneous determination of five isoquinoline alkaloids from Chelidonium majus L. was developed and validated. The analytes (protopine, chelidonine, coptisine, sanguinarine and chelerythrine), together with the internal standard (palmatine), were extracted from acidified rat plasma with ethyl acetate?dichloromethane (4:1, v/v). Chromatographic separation was carried out on a Diamonsil C₁₈ column with an isocratic mobile phase consisting of acetonitrile and water (adjusted to pH 2.3 with formic acid) (30:70, v/v) at a flow rate of 0.4 ml/min. Mass spectrometric detection was performed by selected reaction monitoring mode via electrospray ionization source operating in positive ionization mode. The assay exhibited good linearity (r ≥ 0.9933) for all the analytes. The lower limits of quantification were 0.197?1.27 ng/ml using only 50 µl of plasma sample. The intra‐ and inter‐day precisions were less than 11.9%, and the accuracy was between ?6.3% and 9.3%. The method was successfully applied to the pharmacokinetic study of the five alkaloids in rats after intragastric administration of Chelidonium majus L. extract. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of dried blood spots combined with high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of vincristine and actinomycin-D

A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile–methanol–water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm ID Xbridge C₁₈ column using elution with 1 mM ammonium acetate–acetonitrile (70:30, v/v) adjusted to pH 10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycine-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.     

Nonaqueous CE for Rapid and Sensitive Determination of Matrine and Oxymatrine in Sophora flavescens and Its Medicinal Preparations

A simple, rapid, and sensitive non-aqueous capillary electrophoresis procedure for the quantitative determination of matrine and oxymatrine is established. Optimum separation conditions were obtained when the sample was injected under pressure for 3 s at 50 mbar and separated with the buffer containing 70 mM ammonium acetate, 7.0% (v/v) acetic acid, and 10% (v/v) acetonitrile in methanol medium at 25 kV applied voltage. The analytes were detected at 205 nm. The two alkaloids can be separated within 12 min and quantified with high sensitivity. The method was validated in terms of reproducibility, linearity, and accuracy when applied to the analysis of matrine and oxymatrine in Sophora flavescens and its medicinal preparations.     

Selective sample preparation for the analysis of (fluoro)quinolones in baby food: molecularly imprinted polymers versus anion-exchange resins

In this work, an analytical method for simultaneous analysis of several quinolones (cinoxacin, oxolinic acid, nalidixic acid, and flumequine) and fluoroquinolones (norfloxacin, enrofloxacin, enoxacin, ciprofloxacin, and danofloxacin) in baby-food samples is described for the first time. The method is based on isolation of these analytes by ultrasound-assisted extraction procedure followed by a solid-phase extraction sample clean-up step and final determination of the analytes by HPLC using UV detection. For the extraction step, 2 g baby food was mixed with methanol in a centrifuge tube and one single extraction cycle of 15 min at room temperature was carried out. After centrifugation, supernatant was collected and two different solid-phase extraction procedures were developed and evaluated for sample clean-up. The first was based on use of strong anion-exchange cartridges whereas the second was based on use of a ciprofloxacin-imprinted polymer. Both sample clean-up procedures had their own advantages and drawbacks, and the analytical performance and applicability of each procedure was established and properly discussed. The anion-exchange resin-based method enabled simultaneous determination of quinolones and fluoroquinolones, reaching limits of detection ranging from 0.03 to 0.11 μg g⁻¹. In contrast, the use of a ciprofloxacin-imprinted polymer did provide selectivity towards fluoroquinolones, leading to chromatograms free from co-extractives reaching limits of detection one order of magnitude lower than those obtained by the first approach.     

Catanionic surfactant ambient cloud point extraction and high-performance liquid chromatography for simultaneous analysis of organophosphorus pesticide residues in water and fruit juice samples

A mixed anionic–cationic surfactant cloud point extraction (CPE) has been developed using sodium dodecyl sulfate (SDS) and tetrabutylammonium bromide (TBABr) for the extraction and preconcentration of organophosphorus pesticides (OPPs) at ambient temperature before analysis by high-performance liquid chromatography. The studied OPPs were azinphos-methyl, parathion-methyl, fenitrothion, diazinon, chlorpyrifos, and prothiophos. The optimum conditions of the mixed anionic–cationic CPE were 50 mmol L⁻¹ SDS, 100 mmol L⁻¹ TBABr, and 10% (w/v) NaCl. The extracted OPPs were successfully separated within 11 min using the conditions of a Waters Symmetry C8 column, a flow rate of 0.8 mL min⁻¹, a gradient elution of methanol and water, and detection at 210 nm. Linearity was found over the range 0.05–5 μg mL⁻¹, with the correlation coefficients higher than 0.996. The enrichment factor of the target analytes was in the range 6–11, which corresponds to their limits of detection from 1 to 30 ng mL⁻¹. High precisions (intra-day and inter-day) were obtained with relative standard deviation <1.5% (t _R) and 10% (peak area). Accuracies (% recovery) of the different spiked OPP concentrations were 82.7–109.1% (water samples) and 80.3–113.3% (fruit juice samples). No contamination by the OPPs was observed in any studied samples.     

Selective Determination of Sulfonamide Residues in Honey by SPE-RP-LC with UV Detection

Solid-phase extraction was used to isolate sulfacetamide, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethoxypyridazine and sulfamethoxazole from honey. The optimized procedure used polymeric Abselut Nexus cartridges and the sulfonamides were separated, in the isocratic mode, on an Inertsil ODS-3 (250 × 4 mm I.D., 5 μm) column, using methanol-0.05 M acetate buffer (pH 3.6) (20:80 v/v) with 1% (v/v) of acetic acid, UV detection at 263 nm and a flow-rate of 1 mL min⁻¹. Caffeine was used as internal standard. Average recoveries of the analytes from spiked honey ranged from 80 to 117% and the detection limits based on a spiked honey extract were 20–25 μg kg⁻¹.     

Cloud-point extraction and reversed-phase high-performance liquid chromatography for the determination of carbamate insecticide residues in fruits

A cloud-point extraction (CPE) method using Triton X-114 non-ionic surfactant was developed for the extraction and preconcentration of carbamate insecticide residues (i.e., methomyl, propoxur, carbofuran, carbaryl, isoprocarb, and promecarb) in fruit samples. The optimum conditions of CPE were 1.5% (w/v) Triton X-114, 7.0% (w/v) NaCl and 20 min equilibrated at 45 °C. The surfactant-rich phase was then analyzed by reversed-phase high-performance liquid chromatography with ultraviolet detection at 270 nm, under gradient separation using methanol and 0.1% (v/v) acetic acid. Under the study conditions, six carbamate insecticides were successfully separated within 27 min. Good reproducibility was obtained with the relative standard deviation of <3% for retention time and <9% for peak area. Limits of detection in the studied fruit samples were in the range of 0.1–1.0 mg kg⁻¹. No carbamate insecticides were detected in the studied fruit samples. The high recoveries of the spiked fruit samples were obtained in the range 80.0–107%. The CPE method has been shown to be a potential useful methodology for the preconcentration of the target analytes, with a preconcentration factor of 14. Moreover, the method is simple, has high sensitivity, consumes much less solvent than traditional methods, and is environmental friendly.     

Enantioselective analysis of zopiclone and its metabolites in plasma by liquid chromatography/tandem mass spectrometry

A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid–liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol–methanol–acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min⁻¹. Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5–500 ng mL⁻¹. The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (−)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.     

Simultaneous RP-LC Determination of Ketorolac and its Piperazinylalkyl Ester Prodrugs

A simple, rapid and selective RP-HPLC method was developed and validated for the determination of ketorolac and five piperazinylalkyl ester prodrugs. A binary isocratic mobile phase composed of a mixture of 65:35 (v/v) 0.02 M phosphate buffer (pH 5.4) and acetonitrile was used on a C₁₈ column (125 × 4 mm, 5 μm). The injection volume was 25 μL and the detection wavelength was 314 nm and the flow rate was 1.5 mL min⁻¹. The method exhibited excellent linearity with R ² of no less than 0.999 and intra-assay and inter-assay precision that were less than the maximum amount allowed according to Horwitz equation. The accuracy was found to be within the allowed ±15%. The limits of detection for the analytes were between 0.060 and 0.220 μg mL⁻¹ and the limits of quantification were between 0.183 and 0.667 μg mL⁻¹. This method was used successfully for the study of the solubility, stability and partition coefficients of piperazinylalkyl ester prodrugs of ketorolac.     

On-line determination of aliphatic amines in water using in-tube solid-phase microextraction-assisted derivatisation in in-valve mode for processing large sample volumes in LC

This paper describes a cost-effective procedure for the analysis of short-chain aliphatic amines in water samples using a solid-phase microextraction device. Analyte preconcentration and derivatisation were effected into a capillary column coated with 95% polydimethylsiloxane–5% polydiphenylsiloxane, which was used as the injection loop of a Rheodyne injection valve. The coating was previously loaded with the derivatisation reagent, 9-fluorenylmethyl chloroformate. A volume of 1 mL of samples was then drawn into the capillary column, and the extracted analytes were left to react on the capillary coating for 5 min. Next, the capillary column was cleaned by passing water. Finally, the injection valve was rotated, and the derivatives formed were dynamically desorbed and transferred to the analytical column into the mobile phase. Methylamine, ethylamine, propylamine, n-butylamine and n-pentylamine were selected as model compounds. Excellent sensitivity was achieved, being the limits of detection of 15–200 μg/L when using UV detection and of 0.1–0.4 μg/L by fluorescence.     

Determination of 19 drugs of abuse and metabolites in whole blood by high-performance liquid chromatography–tandem mass spectrometry

A high-performance liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method has been developed and validated for the determination of 19 drugs of abuse and metabolites and used in whole blood. The following compounds were included: amphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methamphetamine, cocaine, benzoylecgonine, morphine, 6-acetylmorphine, codeine, methadone, buprenorphine, norbuprenorphine, ketobemidone, tramadol, O-desmethyltramadol, zaleplone, zolpidem, and zopiclone. The sample pretreatment consisted of solid-phase extraction using mixed-mode columns (Isolute Confirm HCX). Deuterated analogues were used as internal standards for all analytes, except for ketobemidone and O-desmethyltramadol. The analytes were separated by a methanol/ammonium formate gradient using high-performance LC (Agilent HPLC 1100) with a 3 mm × 100 mm Varian Pursuit 3 C₁₈ column, 3-μm particle size, and were quantified by MS/MS (Waters Quattro micro tandem quadrupole mass spectrometer) using multiple reaction monitoring in positive mode. Two transitions were used for all analytes, except for tramadol and O-desmethyltramadol. The run time of the method was 35 min including the equilibration time. For all analytes, responses were linear over the range investigated, with R ² > 0.99. One-point calibration was found to be adequate by validation, thereby saving analysis of multiple calibrators. The limits of quantification (LOQs) for the analytes ranged from 0.0005 to 0.01 mg/kg. Absolute recoveries of the analytes were from 34 to 97%, except for zaleplone (6%). Both the interday precision and the intraday precision were less than 15% (20% at the LOQ) for all analytes, except buprenorphine, norburprenorphine, and zaleplone (less than 18%). Accuracy (bias) was within ±15% (±20% at the LOQ) for all analytes, except MDMA and O-desmethyltramadol (within ±19%). No ion suppression or enhancement was seen nor was suppression from coeluted analytes seen. Matrix effects were found to be less than 23% for all analytes, except zopiclone (64%). High-concentration and low-concentration quality control samples gave acceptable values, and the method has been tried in international proficiency test schemes with good results. The present LC-MS/MS method provides a simple, specific, and sensitive solution for the quantification of some of the most frequent drugs of abuse and their metabolites in whole blood. The quantification by LC-MS/MS was successfully applied to 412 forensic cases from October 2008 to mid February 2009, where 267 cases were related to zero-tolerance traffic legislation.     

A sensitive liquid chromatography–mass spectrometry method for simultaneous determination of alisol A and alisol A 24-acetate from <Emphasis Type="Italic">Alisma orientale</Emphasis> (Sam.) Juz. in rat plasma

A liquid chromatography–mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of alisol A and alisol A 24-acetate from Alisma orientale (Sam.) Juz. in rat plasma using diazepam as an internal standard. A 200-μl plasma sample was extracted by methyl tert-butyl ether and the separation was performed on Kromasil C₁₈ column (150 × 4.6 mm, 5 μm) with the mobile phase of acetonitrile (containing 0.1% of formic acid)–water (73:27, v/v) at a flow rate of 0.8 ml/min in a run time of 10 min. The two analytes were monitored with positive electrospray ionization by selected ion monitoring mode. The lower limit of quantitation for both alisol A and alisol A 24-acetate were 10 ng/ml. The calibration curves were linear in the measured range 10–1,000 ng/ml for alisol A and 10–500 ng/ml for alisol A 24-acetate. The mean extraction recoveries were above 74.7% for alisol A and above 72.4% for alisol A 24-acetate from biological matrixes. The intra- and inter-day precision for all concentrations of quality controls was lower than 14.1% (RSD %) for each analyte. The accuracy ranged from −12.3% to 9.8% (RE %) for alisol A, and −8.6% to 14.2% (RE %) for alisol A 24-acetate. The method was successfully applied to the study on the pharmacokinetics of alisol A and alisol A 24-acetate in rat plasma.     

Simultaneous Determination of the Endogenous Free ��-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

A highly sensitive, precise, and accurate reversed-phase high-performance liquid-chromatography/electrochemical detection method for simultaneous determination of the endogenous free ??-lipoic acid and dihydrolipoic acid in biological matrices was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution_(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C₁₈ (150 × 4.6 mm, 5 ??m) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min^?1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r ² = 0.999 in the range of 0.1?C500 and 0.25?C1,000 ng mL^?1 for ??-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for ??-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for ??-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.     

Determination of betaine,l‐carnitine,and choline in human urine using a self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A simple method for the determination of betaine, l ‐carnitine, and choline in human urine was developed based on column‐switching ion chromatography coupled with nonsuppressed conductivity detection by using a self‐packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate‐divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate‐divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column‐switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r ² ≥ 0.99) was obtained for the concentration range of 0.50–100, 0.75–100, and 0.25–100 μg/mL for betaine, l ‐carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 μg/mL for betaine, l ‐carnitine, and choline, respectively. The intra‐ and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l ‐carnitine, and choline in urine samples from healthy people.