A novel fast method for aqueous derivatization of THC,OH‐THC and THC‐COOH in human whole blood and urine samples for routine forensic analyses

A novel aqueous in situ derivatization procedure with propyl chloroformate (PCF) for the simultaneous, quantitative analysis of Δ⁹‐tetrahydrocannabinol (THC), 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (OH‐THC) and 11‐nor‐Δ⁹‐tetrahydrocannabinol‐carboxylic acid (THC‐COOH) in human blood and urine is proposed. Unlike current methods based on the silylating agent [N,O‐bis(trimethylsilyl)trifluoroacetamide] added in an anhydrous environment, this new proposed method allows the addition of the derivatizing agent (propyl chloroformate, PCF) directly to the deproteinized blood and recovery of the derivatives by liquid–liquid extraction. This novel method can be also used for hydrolyzed urine samples. It is faster than the traditional method involving a derivatization with trimethyloxonium tetrafluoroborate. The analytes are separated, detected and quantified by gas chromatography–mass spectrometry in selected ion monitoring mode (SIM). The method was validated in terms of selectivity, capacity of identification, limits of detection (LOD) and quantification (LOQ), carryover, linearity, intra‐assay precision, inter‐assay precision and accuracy. The LOD and LOQ in hydrolyzed urine were 0.5 and 1.3 ng/mL for THC and 1.2 and 2.6 ng/mL for THC‐COOH, respectively. In blood, the LOD and LOQ were 0.2 and 0.5 ng/mL for THC, 0.2 and 0.6 ng/mL for OH‐THC, and 0.9 and 2.4 ng/mL for THC‐COOH, respectively. This method was applied to 35 urine samples and 50 blood samples resulting to be equivalent to the previously used ones with the advantage of a simpler method and faster sample processing time. We believe that this method will be a more convenient option for the routine analysis of cannabinoids in toxicological and forensic laboratories.     

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.     

Quantitative determination of pravastatin and its metabolite 3α‐hydroxy pravastatin in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α‐hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one‐step sample preparation by liquid–liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α‐hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α‐hydroxy pravastatin. The relative deviation of this method was <10% for intra‐ and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of hormones in human urine by ultra‐high‐performance liquid chromatography/triple‐quadrupole mass spectrometry

Steroid hormones play a critical role in maintaining the homeostasis of human metabolism. Urine as a noninvasive sample has been extensively used in clinical diagnosis for hormones homeostasis. In this study, the simultaneous characterization of fourteen hormones in urine was performed based on ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPHLC/ESI(+)‐MS/MS) with multiple reaction monitoring in the positive ionization mode. The target hormones were cortisone, cortisol, 11‐deoxycortisol, aldosterone, corticosterone, 11‐deoxycorticosterone, progesterone, 17‐OH‐progesterone, pregnenolone, estrone, estradiol, estriol, testosterone and dehydreopiandrosterone. β‐Glucuronidase/sulfatase deconjugation and liquid–liquid extraction (LLE) were conducted for the determination of urinary hormones (free + conjugated forms). The limits of detection (LODs) ranged from 0.2 ng/mL (11‐deoxycortisol and testosterone) to 1 ng/mL (cortisone). The extraction recovery of the targeted compounds ranged from 87% to 127%, indicating sufficient extraction efficiency for the LLE process. Intraday precision was below 10% and the accuracy ranged from 84% to 122%. The profiling analysis of hormones in urine samples helps to understand the metabolic state of biological systems and can be employed as a diagnostic tool in diseases developed by endocrine‐disrupted systems.     

Method validation and determination of lisdexamfetamine and amphetamine in oral fluid,plasma and urine by LC–MS/MS

Lisdexamfetamine (LDX) is a long‐acting prodrug stimulant indicated for the treatment of attention‐deficit/hyperactivity disorder and binge‐eating disorder symptoms. In vivo hydrolysis of LDX amide bond releases the therapeutically active d ‐amphetamine (d ‐AMPH). Since toxicological tests in biological samples can detect AMPH from the use of some legal medications, efficient methods are needed in order to correctly interpret the results. The aim of this study was to develop and validate an LC–MS/MS method for the simultaneous quantification of LDX and its main biotransformation product AMPH in human oral fluid, plasma and urine. Calibration curve range for both analytes was 1–128 ng/mL in oral fluid and plasma and 4–256 ng/mL in urine, being the lowest concentration the limit of quantification. Accuracy of the determined values of the target analytes for the five control levels ranged from 94.8 to 111.7% for oral fluid, from 91.3 to 100.2% for plasma and from 94.8 to 109.8% for urine. Imprecision for the five control levels did not exceeded 12.8% for oral fluid, 16.2% for plasma and 17.1% for urine. The method developed for the three matrices was validated and was also successfully applied to assess real samples, showing for the first time the detection of LDX in oral fluid.     

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5‐HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC‐ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5‐HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R²) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5‐HT, and 5 and 2 ng/mL for DA and NE. Within‐ and between‐run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50–8.36 and 0.54–13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5‐HT, DA and NE in urine by coulometric detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Gas chromatography‐mass spectrometric method for the screening and quantification of illicit drugs and their metabolites in human urine using solid‐phase extraction and trimethylsilyl derivatization

A simple and rapid GC‐MS method has been developed for the screening and quantification of many illicit drugs and their metabolites in human urine by using automatic SPE and trimethylsilylation. Sixty illicit drugs, including parent drugs and their metabolites that are possibly abused in Korea, can be monitored by this method. Among them, 24 popularly abused illicit drugs were selected for quantification. Very delicate optimizations were carried out in SPE, trimethylsilylation derivatization, and GC/MS to enable such remarkable achievements. Trimethylsilylated analytes were well separated within 21 min by GC‐MS. In the validation results, the LOD of all the analytes were in the range of 2–75 ng/mL. The LOQ of the quantified analytes were in the range of 5–98 ng/mL. The linearity (r²) of the quantified analytes ranged 0.990–1.000 in each concentration range between 10 and 1000 ng/mL. The mean recoveries ranged from 62 to 126% at three different concentrations of each analyte. The inter‐day and inter‐person accuracies were within ?13.3～14.9%, and ?10.1～13.0%, respectively, and the inter‐day and inter‐person precisions were less than 12.9%. The method was reliable and efficient for the screening and quantification of abused illicit drugs in routine urine analysis.     

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.     

Analysis of 3‐methoxypterostilbene in biological fluids by high‐performance liquid chromatography: application to pre‐clinical pharmacokinetics

A method of analysis for 3‐methoxypterostilbene [trans‐3,3′5‐trimethoxy‐4′hydroxystilbene] in biological fluids is necessary to study pharmacokinetics. A novel and simple high‐performance liquid chromatographic method was developed for the determination of 3‐methoxypterostilbene in rat serum and urine. The internal standard, pinosylvin, was added to 0.1 mL serum or urine (serum proteins were precipitated with cold acetonitrile at ?20°C). Separation was achieved with a Phenomenex® C₁₈ (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 327 nm. The calibration curves in both matrices were linear ranging from 0.05 to 100 µg/mL, and the mean extraction efficiency was >99%. Precision of the assay for both matrices was <12% (RSD) and was within 13% for all points on the calibration curve. The limit of quantification for this method was 0.05 µg/mL. The assay was successfully applied to a preliminary study of 3‐methoxypterostilbene pharmacokinetics in a rat. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid,simple and reliable HPLC‐triple quadrupole tandem mass spectrometer method for a simultaneous quantification of irinotecan and its active metabolite 7‐ethyl‐10‐hydroxycamptothecin (SN38) in mouse plasma

A simple, fast and reliable high‐performance liquid chromatography–triple quadrupole mass spectrometry method (HPLC‐MS/MS method) was developed, validated and used for the simultaneous quantification of irinotecan and 7‐ethyl‐10‐hydroxycamptothecin (SN38) in heparinized mouse plasma. Camptothecin was used as the internal standard. A single‐step protein precipitation without evaporation and reconstitution steps was adopted as sample processing method. Our bioanalytical method was validated in compliance with the guidelines from the European Medicines Agency. The lower limit of quantification for both irinotecan and SN38 was 5 ng/mL. The calibration curves for both analytes fitted to a 1/x² weighted linear regression model and ranged from 5 to 1000 ng/mL. The intra‐run and inter‐run precisions were within 8.6%, and the intra‐run and inter‐run accuracies were within 96.4?103.9%. Our validated bioanalytical method was successfully applied to the pharmacokinetic study in mice, in which 4 mg/kg irinotecan was intraperitoneally injected. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous quantitative and qualitative analysis of aliskiren,enalapril and its active metabolite enalaprilat in undiluted human urine utilizing LC‐ESI‐MS/MS

The benefit–risk ratio of combined blocking by the direct renin inhibitor aliskiren and an angiotensin‐converting enzyme inhibitor (e.g. enalapril) on the renin–angiotensin–aldosterone system is discussed. No method was available for simultaneous determination of both drugs in urine. A novel sensitive method for simultaneous quantification in undiluted human urine was developed which enables systematic pharmacokinetic investigations, especially in poorly investigated populations like children. Matrix effects were clearly reduced by applying solid‐phase extraction followed by a chromatographic separation on Xselect^TM C₁₈ CSH columns. Mobile phase consisted of methanol and water, both acidified with formic acid. Under gradient conditions and a flow rate of 0.4 mL/min the column effluent was monitored by tandem mass spectrometry with electrospray ionization. Calibration curves were constructed in the range of 9.4–9600 ng/mL regarding aliskiren, 11.6–12000 ng/mL for enalapril and 8.8–9000 ng/mL for enalaprilat. All curves were analyzed utilizing 1/x²‐weighted quadratic squared regression. Intra‐run and inter‐run precision were 3.2–5.8% and 6.1–10.3% for aliskiren, 2.4–6.1% and 3.9–7.9% for enalapril as well as 3.1–9.4% and 4.7–12.7% regarding enalaprilat. Selectivity, accuracy and stability results comply with current international bioanalysis guidelines. The fully validated method was successfully applied to a pharmacokinetic investigation in healthy volunteers. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative analysis of five tobacco‐specific N‐nitrosamines in urine by liquid chromatography–atmospheric pressure ionization tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of five total tobacco‐specific N‐nitrosamines (TSNA), including free and conjugated forms in urine. The limits of detection for 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol, N′‐nitrosonornicotine, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone, N′‐nitrosoanatabine and N′‐nitrosoanabasine were 0.6, 0.6, 10.0, 0.4 and 0.4 pg/mL, respectively, with a linear calibration range of up to 20,000 pg/mL. Intra‐ and inter‐day precision for TSNA measurements ranged from 0.82 to 3.67% and from 2.04 to 7.73% respectively. For total TSNAs, the β‐glucuronidase amount was optimized for hydrolysis time and yield. Different liquid chromatography columns and mobile phases with different pH conditions were evaluated. The validated method was then applied to 50 smoker and 30 nonsmoker urine samples. Our results suggest that this sensitive and relatively simple analytical method is suitable for application to epidemiological investigations of health risks associated with the exposure to tobacco smoke or secondhand smoke in both smokers and nonsmokers. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Liquid chromatography/tandem mass spectrometry method for quantification of trans-stilbene glycoside in rat plasma and its pharmacokinetic application

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of trans‐stilbene glycoside (SG) in rat plasma. As trans‐SG can be rapidly isomerized under light exposure, trans‐SG plasma samples were prepared in the dark and assayed immediately. Trans‐SG and internal standard were extracted by protein precipitation. Chromatographic separation was achieved on a C₁₈ column with a gradient elution program. The detection of analytes was performed by negative ion via multiple reaction monitoring mode. The precursor‐to‐product ions of m/z 405.1 → 242.9 for trans‐SG and m/z 389.1 → 226.9 for polydatin (internal standard) were monitored. No interference of endogenous components was observed for any plasma samples that we studied.The method was linear over the concentration range of 1.0–1000.0 ng/mL with a good correlation coefficient. The lower limit of quantification was 1.0 ng/mL for trans‐SG. The intra and inter‐batch accuracy for trans‐SG in stable rat plasma samples ranged from 93.3 to 102.7% and the variation was less than 8.1%. The extraction recoveries of trans‐SG in rat plasma were from 102.8 to 112.4% and the matrix effects were also acceptable. This method was successfully applied to pharmacokinetic study of trans‐SG in rats after intravenous administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Application of a LC–MS/MS method developed for the determination of p‐phenylenediamine,N‐acetyl‐p‐phenylenediamine and N,N‐diacetyl‐p‐phenylenediamine in human urine specimens

Cases of poisoning by p‐phenylenediamine (PPD) are detected sporadically. Recently an article on the development and validation of an LC–MS/MS method for the detection of PPD and its metabolites, N‐acetyl‐p‐phenylenediamine (MAPPD) and N,N‐diacetyl‐p‐phenylenediamine (DAPPD) in blood was published. In the current study this method for detection of these compounds was validated and applied to urine samples. The analytes were extracted from urine samples with methylene chloride and ammonium hydroxide as alkaline medium. Detection was performed by LC–MS/MS using electrospray positive ionization under multiple reaction‐monitoring mode. Calibration curves were linear in the range 5–2000 ng/mL for all analytes. Intra‐ and inter‐assay imprecisions were within 1.58–9.52 and 5.43–9.45%, respectively, for PPD, MAPPD and DAPPD. Inter‐assay accuracies were within ?7.43 and 7.36 for all compounds. The lower limit of quantification was 5 ng/mL for all analytes. The method, which complies with the validation criteria, was successfully applied to the analysis of PPD, MAPPD and DAPPD in human urine samples collected from clinical and postmortem cases.     

Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on‐line post‐column derivatization

In the present study, we propose the first HPLC method coupled to postcolumn derivatization for the determination of rimantadine in human urine samples. The analyte and amantadine (internal standard) were isocratically separated using an RP monolithic stationary phase (100 × 4.6 mm id) with a mobile phase consisting of CH₃OH/phosphate buffer (25 mmol/L, pH 3.0) at a volume ratio of 50:50. Postcolumn derivatization involved on‐line reaction with o‐phthalaldehyde (20 mmol/L) and N‐acetyl‐cysteine (5 mmol/L) at alkaline medium (100 mmol/L borate pH 11.0). Spectrofluorimetric detection at λ_ex/λ_em = 340/455 nm enabled the selective and sensitive determination of rimantadine in urine samples at a range of 50–500 ng/mL with an LOD of 5 ng/mL. Human urine samples were analyzed successfully after SPE using hydrophilic‐lipophilic balanced RP cartridges (30 mg/mL, Oasis HLB). Recoveries ranged between 89.7 and 102.7%.     

New application of chitosan‐grafted polyaniline in dispersive solid‐phase extraction for the separation and determination of phthalate esters in milk using high‐performance liquid chromatography

Chitosan‐grafted polyaniline was synthesized and applied as a sorbent for the preconcentration of phthalate esters in dispersive solid‐phase extraction. By coupling dispersive solid‐phase extraction with high‐performance liquid chromatography and response surface methodology (central composite design), a reliable, sensitive, and cost‐effective method for simultaneous determination of phthalate esters including dimethyl phthalate, di‐n‐butyl phthalate, and di(2‐ethylhexyl)phthalate was developed. The morphology of sorbent had been studied by scanning electron microscopy and its chemical structure confirmed by Fourier transform infrared spectroscopy. Under optimum condition, good linearity was observed in the range of 5.0–5000.0 ng/mL. The limits of detection (S/N = 3) and limits of quantification (S/N = 10) were 0.1–0.3 and 0.3–1 ng/mL, respectively. The relative standard deviations were less than 8.8%. Finally, this procedure was employed for extraction of trace amounts of phthalic acid esters in milk samples, the relative recoveries ranged from 82 to 103%.     

Identification and quantification of thymol metabolites in plasma,liver and duodenal wall of broiler chickens using UHPLC‐ESI‐QTOF‐MS

In the present study, we aimed to develop a method for thymol sulfate and thymol glucuronide determination in plasma, liver and duodenal wall of broiler chickens after feeding with a Thymus vulgaris essential oil at the different concentrations (0.01, 0.05 and 0.1% w /w). UHPLC coupled with accurate‐mass QTOF‐MS was used for identification and quantification of thymol metabolites. Novel Waters Oasis Prime HLB solid‐phase extraction cartridges were applied to sample clean‐up with extraction recoveries ranged from 85 to 92%. The presence of thymol glucuronide was confirmed by MS software according to molecular formula, score, mass error and double bond equivalent. In terms of validation, calibration curves of thymol sulfate were constructed in matrix samples with linearity from 3.91 to 250.0 ng/mL and correlation coefficients were within the range of 0.9979–0.9995. Limits of detection were 0.97, 0.29 and 0.63 ng/mL and limits of quantification were 3.23, 0.97 and 2.09 ng/mL for plasma, liver and duodenal wall, respectively. Intra‐day and inter‐day precision expressed as relative standard deviation were <4.35%. To highlight, thymol metabolites were directly detected for the first time in liver and duodenal wall and this method was shown to be successfully applicable for investigation of thymol metabolism in chickens after thyme essential oil ingestion.     

High‐sensitivity simultaneous quantification of tacrolimus and 13‐O‐demethyl tacrolimus in human whole blood using ultra‐performance liquid chromatography coupled to tandem mass spectrometry

Blood concentrations of tacrolimus show large variability among patients and the narrow therapeutic range is related to adverse effects. Therefore, therapeutic drug monitoring is needed for strict management. 13‐O‐Demethyl tacrolimus (13‐O‐DMT) was reported as the major metabolite formed by cytochrome P450 (CYP)3A such as CYP3A5. In previous studies, the best lower limit of quantification (LLOQ) was 0.1 ng/mL for both substances. However, this LLOQ may not be low enough now because the dosage of tacrolimus has decreased in recent years. The purpose of this study was to develop and validate a high‐sensitivity and high‐throughput assay for simultaneous quantification of tacrolimus and 13‐O‐DMT in human whole blood using ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Thirty‐five stable kidney transplant recipients receiving tacrolimus were recruited in this study. The calibration curve range was 0.04–40 ng/mL. All calibration samples and quality control samples fulfilled the requirements of the US Food and Drug Administration and the European Medicines Agency guidelines for assay validation. Trough concentrations of tacrolimus and 13‐O‐DMT in 35 stable kidney transplant recipients receiving tacrolimus were within the range of the respective calibration curve. Our novel UPLC–MS/MS method is more sensitive than previous methods for quantification of tacrolimus and 13‐O‐DMT.     

Stereoselective determination of ginsenosides Rg3 and Rh2 epimers in rat plasma by LC‐MS/MS: Application to a pharmacokinetic study

We developed and validated an accurate and sensitive LC–MS/MS method for the simultaneous quantitation of ginsenoside Rg3 and Rh2 epimers (R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2) in rat plasma. Analytes were extracted from 0.1 mL aliquots of rat plasma by liquid–liquid extraction, using 2 mL of ethyl acetate. In this assay, dioscin (500 ng/mL) was used as an internal standard. Chromatographic separation was conducted using an Acclaim RSLC C₁₈ column (150 × 2.1 mm, 2.2 μm) at 40°C, with a gradient mobile phase consisting of 0.1% formic acid in distilled water and in acetonitrile, a flow rate of 0.35 mL/min, and a total run time of 20 min. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with negative electrospray ionization at m/z 783.4 → 161.1 for R‐Rg3 and S‐Rg3, m/z 621.3 → 161.1 for R‐Rh2 and S‐Rh2, and m/z 867.2 → 761.5 for the internal standard. For R‐Rg3 and S‐Rg3, the lower limit of quantification was 5 ng/mL, with a linear range up to 500 ng/mL; for R‐Rh2 and S‐Rh2, the lower limit of quantification was 150 ng/mL, with a linear range up to 6000 ng/mL. The coefficient of variation for assay precision was less than 10.5%, with an accuracy of 86.4–112%. No relevant cross‐talk or matrix effect was observed. The method was successfully applied to a pharmacokinetic study after oral administration of 400 mg/kg and 2000 mg/kg of BST204, a fermented ginseng extract, to rats. We found that the S epimers exhibited significantly higher plasma concentrations and area under curve values for both Rg3 and Rh2. This is the first report on the separation and simultaneous quantification of R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2 in rat plasma by LC‐MS/MS. The method should be useful in the clinical use of ginseng or its derivatives.     

Development of hollow fiber‐supported liquid‐phase microextraction and HPLC‐DAD method for the determination of pyrethroid metabolites in human and rat urine

A simple hollow fiber liquid‐phase microextraction method for the determination of synthetic pyrethroid metabolites, 3‐phenoxybenzoic acid and 4‐hydroxy‐3‐phenoxybenzoic acid, in human and rat urine was developed and validated. A polypropylene hollow fiber tightly fitted onto a Nylon rod and impregnated with organic solvent served as a disposable extraction device. Desorption of analytes was carried out in NaOH solution, analyzed further by gradient HPLC and diode array detection method. Important factors were identified using Taguchi OA16 (4⁵) orthogonal array design and further optimized using univariate approach. The optimum method performance was observed when 1 mL of urine hydrolyzed with 0.2 mL of concentrated HCl was further supplemented with 100 mg of NaCl and extracted for 120 min into dihexyl ether immobilized in the pores of the hollow fiber. Metabolites were desorbed into 0.1 mL of 0.1 M NaOH for another 120 min. Limits of detection and quantitation of 15 and 50 ng/mL were obtained for both analytes. Relative standard deviations of 1.6–12.6% over the linear range (50–10,000 ng/mL, r > 0.9906) were observed. Intra‐ and inter‐day accuracies of the method ranged from 98.3 to 109.5% and from 93.3 to 110.9%, respectively. The optimized method was applied to the analysis of real urine samples collected from rats exposed orally to cypermethrin. Copyright © 2013 John Wiley & Sons, Ltd.