Analysis of cholesterol oxidation products by Fast gas chromatography/mass spectrometry

The aim of the present study was to set‐up a Fast gas chromatography/mass spectrometry method for the analysis of cholesterol oxidation products (COPs). A silylated mixture of seven oxysterol standards was injected into a Fast GC/MS system. A capillary GC column (10 m×0.1 mm internal diameter×0.1 μm film thickness) coated with 95% dimethyl‐ and 5% diphenyl‐polysiloxane, was used. The method gave a fast (total analysis time=3.5 min) and satisfactory resolution (R>1.2) of the COPs standards, with a good repeatability and sensitivity, similar to those of conventional GC/MS; recoveries were tested on mice liver. Fast GC/MS method suitability for COPs analysis in food was also tested on an oxidized sardine fillet, which had been previously saponified and purified by NH₂ solid‐phase extraction (SPE); a good repeatability and sensitivity was also obtained. The analytical performance of the Fast GC/MS method for the determination of COPs, together with the consequent significant reduction of the analysis time and consumables, demonstrates that Fast GC/MS represents a valid alternative to conventional GC/MS and evinces the great potential of such an analytical technique, which could be applied for both food and biological samples.     

Development of a fast sample treatment for the analysis of free and bonded sterols in human serum by LC‐MS

The analysis of sterols in biological fluids allows the clinical study of cholesterol related diseases. This research is focused on reducing the sample processing time of the determination of free and bonded sterols in human serum. Ten sterols were studied: cholesterol precursors (desmosterol, lanosterol, and cholestanol); phytosterols (stigmasterol, campesterol, sitosterol, and sitostanol) and oxysterols (7‐α‐hydroxy‐4‐cholesten‐3‐one, 24‐hydroxycholesterol, and 27‐hydroxycholesterol). Ultrasound assistance was used to diminish the reaction time during the alkaline hydrolysis for determining total sterols. Different retention mechanisms of solid‐phase extraction were compared, two reversed‐phase sorbents DSC‐18 and polymeric Oasis‐HLB and a novel zirconia‐coated silica phase. DSC‐18 and zirconia‐coated silica were the most suitable sorbents to analyze these metabolites. The resulting extracts were analyzed by liquid chromatography coupled to mass spectrometry. The analytical parameters were determined and better values were observed with DSC‐18 cartridges for most sterols. LOQ were in the low ng/mL level. Recoveries were in the range 85–99%. Average intermediate precision was 15%. Accuracy for both cartridges was more than 92%. Zirconia‐coated silica showed better performance for the oxysterols, with recoveries around 90%. The procedure allows the determination of free and bonded sterol precursors, phytosterols, and oxysterols in human serum.     

Determination of the deuterium/hydrogen ratio of endogenous urinary steroids for doping control purposes

The development and application of a combined gas chromatography/thermal conversion/isotope ratio mass spectrometry (GC/TC/IRMS) method for D/H ratio determination of endogenous urinary steroids are presented. The key element in sample preparation was the consecutive cleanup with high‐performance liquid chromatography of initially native and subsequently acetylated steroids. This strategy enabled sufficient cleanup off all target analytes for determination of their respective D/H values. Ten steroids (11β‐hydroxyandrosterone, 5α‐androst‐16‐en‐3α‐ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5α‐androstan‐3α,17β‐diol, 5β‐androstan‐3α,17β‐diol and dehydroepiandrosterone) were measured from a single urine specimen. Depending on the biological background, the determination limit for all steroids ranged from 10 to 15 ng/mL for a 20 mL specimen. The method was validated by application of linear mixing models on each steroid and covered repeatability and reproducibility. The specificity of the procedure was ensured by gas chromatography/mass spectrometry (GC/MS) analysis of the sample using equivalent chromatographic conditions to those employed in the GC/TC/IRMS measurement. Within the sample preparation, no isotopic fractionation was observed, and no amount‐dependent shift of the D/H ratios during the measurement was noticed. Possible memory effects occurring during IRMS measurements were corrected by applying a simple rule of proportion. In order to determine the naturally occurring D/H ratios of all implemented steroids, a population of 18 male subjects was analyzed. Relevant mean Δ values among selected steroids were calculated which allowed us to study the metabolic pathways and production sites of all the implemented steroids with additional consideration of the corresponding ¹³C/¹²C ratios. Copyright © 2009 John Wiley & Sons, Ltd.     

The detection of various opiates and benzodiazepines by comprehensive two‐dimensional gas chromatography/time‐of‐flight mass spectrometry

A technique using comprehensive two‐dimensional gas chromatography/time‐of‐flight mass spectrometry (GC × GC/TOFMS) is applied to qualitative and quantitative drug testing. Human serum was ‘spiked’ with known quantities of benzodiazepines and a ‘street heroin’ mixture including some of the major metabolites and impurities. The sample components were extracted from the matrix by solid‐phase extraction (SPE). Constituents containing polar hydroxyl and/or secondary amine groups were derivatised with N‐methyl‐N‐(tert‐butyldimethyl)trifluoroacetamide (MTBSTFA) to improve the chromatographic performance. An orthogonal separation of the matrix constituents was achieved by coupling a DB‐5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The eluant was focused onto the second column by a twin‐stage cryo‐modulator. Rapid 6 s modulation times were achieved by transfer from a 30 m × 0.25 mm (length × internal diameter) to a 2 m × 0.1 mm column. TOFMS with rapid spectral acquisition (≤500 spectra/s) was employed in the mass range m/z 40–650. A clean mass spectrum was obtained for each analyte using mass spectral deconvolution software. The sensitivity and repeatability of the method were evaluated by the preparation of calibration standards for two benzodiazepines, flunitrazepam and its major metabolite 7‐aminoflunitrazepam (7‐amino‐FN), in the concentration range 5–1000 ng/mL. The limits of detection (LODs) and limits of quantitation (LOQs), calculated by repeat injections (×10) of the lowest standard, were 1.6 and 5.4 ng/mL (flunitrazepam); 2.5 and 8.5 ng/mL (7‐amino‐FN), respectively. There is scope to extend this protocol to screen a large number of drugs and metabolites stored in a library database. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of the enantiomeric purity of amphetamine after derivatization with Sanger’s reagent (2,4-dinitrofluorobenzene [DNFB]) by simultaneous dual circular dichroism and ultraviolet spectroscopy

Determination of Se in biological materials was attempted by microwave-induced plasma mass spectrometry (MIP-MS). (1) Serum samples were available after 10 times dilution with 0.5% nitric acid solution containing 0.1% Triton X-100. When oxygen gas was inserted into the plasma gas (nitrogen) in order to improve the combustion, the sensitivity was reduced to 45%. The detection limit of this method was 0.5 ng/mL. (2) Standard reference materials on commercial base were used to evaluate the accuracy of the Se determination by MIP-MS after microwave digestion. In samples like bovine liver and human hair with Se concentrations of more than 0.7 μg/g, the standard curve method after internal standard (IS) correction was acceptable. This procedure was unsuitable for samples with low Se concentrations such as milk powder (certified value of Se 0.11 μg/g), or plant leaf samples. (3) Instead of IS correction, the peak height of the spectrum was used for calculations from the matrix matched calibration curve. The results of all materials were close to the certified values, even at 25 ng/g. The detection limit of the MIP-MS with microwave digestion and IS correction was 0.05 ng/mL in standard solutions. The detection limit of the peak height method was 0.1 ng/mL and was estimated to be < 20 ng/g in plant materials. Received: 25 September 1998 / Revised: 15 February 1999 / Accepted: 18 February 1999     

Analyzing multiple pesticides in tobacco leaf using gas chromatography with quadrupole time‐of‐flight mass spectrometry

A method combining gas chromatography with quadrupole time‐of‐flight mass spectrometry has been developed for the simultaneous analysis of multiple pesticide residues in tobacco leaf. The retention index and high accurate masses of ions from the first‐stage and the second‐stage mass spectra of each pesticide were collected for qualitation and quantification. A total of 115 pesticides were evaluated. The extract from organic tobacco leaf was used as a model matrix. The limit of detection was <10 ng/mL, and the limit of quantification was in the range of 1–20 ng/mL for 95% of the tested pesticides. The correlation coefficients were >0.9900 for all tested pesticides. At three concentrations (10, 50, and 100 ng/mL), most compounds presented satisfactory recoveries ranging from 70 to 120% and good precision <20%. Finally, three tobacco leaf samples collected from a local market were analyzed. A total of three pesticides were found, including dimethachlon, triadimenol, and flumetralin. Each pesticide was confirmed by the presence of three ions at the expected retention index and mass. In conclusion, gas chromatography with quadrupole time‐of‐flight mass spectrometry appears to be one of the most efficient tools for the analysis of pesticide residues in tobacco leaf.     

Comparison of multiple API techniques for the simultaneous detection of microconstituents in water by on‐line SPE‐LC‐MS/MS

This study described a fully automated method using on‐line solid phase extraction of large volume injections coupled with high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) to simultaneously detect a group of recalcitrant microconstituents (pharmaceuticals and personal care products, steroid hormones and sterols) in aqueous matrices. Samples (1 mL to 20 mL) were loaded to the preconcentration column at 1 mL/min, and the column was washed with 1000 μL of 25% methanol in LC/MS water to remove polar and ionic interferences before LC‐MS/MS analysis. Three different atmospheric pressure ionization (API) techniques, including photoionization (APPI) with four different dopants (acetone, anisole, chlorobenzene and toluene), heated electrospray ionization (HESI) and atmospheric pressure chemical ionization (APCI), were evaluated on the basis of method detection limits (MDLs) and recoveries from different aqueous matrixes. Results indicated that APPI with toluene as dopant was the most sensitive ionization method for the majority of the analytes. When using 5 mL of sample, MDLs for pharmaceuticals and personal care products, including carbamazepine, DEET, caffeine, naproxen, acetaminophen and primidone, were between 0.3 ng/L and 15 ng/L. MDLs of hormones, including testosterone, equilenin, progesterone, equilin, 17β‐estradiol, 17α‐ethynylestradiol, estrone, androsterone, mestranol and estriol, were between 1.2 ng/L and 37 ng/L. The combination of APPI with dopant allowed the detection of two difficult to ionize fecal related sterols, such as coprostan‐3‐ol and coprostan‐3‐one with MDLs of 5.4 ng/L and 11 ng/L, respectively. Calculated MDLs are more than adequate for analysis of wastewater using 1 to 5 mL sample size and for surface waters using up to 20 mL sample size. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of β‐sitosterol,campesterol, and stigmasterol in rat plasma by using LC–APCI‐MS/MS: Application in a pharmacokinetic study of a titrated extract of the unsaponifiable fraction of Zea mays L.

A liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated to investigate the pharmacokinetic properties of β‐sitosterol, campesterol, and stigmasterol in rat plasma. Cholesterol‐d₆ was used as an internal standard. To avoid interference of the three phytosterols in rat plasma and minimize matrix effects, a small volume (10 μL) of 4% bovine serum albumin was used as a surrogate matrix for making calibrators and quality control samples. Rat plasma (10 μL) samples were extracted by liquid–liquid extraction with methyl tert‐butyl ether and separated on a Kinetex C₁₈ column. The detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode using positive atmospheric pressure chemical ionization. This assay was linear over concentration ranges of 250–5000 ng/mL (β‐sitosterol), 250–5000 ng/mL (campesterol), and 50–2000 ng/mL (stigmasterol). Additionally, a second set of quality controls made in rat plasma was also evaluated against calibration curves made using the surrogate matrix. All the validation data, including the specificity, precision, accuracy, recovery, matrix effect, stability, and incurred sample reanalysis conformed to the acceptance requirements. Our method was successfully applied to study the pharmacokinetics of three phytosterols in rats.     

Simultaneous determination of three organophosphorus pesticides in different food commodities by gas chromatography with mass spectrometry

A sensitive and selective gas chromatography with mass spectrometry method was developed for the simultaneous determination of three organophosphorus pesticides, namely, chlorpyrifos, malathion, and diazinon in three different food commodities (milk, apples, and drinking water) employing solid‐phase extraction for sample pretreatment. Pesticide extraction from different sample matrices was carried out on Chromabond C₁₈ cartridges using 3.0 mL of methanol and 3.0 mL of a mixture of dichloromethane/acetonitrile (1:1 v/v) as the eluting solvent. Analysis was carried out by gas chromatography coupled with mass spectrometry using selected‐ion monitoring mode. Good linear relationships were obtained in the range of 0.1–50 μg/L for chlorpyrifos, and 0.05–50 μg/L for both malathion and diazinon pesticides. Good repeatability and recoveries were obtained in the range of 78.54–86.73% for three pesticides under the optimized experimental conditions. The limit of detection ranged from 0.02 to 0.03 μg/L, and the limit of quantification ranged from 0.05 to 0.1 μg/L for all three pesticides. Finally, the developed method was successfully applied for the determination of three targeted pesticides in milk, apples, and drinking water samples each in triplicate. No pesticide was found in apple and milk samples, but chlorpyrifos was found in one drinking water sample below the quantification level.     

Sensitive and simultaneous quantitation of 6β‐hydroxycortisol and cortisol in human plasma by LC‐MS/MS coupled with stable isotope dilution method

CYP3A phenotyping provides a means for personalized drug therapy. We focused our attention on the plasma 6β‐hydroxycortisol (6β‐OHF) to cortisol ratio as an index for CYP3A phenotyping. In the present study, we developed a sensitive and reliable method for the simultaneous determination of 6β‐OHF and cortisol in human plasma using high‐performance liquid chromatography/tandem mass spectrometry together with picolinylester derivatization or nonderivatization methods and 6β‐[9,11,12,12‐²H₄]hydroxycortisol and [1,2,4,19‐¹³C₄]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513 pg/mL for 6β‐OHF and 38.100 pg/mL for cortisol. The relative error and relative standard deviation of the lower limits of quantification were <5% for both methods. The intra‐day and inter‐day assay reproducibilities of the determined 6β‐OHF and cortisol concentrations were consistent with the actual amounts added as relative errors and relative standard deviations for both methods, which were <5.4% and <3.9%, respectively. Both methods were applied for the quantification of plasma 6β‐OHF and cortisol concentrations in healthy subjects taking oral contraceptives. The absolute concentrations and time course of 6β‐OHF and cortisol were found to be consistent when measured using the 2 methods. The ratio as an index for in vivo CYP3A activity decreased after 21 days of taking oral contraceptives for both methods. To the best of our knowledge, this is the first study reporting the detailed investigation of accuracy and precision in the simultaneous measurement of 6β‐OHF and cortisol in human plasma using liquid chromatography/tandem mass spectrometry coupled with stable isotope dilution method, which can be applied to CYP3A phenotyping.     

Determination of the enantiomeric purity of amphetamine after derivatization with Sanger’s reagent (2,4-dinitrofluorobenzene [DNFB]) by simultaneous dual circular dichroism and ultraviolet spectroscopy

Determination of Se in biological materials was attempted by microwave-induced plasma mass spectrometry (MIP-MS). (1) Serum samples were available after 10 times dilution with 0.5% nitric acid solution containing 0.1% Triton X-100. When oxygen gas was inserted into the plasma gas (nitrogen) in order to improve the combustion, the sensitivity was reduced to 45%. The detection limit of this method was 0.5 ng/mL. (2) Standard reference materials on commercial base were used to evaluate the accuracy of the Se determination by MIP-MS after microwave digestion. In samples like bovine liver and human hair with Se concentrations of more than 0.7 μg/g, the standard curve method after internal standard (IS) correction was acceptable. This procedure was unsuitable for samples with low Se concentrations such as milk powder (certified value of Se 0.11 μg/g), or plant leaf samples. (3) Instead of IS correction, the peak height of the spectrum was used for calculations from the matrix matched calibration curve. The results of all materials were close to the certified values, even at 25 ng/g. The detection limit of the MIP-MS with microwave digestion and IS correction was 0.05 ng/mL in standard solutions. The detection limit of the peak height method was 0.1 ng/mL and was estimated to be < 20 ng/g in plant materials.     

A high‐performance liquid chromatography–tandem mass spectrometry method coupled with protein precipitation for determination of granisetron in human plasma and its application to a comparative pharmacokinetic study

A rapid, simple and validated method based on liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) has been developed for the determination of granisetron in human plasma. Plasma samples were pre‐purified by protein precipitation procedure. The chromatographic separation was achieved with Synergi Polar‐RP (75 × 2 mm, 4 µm) column using a mixture of 5 mm pH4.0 ammonium formate and methanol (300:316, v/v) under isocratic conditions at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The analysis time was about 2.5 min. The method was fully validated over the concentration range 0.1–10 ng/mL. The lower limit of quantification was 0.1 ng/mL. Inter‐ and intra‐batch precision was <6.1% and the accuracy was within 95.6–100.0%. The mean extraction recovery was 96.3%. Selectivity, matrix effect and stability were also validated. The method was applied to the comparative pharmacokinetic study of granisetron in Chinese healthy subjects. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of selected estrogenic endocrine disrupting chemicals and bisphenol A residues in whole milk using fabric phase sorptive extraction coupled to HPLC‐UV detection and LC‐MS/MS

A simple and sensitive analytical methodology is developed for rapid screening and quantification of selected estrogenic endocrine disrupting chemicals and bisphenol A from intact milk using fabric phase sorptive extraction in combination with high‐performance liquid chromatography coupled to ultraviolet detection/tandem mass spectrometry. The new approach eliminates protein precipitation and defatting step from the sample preparation workflow. In addition, the error prone and time‐consuming solvent evaporation and sample reconstitution step used as the sample post‐treatment has been eliminated. Parameters with most significant impact on the extraction efficiency of fabric phase sorptive extraction including sorbent chemistry, sample volume, extraction time have been thoroughly studied and optimized. Separation of the selected estrogenic endocrine disrupting chemicals including α‐estradiol, hexestrol, estrone, 17α‐ethinyl estradiol, diethylstilboestrol, and bisphenol A were achieved using a Zorbax Extend‐C18 high‐performance liquid chromatography column (15 cm × 4.6 mm, 5 μm particle size). The limit of detection values obtained in fabric phase sorptive extraction with high‐performance liquid chromatography with ultraviolet detection ranged from 25.0 to 50.0 ng/mL. The method repeatability values were 3.6–13.9 (relative standard deviation, %) and intermediate precision values were 4.6–12.7 (relative standard deviation, %). The fabric phase sorptive extraction method was also coupled to liquid chromatography with tandem mass spectrometry for identifying each endocrine disrupting chemical at 10 ng/mL.     

Quantification of lipid modified estrogenic derivative (ESC8) in rat plasma by LC‐MS: application to a pharmacokinetic study

A lipid‐conjugated, estrogenic derivative molecule, ESC8, compared with other estrogenic molecules, encourages cell death in both ER‐positive and ER‐negative breast cancer cells. A rapid and highly sensitive assay method has been developed and validated for the estimation of a ESC8 in rat plasma using liquid chromatography coupled with mass spectrometry under positive‐ion mode with electrospray ionization. The sample process includes using methanol for precipitation of ESC8 and dextromethorphan (internal standard, IS) from plasma. Chromatographic separation was achieved with methanol–water–formic acid (70:30:0.1% v/v/v) pumped at a flow rate of 0.3mL/min and a C₁₈ column (50 × 2.1 mm i.d., 1.7 μm particle size) with a total run time of 5 min. The m/z ions monitored were 568.5 and 272.1 for ESC8 and IS, respectively. The lower limit of quantitation achieved was 1.08 ng/mL and linearity was observed from 5 to 500 ng/mL. The intra‐ and inter‐day precisions were <4%. The proposed method was successfully applied to a preliminary pharmacokinetic study of ESC8 liposomal formulation following an intraperitoneal administration of 3.67 mg/kg in rats. The concentrations of ESC8 in plasma were quantifiable up to 36 h. The peak concentration of ESC8 was found to be 110.72 ng/mL, the area under the concentration–time curve was 1625.23ng/mL h and the half‐life was 11.72 h.     

A rapid and sensitive LC‐MS/MS method for quantification of 3,29‐dibenzoyl rarounitriol in rat plasma: application to a pharmacokinetic study

A rapid, sensitive and high‐throughput liquid chromatography–tandem mass spectrometry was established and validated to assay the concentrations of 3,29‐dibenzoyl rarounitriol in rat plasma. Plasma samples were processed by liquid–liquid extraction with ethyl acetate and separated on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 3 µm) at an isocratic flow rate of 0.5 mL/min using methanol–10 mm ammonium acetate–formic acid (90:10:0.1, v/v/v) as mobile phase. The total run time was 5 min for each sample. MS/MS detection was accomplished in selected reaction monitoring mode with positive electrospray ionization. The calibration curve was linear over the concentration range of 0.125–50 ng/mL with lower limit of quantification of 0.125 ng/mL. The intra‐ and inter‐day precisions were <10.1% in terms of coefficient of variation, and the accuracy was within ±11.7% in terms of relative error. The developed method was successfully applied to a pharmacokinetic study of 3,29‐dibenzoyl rarounitriol following intragastric administration of 3.65 mg/kg to Wistar rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of the bioactive components in rat plasma by UPLC‐MS/MS and application in pharmacokinetic studies after oral administration of radix Scutellariae extract

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantification of the four main bioactive compounds, i.e. baicalin, baicalein, wogonoside and wogonin, in rat plasma after oral administration of Radix Scutellariae extract. Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 447.5 → 270.1 for baicalin, 270.1 → 168.1 for baicalein, 461.2 → 284.0 for wogonoside, 284.2 → 168.1 for wogonin and 748.5 → 158.1 for IS. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 1.13 ng/mL for baicalin, 1.23 ng/mL for baicalein, 0.82 ng/mL for wogonoside and 0.36 ng/mL for wogonin. The calibration curves obtained were linear (r > 0.99) over the concentration range ~ 1–1000 ng/mL. The intra‐ and inter‐day precision was <15% and the accuracy was within ±14.7%. After validation, this method was successfully applied to a pharmacokinetic study of Radix Scutellariae extract.     

Development and validation of a UPLC‐MS/MS method for the determination of 7‐hydroxymitragynine,a μ‐opioid agonist,in rat plasma and its application to a pharmacokinetic study

A simple, sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated to determine the concentrations of 7‐hydroxymitragynine in rat plasma. Following a single‐step liquid–liquid extraction of plasma samples using chloroform, 7‐hydroxymitragynine and the internal standard (tryptoline) were separated on an Acquity UPLC^TM BEH C₁₈ (1.7 µm, 2.1 × 50 mm) column using an isocratic elution at a flow rate of 0.2 mL/min. The mobile phase consisted of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile (10:90, v/v). The run time was 2.5 min. The analysis was carried out under the multiple reaction‐monitoring mode using positive electrospray ionization. Protonated ions [M + H]⁺ and their respective product ions were monitored at the following transitions: 415 → 190 for 7‐hydroxymitragynine and 173 → 144 for the internal standard. The calibration curve was linear over the range of 10–4000 ng/mL (r² = 0.999) with a lower limit of quantification of 10 ng/mL. The extraction recoveries ranged from 62.0 to 67.3% at concentrations of 20, 600 and 3200 ng/mL). Intra‐ and inter‐day assay precisions (relative standard deviation) were <15% and the accuracy was within 96.5–104.0%. This validated method was successfully applied to quantify 7‐hydroxymitragynine in rat plasma following intravenous administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a method for the determination of cefovecin in plasma by HPLC

A simple high‐performance liquid chromatography method for the determination of cefovecin in small volume plasma has been developed. Following solid‐phase extraction using Oasis HLB cartridges, samples were separated by reverse‐phase high‐performance liquid chromatography on an XBridge C₈ (3.5 µm) 4.6 × 250 mm column and quantified using ultraviolet detection at 280 nm. The mobile phase was a mixture of 10 mm ammonium acetate (pH 3.5) and acetonitrile (89:11), with a flow rate of 0.85 mL/min. The standard curve ranged from 0.1 to 200 µg/mL. Intra‐ and Inter‐assay variability for cefovecin was <10%, and the average recovery was >90%. The lower limit of quantitation was 0.1 µg/mL. This method was successfully applied to the analysis of cefovecin samples at our institution. This is also the first fully validated method with an internal standard that does not use mass spectrometry. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of paeoniflorin,calycosin‐7‐O‐β‐d‐glucoside,ononin, calycosin and formononetin in rat plasma after oral administration of Buyang Huanwu decoction for their pharmacokinetic study by liquid chromatography–mass spectrometry

The purpose of this study was to simultaneously investigate the pharmacokinetics of five bioactive compounds in rat plasma after oral administration of Buyang Huanwu decoction (BYHWD) using high‐performance liquid chromatography coupled with mass spectrometry (HPLC‐MS). The separations were performed on a Thermo Hypersil Gold C₁₈ analytical column (50 × 2.1 mm, 3 µm) with the column temperature kept at 30°C. The quantitative analysis was performed using a quadrupole mass spectrometer detector operated under selected ion monitoring mode. A linear gradient elution of A (0.1% formic acid solution) and B (100% acetonitrile) was used at a flow rate of 0.2 mL/min. The method was validated within the concentration ranges 1.8–450, 6.0–1500, 2.0–500, 1.2–300 and 1.2–150 ng/mL for paeoniflorin, calycosin‐7‐O‐β‐d ‐glucoside, ononin, calycosin and formononetin, respectively. The calibration curves were linear with correlation coefficients > 0.99. The lower limits of quantitations were < 6.0 ng/mL. The method was further applied to assess the pharmacokinetics of the five bioactive constituents of BYHWD in rat plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Free mycophenolic acid determination in human plasma ultrafiltrate by a validated liquid chromatography–tandem mass spectrometry method

The aim of this study was to develop and validate fully the liquid chromatography–tandem mass spectrometry method for free mycophenolic acid (MPA) concentration measurements in plasma ultrafiltrate that will be reliable and simple in preparation with deuterated MPA (MPA‐d3) chosen as an internal standard. The chromatographic separation was made with Zorbax Eclipse XDB‐C18 column (4.6 × 150 mm) using a gradient of two solutions as a mobile phase: (A) water and (B) methanol, each containing 0.1% formic acid and 2.5 mm ammonium acetate. Satisfactory repeatability of retention times was achieved with average values of 7.54 ± 0.20 min and 7.50 ± 0.19 min for MPA and MPA‐d3, respectively. The method was selective, with no carry‐over or matrix effect observed. The analytical range was proven for MPA ultrafiltrate concentrations of 1–500 ng/mL. The accuracy and precision fell within the acceptance criteria for intraday (accuracy: 100.63–110.46%, imprecision: 6.23–7.76%), as well as interday assay (accuracy: 98.81–110.63%; imprecision: 5.36–10.22%). The method was used for free MPA determination in plasma samples from patients treated with mycophenolate mofetil. To the best of our knowledge this is the first liquid chromatography–tandem mass spectrometry method for free MPA monitoring using MPA‐d3 that allows to measure plasma ultrafiltrate concentrations as low as 1 ng/mL.