Comparative evaluation of new Cookson‐type reagents for LC/ESI‐MS/MS assay of 25‐hydroxyvitamin D3 in neonatal blood samples

The screening of vitamin D deficiency in neonatal infants, which is based on the blood 25‐hydroxyvitamin D₃ [25(OH)D₃] quantification, is important for the early detection, diagnosis and health risk assessment of several diseases. In this study, two new Cookson‐type reagents, 4‐(4‐diethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DEAPTAD) and 4‐(6‐quinolyl)‐1,2,4‐triazoline‐3,5‐dione, were designed and synthesized, then compared with the previous reagents, 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) and 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD), in terms of sensitivity and specificity in the assay of 25(OH)D₃ in neonatal blood samples by liquid chromatography/electrospray ionization–tandem mass spectrometry. Among the reagents, DEAPTAD was found to be the most promising. The limit of detection (0.38 fmol on the column) of the DEAPTAD‐derivatized 25(OH)D₃ was 60 and 2 times lower than those of the intact 25(OH)D₃ and the PTAD derivative, respectively. 25(OH)D₃ was more clearly detected in the plasma sample as the DEAPTAD derivative than the DAPTAD derivative owing to the lower background noise. DEAPTAD derivatization was also useful for the separation of 25(OH)D₃ from a potent interfering metabolite, 3‐epi‐25‐hydroxyvitamin D₃. By using DEAPTAD, a trace amount of 25(OH)D₃ in dried blood spots was reproducibly determined without interference from coexisting compounds. Thus, DEAPTAD was proved useful in the measurement of 25(OH)D₃ in neonatal blood samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Supported liquid extraction in combination with LC‐MS/MS for high‐throughput quantitative analysis of hydrocortisone in mouse serum

A high‐throughput LC–MS/MS bioanalytical method was developed and validated for the determination of hydrocortisone in mouse serum via supported liquid extraction (SLE) in a 96‐well plate format. Although sample extracts from SLE result in similar matrix effects compared with conventional liquid–liquid extraction (LLE), greater analyte extraction recovery and much higher analysis throughput for the quantitative analysis of hydrocortisone in mouse serum were obtained. The current LC‐MS/MS method was validated for a concentration range of 2.00–2000 ng/mL for hydrocortisone using a 0.100 mL volume of mouse serum. The intra‐ and inter‐day precision and accuracy of the quality control samples at low, medium and high concentration levels showed ≤12.9% CV and ?3.4–6.2% bias for the analyte in mouse serum. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid MCM‐41 dispersive micro‐solid phase extraction coupled with LC/MS/MS for quantification of ketoconazole and voriconazole in biological fluids

A rapid dispersive micro‐solid phase extraction (D‐μ‐SPE) combined with LC/MS/MS method was developed and validated for the determination of ketoconazole and voriconazole in human urine and plasma samples. Synthesized mesoporous silica MCM‐41 was used as sorbent in d ‐μ‐SPE of the azole compounds from biological fluids. Important D‐μ‐SPE parameters, namely type desorption solvent, extraction time, sample pH, salt addition, desorption time, amount of sorbent and sample volume were optimized. Liquid chromatographic separations were carried out on a Zorbax SB‐C₁₈ column (2.1 × 100 mm, 3.5 μm), using a mobile phase of acetonitrile–0.05% formic acid in 5 mm ammonium acetate buffer (70:30, v /v). A triple quadrupole mass spectrometer with positive ionization mode was used for the determination of target analytes. Under the optimized conditions, the calibration curves showed good linearity in the range of 0.1–10,000 μg/L with satisfactory limit of detection (≤0.06 μg/L) and limit of quantitation (≤0.3 μg/L). The proposed method also showed acceptable intra‐ and inter‐day precisions for ketoconazole and voriconazole from urine and human plasma with RSD ≤16.5% and good relative recoveries in the range 84.3–114.8%. The MCM‐41‐D‐μ‐SPE method proved to be rapid and simple and requires a small volume of organic solvent (200 μL); thus it is advantageous for routine drug analysis.     

Determination of main taxoids in Taxus species by microwave‐assisted extraction combined with LC‐MS/MS analysis

A method based on microwave‐assisted extraction (MAE) has been developed for the determination of paclitaxel and five related taxoids, namely 10‐deacetylbaccatin III (10‐DAB III), cephalomannine, 10‐deacetylpaclitaxel (10‐DAT), 7‐xyl‐10‐ deacetylpaclitaxel (7‐xyl‐10‐DAT), and 7‐epi‐10‐deacetylpaclitaxel (7‐epi‐10‐DAT) in Taxus species in this study. The influential parameters of the MAE procedure were optimized, and the optimal conditions were as follows: extraction solvent 80% ethanol solution, solid/liquid ratio 1:10 (g/mL), temperature 50°C, and three extraction cycles, each cycle 10 min. The method validation for LC‐MS/MS analysis was performed. The LOD and LOQ were 3.16–9.20 and 12.20–30.45 ng/mL, respectively. Repeatability and reproducibility for the six taxiods with RSD ranged from 2.78 to 3.85% and from 5.26 to 6.60%. The recoveries of the method for the six taxoids were 92.6–105.6%. The developed MAE‐LC‐MS/MS method was also successfully applied to determine the contents of six taxoids in different Taxus species.     

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D₃ [25(OH)D₃, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D₃ in ESI‐MS/MS and to separate 25(OH)D₃ from 3‐epi‐25‐hydroxyvitamin D₃ [3‐epi‐25(OH)D₃], a potent interfering metabolite. 25(OH)D₃ was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB^® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D₄ was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D₃ in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D₃.     

LC‐MS/MS determination of bakkenolide D in rats plasma and its application in pharmacokinetic studies

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determination of bakkenolide D (BD), which was further applied to assess the pharmacokinetics of BD. In the LC‐MS/MS method, the multiple reaction monitoring mode was used and columbianadin was chosen as internal standard. The method was validated over the range of 1–800 ng/mL with a determination coefficient >0.999. The lower limit of quantification was 1 ng/mL in plasma. The intra‐ and inter‐day accuracies for BD were 91–113 and 100–104%, respectively, and the inter‐day precision was <15%. After a single oral dose of 10 mg/kg of BD, the mean peak plasma concentration of BD was 10.1 ± 9.8 ng/mL at 2 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 72.1 ± 8.59 h ng/mL, and the elimination half‐life (T_1/2) was 11.8 ± 1.9 h. In case of intravenous administration of BD at a dosage of 1 mg/kg, the AUC_{0–24 h} was 281 ± 98.4 h?ng/mL, and the T_1/2 was 8.79 ± 0.63 h. Based on these results, the oral bioavailability of BD in rats at 10 mg/kg is 2.57%. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi‐automated LC/LC‐MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back‐flushed onto the analytical column. Ion transitions [M + H]⁺ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter‐day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi‐automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25‐hydroxyvitamin and 1,25‐dihydroxyvitamin D₃. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D₃ in human plasma. A method for the measurement of 25‐hydroxyvitamin D₃ using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125‐4, Purospher RP‐18e, 5 μm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra‐ and inter‐assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0–103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25‐hydroxyvitamin D₃ in a group of blood donors is 62 ± 26 nmol/L.     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.     

A simple and sensitive LC‐MS/MS method for the determination of sotalol in rat plasma

A sensitive, rapid and robust HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the quantification of sotalol in rat plasma. Plasma samples were precipitated with acetonitrile before analysis. The chromatographic separation was performed on an Atlantis hydrophilic interaction liquid chromatography Silica column (50 × 2.1 mm, 3 µm) with a gradient mobile phase of 10 mm NH₄COOH (containing 0.2% of formic acid) as buffer A and acetonitrile as mobile phase B. Sotalol (m/z 273.2 → 255.1) and atenolol (the internal standard, IS, m/z 267.2 → 190.1) were monitored under positive ionization mode with 5500 QTRAP. Retention time of sotalol and the IS were 2.69 and 3.43 min, respectively. The linear range was 5–500 nm based on the analysis of 0.1 mL of plasma. The intrabatch precision ranged from 1.2 to 6.1%, and the inter‐batch precision was from 3.3 to 6.5%. The coefficient of variation of IS‐normalized matrix factor was 7.6%. Experiments for stability were performed and the analyte was sufficiently stable. A run time of 6 min for each injection made it possible to analyze a high throughput of plasma samples. The assay was successfully applied to the determination of sotalol in rat plasma after a micro‐dose oral administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Two‐dimensional LC‐MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver

A two‐dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse‐phase (RP) liquid chromatography (LC) system coupled with triple‐quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC‐RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C₁₈ column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC‐MS/MS system. Limits of detection of 2D LC‐MS/MS system were 2‐ to 3‐fold lower compared with one‐dimensional RPLC‐MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures‐discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC‐MS/MS is a promising method to assist lipidomic studies of complex biological samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of three capsaicinoids in Capsicum annuum by pressurized liquid extraction combined with LC‐MS/MS

A new method based on pressurized liquid extraction followed by LC‐MS/MS analysis has been developed for the identification and quantification of three capsaicinoids (capsaicin, dihydrocapsaicin, and nordihydrocapsaicin) in extracts of Capsicum annuum. For the recovery of three capsaicinoids, the efficiency levels of ultrasonic‐assisted extraction, microwave‐assisted extraction, Soxhlet extraction, and pressurized liquid extraction were compared under different conditions. Pressurized liquid extraction resulted in higher yields. Pressurized liquid extractions were performed using methanol; temperature was set at 100°C and pressure at 1500 psi. LC analysis was performed on a Waters XBridge? C₁₈ column (150 × 2.1 mm, id 3.5 μm) eluted by a mobile phase of 0.1% formic acid and ACN. Data acquisition was carried out in multiple reaction monitoring transitions mode, monitoring two‐reaction monitoring transitions to ensure an accurate identification of target compounds in the samples. The proposed method is rapid, simple, and could be utilized for the routine analysis of three capsaicinoids in C. annuum samples.     

Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of hexabromocyclododecane isomers in sewage sludge by LC‐MS/MS

A method has been developed to determine α, β and γ diastereoisomers of hexabromocyclododecane (HBCD), a brominated flame retardant, in sewage sludge, based on the ultrasonic‐assisted extraction (UAE) of samples with dichloromethane–ACN (1:1) and the subsequent clean‐up of extracts by dispersive solid phase extraction with primary–secondary amine. Levels of HBCD diastereoisomers were determined by LC coupled with ESI MS/MS. Evaluation of the matrix effect showed a high ion suppression for all the diastereoisomers studied, which was counteracted by using a mixture of labelled HBCD diastereoisomers as internal standards. This method yielded recoveries in the range of 79.6–112.5% with SDs equal or lower than 9.1 The limits of detection were 0.3 ng/g for α‐ and β‐HBCD and 0.2 ng/g for γ‐HBCD. The developed method was successfully applied to 19 sludge samples collected from the province of Madrid (Spain). In most of the samples, β‐HBCD was below the method detection limit, whereas α‐ and γ‐HBCD were quantified in all of the sludge samples, and γ‐HBCD was the predominant diastereoisomer in 63% of the analyzed samples and α‐HBCD predominated in the rest.     

A simple LC‐MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development,validation and application in the CUMS rat model

Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC‐MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4‐aminohippurate, 5‐HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25–12,800 ng mL^?1 with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research. Copyright © 2015 John Wiley & Sons, Ltd.     

LC‐MS/MS analysis of Δ9‐tetrahydrocannabinolic acid A in serum after protein precipitation using an in‐house synthesized deuterated internal standard

An assay based on liquid chromatography/tandem mass spectrometry is presented for the fast, precise and sensitive quantitation of Δ9‐tetrahydrocannabinolic acid A (THCA) in serum. THCA is the biogenetic precursor of Δ9‐tetrahydrocannabinol in cannabis and has aroused interest in the pharmacological and forensic field especially as a potential marker for recent cannabis use. After addition of deuterated THCA, synthesized from D₃‐THC as starting material, and protein precipitation, the analytes were separated using gradient elution on a Luna C18 column (150 × 2.0 mm × 5 µm) with 0.1% formic acid and acetonitrile/0.1% formic acid. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode with negative electrospray ionization. After optimization, the following sample preparation procedure was used: 200 μL serum was spiked with internal standard solution and methanol and then precipitated ‘in fractions’ with 500 μL ice‐cold acetonitrile. After storage and centrifugation, the supernatant was evaporated and the residue redissolved in mobile phase. The assay was fully validated according to international guidelines including, for the first time, the assessment of matrix effects and stability experiments. Limit of detection was 0.1 ng/mL, and limit of quantification was 1.0 ng/mL. The method was found to be selective and proved to be linear over a range of 1.0 to 100 ng/mL using a 1/x weighted calibration model with regression coefficients >0.9996. Accuracy and precision data were within the required limits (RSD ≤ 8.6%, bias: 2.4 to 11.4%), extractive yield was greater than 84%. The analytes were stable in serum samples after three freeze/thaw cycles and storage at ?20 °C for one month. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC–MS/MS method

Fat‐soluble vitamins play a pivotal role in the progression of atherosclerosis and the development of cardiovascular disease. Therefore, plasma monitoring of their concentrations may be useful in the diagnosis of these disorders as well as in the process of treatment. The study aimed to develop and validate an HPLC–MS/MS method for determination of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease. The analytes were separated on an HPLC Kinetex F5 column via gradient elution with water and methanol, both containing 0.1% (v/v) formic acid. Detection of the analytes was performed on a triple‐quadrupole MS with multiple reaction monitoring via electrospray ionization. The analytes were isolated from plasma samples with liquid–liquid extraction using hexane. Linearity of the analyte calibration curves was confirmed in the ranges 0.02–2 μg/mL for retinol, 0.5–20 μg/mL for α‐tocopherol, 5–100 ng/mL for 25‐hydroxyvitamin D2 and 2–100 ng/mL for 25‐hydroxyvitamin D3. Intra‐ and inter‐assay precision and accuracy of the method were satisfactory. Short‐ and long‐term stabilities of the analytes were determined. The HPLC‐MS/MS method was applied for the determination of the above fat‐soluble vitamin concentrations in patient plasma as potential markers of the cardiovascular disease progression.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by LC‐MS/MS using supported liquid extraction

A rapid, sensitive and selective bioanalytical method was developed for the simultaneous determination of fluoxetine and its primary metabolite norfluoxetine in human plasma. Sample preparation was based on supported liquid extraction (SLE) using methyl tert‐butyl ether to extract the analytes from human plasma. Chromatography was performed on a Synergi 4 μ polar‐RP column using a fast gradient. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, m/z 310 → 44 for fluoxetine, m/z 296 → 134 for norfluoxetine and m/z 315 → 44 for fluoxetine‐d₅ (internal standard). The method is linear over the range of 0.05–20 ng/mL (using a human plasma sample volume of 0.1 mL) with a coefficient determination of greater than 0.999. The method is accurate and precise with intra‐batch and inter‐batch accuracy (%bias) of <±15% and precision (%CV) of <15% for both analytes. A run time of 4 min means a high throughput of samples can be achieved. To our knowledge, this method appears to be the most sensitive one reported so far for the quantitation of fluoxetine and norfluoxetine and can be used for routine therapeutic drug monitoring or pharmacokinetic studies. Copyright © 2011 John Wiley & Sons, Ltd.