Main Metabolites In Plasma Research Articles

Tamoxifen in horses: pharmacokinetics and safety study

BackgroundTamoxifen (TAM), a selective modulator of estrogen receptors (SERMs) has been recently explored as a therapeutic option for the oral treatment of airway inflammation in the horse. The objective of this work was to establish pharmacokinetic parameters of TAM and its main metabolites in equines, as well as to determine its clinical safety in short-term treatments.ResultsWe determined TAM and its three main metabolites (4-OH tamoxifen, endoxifen, and N-desmethyl tamoxifen) in plasma after single administration of 0.25 mg/kg in healthy adult horses (n = 12). A maximum concentration of TAM was achieved 3 h after the oral administration (4.65 pg/mL ± 1.69); 4-OH tamoxifen was the metabolite that reached the highest concentration (78 pg/mL ± 70), followed by N-desmethyl tamoxifen (0.43 pg / mL ± 0.48), and finally endoxifen (0.17 pg/mL ± 0.17). All metabolites showed peak concentration 2 h after oral administration of the drug. Oral TAM bioavailability was 13,15% ± 4,18, with a steady state volume of distribution of 7831 ± 2922 (L/kg). Elimination half-life was 15.40 ± 5.80 h, and clearance was 5876 ± 699 (mL/kg/min). Clinical safety of TAM was determined over a 7-day course of treatment (0.25 mg/kg, orally q 24 h, n = 20). No adverse effects were observed through clinical examination, blood hematology, serum biochemistry, ophthalmological and reproductive examinations. Endometrial edema observed in some mares was attributed to normal cyclic activity.ConclusionsTamoxifen has moderate oral bioavailability and a large volume of distribution, with three main metabolites in horses. Additionally, oral TAM administration over a 7-day treatment period demonstrated to be clinically safe, without adverse effects on clinical, hematological or serum biochemical parameters. These data could contribute to the continued research into this drug’s potential for the treatment of different inflammatory conditions in equine species.

Characterization of TPN171 metabolism in humans via ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

TPN171 is a novel potent pyrimidinone phosphodiesterase type 5 (PDE5) inhibitor with high selectivity and long duration of action. It has been used to treat patients suffering from pulmonary arterial hypertension and entered phase I clinical trials in 2016. Considering the potential therapeutic value of TPN171, its metabolism in humans is necessary to be elucidated during early-stage of drug development. This study aimed to establish a rapid and reliable method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry for the characterization of TPN171 metabolites in human plasma, urine and stool samples. A total of 17 metabolites, including 14 from phase I and 3 from phase II metabolic reactions, were identified and characterized. TPN171 was found to be the predominant component in all plasma, urine and stool samples. Seven proposed metabolites were validated by comparing with synthetic reference standards. N-demethylation, O-depropylation, N-oxidation and dehydrogenation were demonstrated to be the main metabolic pathways of TPN171 in humans, yielding metabolites M4, M3, M7-2 and M5-3, respectively. Notably, M5-3 (a dehydrogenation product) and M3 (an O-depropylation product) were the main metabolites in human plasma while M5-3 (produced via dehydrogenation) and M7-2 (produced via N-oxidation) were the major metabolites in human urine. Besides, O-depropylation product M3 and N-demethylation product M4 were the main metabolites in human stool. In overall, this study assessed the metabolic fate of TPN171 in humans, which may yield considerably benefits for subsequent studies focusing on TPN171 metabolism and development of other PDE5 inhibitors.

Determination of BPI15086 and its metabolite in human plasma by ultra-high performance liquid chromatography-MS/MS and its application to a pharmacokinetic study.

Aim: BPI15086 is a potent, irreversible mutant-selective inhibitor of both EGFR (EGFR tyrosine kinase inhibitor) and the T790M resistance mutations tyrosine kinase. A simultaneous quantification method of BPI15086 and its main metabolite in human plasma using LC-MS/MS is documented and fully validated in this study. Methodology & results: Plasma samples were extracted and chromatographed on an Acquity ultra-high performance liquid chromatography BEH C18 column with a gradient elution. Detection was performed on a Sciex 5500 QTRAP® mass spectrometer using positive electrospray ionization. The results indicated that the method had excellent sensitivity and specificity. Conclusion: For the first time a sensitive and robust ultra-high performance liquid chromatography-MS/MS method was established and validated of BPI15086 in human plasma, this method was successfully applied in a first-in-human Phase I clinical trial studying the pharmacokinetics of the BPI15086 tablet in Chinese non-small-cell lung cancer patients.

Validated Microemulsion Liquid Chromatography-Fluorescence Method for the Quantification of Duloxetine and its Two Main Metabolites in Plasma: Application to Clinical Pharmacokinetic Studies

Background: Duloxetine (DL) is a selective serotonin and norepinephrine reuptake inhibitor. The drug is used in the treatment of major depression, anxiety, pain related to diabetic peripheral neuropathy and stress urinary incontinence. </P><P> Objective: This study described, for the first time, the development and validation of a highly selective and sensitive microemulsion liquid chromatography-fluorescence (MELC-FL) method with low environmental pollution and without extraction steps for the simultaneous quantification of DL, and its two main metabolites; 5-hydroxy-6-methoxy duloxetine (5-HDL) and 4-hydroxy duloxetine glucuronide (4- HDLG) in plasma. Methods: The studied analytes and methyl paraben (an internal standard) were detected using excitation and emission wavelengths of 280 and 340 nm, respectively. The analysis was performed on Water Symmetry C18 analytical column (100 Å, 150 mm x 3.9 mm, 5 µm) by directly injecting the plasma after appropriate dilution with microemulsion mobile phase. Total analytical run time was 4 min. Results: The MELC-FL method was statistically validated according to the FDA guidelines for bioanalytical methods for linearity, accuracy, precision, specificity, robustness, and stability. Linear calibration plots were achieved in the ranges of 25-1200 ng/mL for DL and 50-1500 ng/mL for 5-HDL and 4- HDLG (r2 ≥ 0.997) in rat plasma. The intra- and inter- assay precisions and accuracy were acceptable. The overall recoveries of DL and its two main metabolites from rat plasma were between 97.12% and 103.12% with an RSD value between 0.34% and 4.57%. Conclusion: The present study supports the possible use of the microemulsion mobile phase in LC as a “greener ” mobile phase. The developed method offered an advantage in the form of direct analysis of biological samples after appropriate dilution with eco-friendly microemulsion mobile phase, which decreased the possibility of sample loss during analysis. The developed assay was successfully applied in a pharmacokinetic study and it established the applicability of the method for the determination of concentration-time profiles of DL and its two main metabolites in rat plasma after systemic administration.

A validated UPLC-MS/MS method to determine free and total irinotecan and its two metabolites in human plasma after intravenous administration of irinotecan hydrochloride liposome injection

Irinotecan hydrochloride liposome injection (IHLI) is a formulation of anticancer drug irinotecan hydrochloride (CPT-11) entrapped in the aqueous core of liposomes. To understand the pharmacokinetic property and evaluate the relationship between pharmacokinetics and pharmacodynamics/toxicity of IHLI, it is of prime importance to determine the concentrations of free CPT-11, total CPT-11 and its main metabolites (SN-38 and SN-38 G) in human plasma. In this paper, we developed and validated a sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to quantify the concentrations of these related substances in human plasma. Free CPT-11, SN-38 and SN-38 G in human plasma were simultaneously separated and extracted by 96-well solid phase extraction (SPE) plate, while total CPT-11 was extracted by protein precipitation (PP) method. The analytes were chromatographed on an Acquity UPLC BEH C18 column and then detected on a Xevo TQ-S tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The UPLC-MS/MS method combined with SPE and PP techniques were fully validated in line with existing guidelines issued by regulatory agencies. In brief, all the analytes achieved a satisfactory selectivity and sensitivity in this method. The calibration curves were proved to be linear over the concentration range of 10–10000 ng/mL for total CPT-11, 0.5–1000 ng/mL for free CPT-11, 0.5–200 ng/mL for SN-38 and SN-38 G, respectively. For all the analytes, the intra- and inter-run precisions were less than 11.4% and the accuracies (in terms of RE%) were within -7.7–7.3% except for accuracies of LLOQs were within -15.8 to 7.2%. Besides, carry-over, extraction recovery, matrix effect, dilution integrity, stability and special matrices were also assessed. Finally, the method was successfully applied to a phase I clinical pharmacokinetic study of IHLI in Chinese subjects with advanced solid tumors.

New insights into antioxidant activity of Prunus spinosa flowers: Extracts, model polyphenols and their phenolic metabolites in plasma towards multiple in vivo-relevant oxidants

Prunus spinosa L. flowers are a traditional herbal medicine recommended for the adjunctive treatment of oxidative stress-related diseases. In search of the main bioactive constituents of the flowers, the antioxidant activity of dry extracts obtained thereof by fractionated extraction was tested in vitro towards primary in vivo-relevant reactive oxygen species (O2–, OH, NO, H2O2, ONOO–, HOCl) and juxtaposed with the activity of eight model native polyphenols and five compounds considered to be their main metabolites in human plasma. All extracts exhibited concentration- and phenolic-dependent scavenging potential towards the tested oxidants, and the highest responses were observed for O2–, OH, ONOO–, and HOCl. The total antioxidant capacity of the extracts towards the six oxidants, expressed as sum of ascorbic acid (AA) equivalents, was in the range of 4.8–29.2 μmol AA/mg dw. The most active model polyphenols were quercetin, avicularin, procyanidin A2 and chlorogenic acid (44.5–72.5 μmol AA/mg), while the prevalent in the extracts kaempferol derivatives exhibited relatively weak capacity (11.8–27.0 μmol AA/mg). Among the metabolites, dihydrocaffeic acid (68.8 μmol AA/mg) and 2-(3′,4′-dihydroxyphenyl)acetic acid (63.2 μmol AA/mg) were particularly active – in some tests exhibited stronger activity than their parent compounds. The work gives an insight into the role of native and metabolised phenols as contributory factors to systemic activity of flower extracts and proposes the analytical markers for their standardisation.

Determination of valproic acid and 3-heptanone in plasma using air-assisted liquid-liquid microextraction with the assistance of vortex: Application in the real samples.

Introduction: Valproic acid (VPA) is an antiepileptic drug used to treat epilepsy and bipolar disorder. Adverse effects of VPA were studied in many reports, however, a dose-response relationship between VPA and its metabolites in epilepsy patients are extremely limited. In this paper, a high efficient method was developed for the preconcentration and determination of VPA and its main metabolite in plasma. Methods: For the extraction and preconcentration of the selected analytes, a volume of an extractant was placed at the bottom of the microtube containing pretreated plasma. The mixture was repeatedly withdrawn from the microtube and pushed-out into it using a 1.0-mL glass syringe and resulted in a cloudy mixture. For further turbidity, the mixture was shaken on a vortex agitator. This procedure was used to analyze the plasma samples of patients with epilepsy (n = 70). Results: The results revealed that in most patients with a low level of VPA relative to its expected level, 3-heptanone concentrations were high. The limits of quantification of 3-heptanone and VPA were 0.04 mg L–1 and 0.2 mg L–1, respectively. A suitable precision at a concentration of 2 mg L-1 for each analyte was obtained (relative standard deviation ≤ 9%). Conclusion: The obtained results indicated that this procedure is easy, sensitive, and reliable, and can be used for the analysis of the selected analytes in the plasma samples of patients with epilepsy.

Simultaneous determination of cinobufagin and its five metabolites in rat plasma by LC-MS/MS for characterization of metabolic profiles and pharmacokinetic study

Cinobufagin, an ingredient ofVenenum Bufonishas various pharmacological activities and toxicities. In this study we simultaneously determine cinobufagin (quantification) and its five main metabolites (semi-quantification) in rat plasma.

Rapid characterization of chemical constituents and rat metabolites of Fufang Gancao Tablets by UHPLC-LTQ-Orbitrap mass spectrometer

Ultra-high-performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HR-MSn ) method was used to analyze the constituents of Fufang Gancao Tablets and its main metabolites in rat plasma. Rat plasma was collected both before and after oral administration of Fufang Gancao Tablets. After solid phase extraction, ACQUITY UHPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. The chemical components in Fufang Gancao Tablets and their prototypes and metabolites in plasma samples were analyzed by LTQ-Orbitrap equipped with an ESI ion source in a positive ion mode. Based on the accurate mass measurements, the retention time and mass fragmentation patterns, a total of 55 compounds were tentatively identified from Fufang Gancao Tablets, including 42 flavonoids, 9 triterpenes and 4 alkaloids. Furthermore, metabolites in rat plasma after oral administration of Fufang Gancao Tablets were also analyzed. A total of 26 compounds were identified, including 20 prototypes and 6 metabolites mainly through metabolic pathways of hydroxylation, glucuronide conjugation, and sulfate conjugation, etc. Our results showed that the ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap) could comprehensively elucidate the chemical constituents of Fufang Gancao Tablets and their migrating components in rat plasma, providing scientific basis for further studying the metabolism process and pharmacodynamic substance of Fufang Gancao Tablets.

Simultaneous determination of valproic acid and its main metabolite in human plasma using a small scale dispersive liquid–liquid microextraction followed by gas chromatography–flame ionization detection

A simple, sensitive, and rapid analytical method has been developed and validated for the extraction and quantification of valproic acid and its main metabolite (3-heptanone) in human plasma. Initially, the proteins of plasma were precipitated with trifluoroacetic acid. Then a very small volume of a water-immiscible extractant and acetonitrile was mixed and rapidly injected into the pre-treated plasma sample. For further turbidity (dispersion of the extractant into sample solution), the cloudy solution was vortexed. After centrifugation, the settled phase was injected into gas chromatography-flame ionization detection. The effective parameters, such as type and volume of extraction and disperser solvents, vortex time, and pH were studied and optimized. The limits of detection of valproic acid and 3-heptanone were obtained, 0.065 and 0.015 mg L−1, respectively. An acceptable precision was obtained for a concentration of 2 mg L−1 of each analyte (relative standard deviation ≤ 8%). The average absolute recoveries (n = 3) of valproic acid and 3-heptanone were 52 ± 2 and 42 ± 1%, respectively. The validated method has been successfully used in analysis of the analytes in human plasma samples.

Pharmacokinetics and tissue distribution of eupatilin and its metabolite in rats by an HPLC-MS/MS method

Eupatilin, a major pharmacologically active ingredient in StillenTM, has been known to possess anti-peptic, anti-cancer and anti-allergy activities. A rapid, simple, sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of eupatilin and its main metabolite (eupatilin-7β-O-glucuronide, E-7-G) in rat plasma and tissues was established and validated. The linear range of eupatilin and E-7-G was 0.20∼500 ng/mL and 1.00–2500 ng/mL, and the lowest limit of quantification (LLOQ) of eupatilin and E-7-G was 0.20 and 1.00 ng/mL, respectively. The inter-day and intra-day precision of this assay was restricted to within 10%, with a highest accuracy of more than 90%. The matrix effect, recovery and stability of both eupatilin and E-7-G were all demonstrated to be within acceptable limits. The validated method was then successfully applied to a pharmacokinetics and tissue distribution study. The absolute bioavailability (F) of eupatilin was estimated to be 2.7%. After intravenous administration, eupatilin was degraded with high clearance (14.82 L/kg/h) and a short half-life t1/2 (0.29 h). Eupatilin was rapidly metabolized to E-7-G with systemic exposure at 1288.8 ng h ml-1, while the levels of the latter declined more slowly, with a longer t1/2 (4.15 h). Moreover, both eupatilin and E-7-G were widely distributed across various tissues, including the liver, kidney and intestine. Taken together, eupatilin showed poor absorption, extensive metabolism into E-7-G and a wide tissue distribution, especially in the intestine. These pharmacokinetic results yield helpful insights into the pharmacological actions of eupatilin.

Development and validation of an analytical method for regorafenib and its metabolites in mouse plasma

An analytical method was developed for measuring the effect of OATP1B2 deficiency on plasma levels of the kinase inhibitor regorafenib and its metabolites regorafenib-N-oxide, N-desmethyl-regorafenib-N-oxide, and regorafenib-N-β-glucuronide (RG) in mice. Compounds were separated by liquid chromatography and monitored by a triple quadrupole mass spectrometer in the selected reaction monitoring mode after positive electrospray ionization. All calibration curves were linear in the selected concentration range (R2 ≥ 0.99). The lower limit of quantification was 5 ng/mL for the four analytes. Within-day precisions, between-day precisions, and accuracies were 2.59–6.82%, 3.97–11.3%, and 94.5–111%, respectively. The identification and structure elucidation of RG, isolated from human urine, was performed by NMR. Compared with wild-type mice given regorafenib (10 mg/kg), deficiency of the drug transporter OATP1B2 in vivo had minimal effects on plasma levels of parent drug and the metabolite regorafenib-N-oxide, and N-desmethyl-regorafenib-N-oxide. However, the area under the curve and peak levels of RG were increased by 5.6-fold and 5.1-fold, respectively, in OATP1B2-knockout mice. In conclusion, our analytical method allowed accurate and precise quantitation of regorafenib and its main metabolites in mouse plasma, and is suitable for evaluation of transporter-dependent pharmacokinetic properties of these agents in vivo.

Simultaneous determination of tianeptine and its active metabolite tianeptine MC5 in rat plasma and brain tissue using high performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

Validated LC-ESI-MS/MS method for tianeptine and its main metabolite in plasma and brain.

Simultaneous quantification of endothelin receptor antagonists and phosphodiesterase 5 inhibitors currently used in pulmonary arterial hypertension

Combination treatment with endothelin receptor antagonists (ERA) and phosphodiesterase 5 inhibitors (PDE5I) improved efficacy of pulmonary arterial hypertension (PAH) therapy. However, drug–drug interactions, variable exposure, non-adherence can influence plasma levels. For these reasons, drug quantification may be advantageous particularly in patients with poor treatment responses. We developed, validated, and applied an assay for the simultaneous quantification of ambrisentan, bosentan, macitentan, sildenafil, and tadalafil as well as their main (and partly active) metabolites in human plasma. This method is based on LC–MS/MS separation for a rapid and sensitive quantification with stable isotopically labelled analogues as internal standards for each drug and metabolite. Sample preparation was carried out using a solid phase extraction protocol based on Oasis HLB material. The separation was achieved on a Kinetex C18 column and multiple reaction monitoring in negative ionization mode was used for sensitive detection. The calibrations were linear for all analytes with correlation coefficients >0.99 within the concentration range observed under a therapeutic PAH dosing scheme with lower limits of quantification between 0.34ng/mL (OH-ambrisentan) and 10ng/mL (despropyl-macitentan). Intra- and inter-day precision at LLOQ and QC levels ranged between 2.03% and 19.8%, and 0.65% and 14.0%, respectively. The sample turnover time was 12min. The applicability of this versatile LC/MS/MS assay was verified by the successful analysis of clinical routine samples of patients on PAH medication. This new method allows for the first time to assess trough drug and metabolite levels of the currently approved PDE5I and ERAs in PAH patients, thus enabling for measurement of samples in clinical routine.

A novel LC–MS/MS method for the simultaneous quantification of topiramate and its main metabolites in human plasma

The aim of the present report was to develop and validate simple, sensitive and reliable LC–MS/MS method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM, 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and afterwards used as an authentic standard for method development and validation. Sample preparation method employs 100μL of plasma sample and liquid–liquid extraction with a mixture of ethyl acetate and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using Kinetex C18 column (50×2.1mm, 2.6μm) by gradient elution using water and methanol as a mobile phase and stable isotope labeled TPM as internal standard. The method showed to be selective, accurate, precise and linear over the concentration ranges of 0.10–20μg/mL for TPM, 0.01–2.0μg/mL for 2,3-desisopropylidene TPM, and 0.001–0.200μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM. The described method is the first fully validated method capable of simultaneous determination of TPM and its main metabolites in plasma over the selected analytical range. The suitability of the method was successfully demonstrated by the quantification of all analytes in plasma samples of patients with epilepsy and can be considered as reliable analytical tool for future investigations of the TPM metabolism.

UPLC-MS/MS method for the determination of tobacco-specific biomarker NNAL, tamoxifen and its main metabolites in rat plasma.

Cigarette smoke is known to interact with tamoxifen-metabolizing enzymes and transporters and potentially affect its treatment outcome. 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important metabolite of 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) because it is frequently used as a biomarker to assess human smoke exposure. In order to study the potential pharmacokinetic interaction between cigarette smoke and tamoxifen in rats a UPLC-MS/MS method for the simultaneous determination of NNAL and tamoxifen along with its metabolites in rat plasma has been developed and validated. Analytes were extracted with methanol and separated on a HSS T3 column by a gradient elution with the mobile phase consisting of acetonitrile and water. The lower limits of quantitation ranged from 0.05 to 0.62 ng/mL. Precisions showed RSD <15.8% and accuracy in the range 80.6-116.0%. Mean analyte recoveries ranged from 76.9 to 108.4%. The method was successfully applied to study the effects of cigarette smoke condensate (CSC), NNK and benzo(a)pyrene pre-treatment on the pharmacokinetics of tamoxifen and its metabolites in rats. Significant effects of CSC, NNK, benzo(a)pyrene were observed on pharmacokinetics of tamoxifen and its metabolites. We also found that plasma NNAL levels are statistically significant correlated with plasma 4-hydroxy-tamoxifen and endoxifen.

Determination of pharmacokinetics of fisetin and its main metabolite in mouse plasma by LC–MS/MS

Determination of pharmacokinetics of fisetin and its main metabolite in mouse plasma by LC–MS/MS

Development of LC–MS/MS Method for Simultaneous Determination of Irinotecan and Its Main Metabolites in Rat Plasma, and Its Application in Pharmacokinetic Studies

The anticancer agent irinotecan (CPT-11) is widely used in several cancer regimens. However, drug–drug interaction effects and considerable interindividual variability in the pharmacokinetics of CPT-11 and its metabolites have been observed, leading to side effects. To solve these problems and promote CPT-11 treatment, we established and validated a simple, rapid, and sensitive LC–MS/MS method for the simultaneous quantitative determination of CPT-11 and its main metabolites (SN-38, SN-38G, and APC). Protein precipitation with methanol was selected for sample preparation. All separations were performed on an Agilent ZORBAX Eclipse XDB-C18 column (2.1 × 50 mm, 3.5 μm) under isocratic elution with a mobile phase consisting of acetonitrile and 0.1% formic acid. The mass spectrometer was operated with multiple reaction monitoring in positive ion mode. The method was validated as linear over the concentration ranges 16.9–6760 ng mL−1 for CPT-11, 2.3–920 ng mL−1 for SN-38, 2.5–1000 ng mL−1 for SN-38G, and 1.25–250 ng mL−1 for APC. The intra- and interbatch precisions and accuracies of the validated method were within acceptable limits (<15%). This method was successfully applied to a pharmacokinetic study of CPT-11 and its main metabolites in rats.

Development and validation of a quantitative assay for the determination of cinacalcet and its main metabolites in human plasma using RP-HPLC method

A novel and sensitive methodology for the quantitative determination of cinacalcet (CIN), and its main metabolites (M2a-Glu, M2b-Glu, M5, and M6) in plasma sample is suggested. The analytical assay is based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. The method has been fully validated at range covering the plasma concentrations in patients receiving therapeutic dosages of CIN. In this investigation, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (NI-ETFMS) was used as a pre-column derivation agent according to interaction of amine and carboxylate groups of CIN and its metabolites with NI-ETFMS. The sample pretreatment consists of protein precipitation with acetonitrile using only 100μL of plasma, and chromatographic separation was achieved in isocratic mode on Kinetex C18 100Å column with mobile phase of acetonitrile, methanol, and 50mM phosphate buffer (40:20:40v/v). The analytical method was fully validated in terms of selectivity, linear range (0.2 to 5.5ngmL−1), linearity (r2>0.997), sensitivity (LOD=0.05 to 0.75ngmL−1 and LOQ=0.36 to 2.52ngmL−1), intra and inter-days accuracy (−4.6 to 8.1%), precision (<7.4%), and robustness (<3.5%). As results, the proposed method can be useful in the routine analysis for the determination of CIN and its main metabolites in human plasma samples.

Sensitive determination of THC and main metabolites in human plasma by means of microextraction in packed sorbent and gas chromatography-tandem mass spectrometry.

Cannabis is one of the most available and consumed illicit drug in the world and its identification and quantification in biological specimens can be a challenge given its low concentrations in body fluids. The present work describes a fast and fully validated procedure for the simultaneous detection and quantification of ▵9-tetrahydrocannabinol (▵9_THC) and its two main metabolites 11-hydroxy ▵9_tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-▵9- tetrahydrocannbinol (THC-COOH) in plasma samples using microextraction by packed sorbent (MEPS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). A small plasma volume (0.25mL) pre-diluted (1:20), was extracted with MEPS M1 sorbent as follows: conditioning (4 cycles of 250μL methanol and 4 cycles of 250μL 0.1% formic acid in water); sample load (26 cycles of 250μL); wash (100μL of 3% acetic acid in water followed by 100μL 5% methanol in water); and elution (6 cycles of 100μL of 10% ammonium hydroxide in methanol). The procedure allowed the quantification of all analytes in the range of 0.1-30ng/mL. Recoveries ranged from 53 to 78% (THC), 57 to 66% (11-OH-THC) and 62 to 65% (THC-COOH), allowing the limits of detection and quantification to be set at 0.1ng/mL for all compounds. Intra-day precision and accuracy revealed coefficients of variation (CVs) lower than 10% at the studied concentrations, with a mean relative error within±9%, while inter-day precision and accuracy showed CVs lower than 15% for all analytes at the tested concentrations, with an inaccuracy within±8%.