Carboxylesterase and UDP‐glucuronosyltransferases mediated metabolism of irinotecan: In vitro and in vivo insights from quantitative ultra‐performance liquid chromatography–mass spectrometry analysis

Carboxylesterase and UDP‐glucuronosyltransferase‐mediated metabolism of irinotecan (CPT‐11) has long been proposed to be responsible for its anti‐tumor activity and toxicity, like delayed‐onset diarrhea. However, recent studies failed to gain more comprehensive in vivo and in vitro pharmacokinetic profiles of irinotecan. Herein, we use rat plasma, human liver microsomes and immortalized HepG2 cell as experimental subjects to describe a sensitive and versatile UHPLC–MS/MS method for simultaneously quantifying CPT‐11 and its metabolites, including SN‐38 and SN‐38G. The method was applied to investigate the pharmacokinetic and metabolic behavior of CPT‐11 in the biological samples. Calibration curves for all bio‐matrices showed acceptable linearity (r² > 0.99). The intra‐ and inter‐day precisions (RSD, %) were within 15% and the excellent accuracy (RE) was between 2.96 and 14.12%. In addition, the specificity, matrix effect and extraction recovery all met the requirements of biological sample analysis. We successfully applied this method to investigate the pharmacokinetics of irinotecan in various biological samples, mediated by carboxylesterase and UDP‐glucuronosyltransferase. This method could be employed in monitoring the metabolic status and clinical efficacy of irinotecan in the future.     

A simple and rapid ultra‐high‐performance liquid chromatography–tandem mass spectrometry method to determine plasma biotin in hemodialysis patients

A simple, rapid, and selective method for determination of plasma biotin was developed using ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). After single‐step protein precipitation with methanol, biotin and stable isotope‐labeled biotin as an internal standard (IS) were chromatographed on a pentafluorophenyl stationary‐phase column (2.1 × 100 mm, 2.7 μm) under isocratic conditions using 10 mm ammonium formate–acetonitrile (93:7, v/v) at a flow rate of 0.6 mL/min. The total chromatographic runtime was 5 min for each injection. Detection was performed in a positive electrospray ionization mode by monitoring selected ion transitions at m/z 245.1/227.0 and 249.1/231.0 for biotin and the IS, respectively. The calibration curve was linear in the range of 0.05–2 ng/mL using 300 μL of plasma. The intra‐ and inter‐day precisions were all <7.1%. The accuracy varied from ?0.7 to 8.2%. The developed UHPLC–MS/MS method was successfully applied to determine plasma biotin concentrations in hemodialysis patients. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous determination of l‐tetrahydropalmatine and its active metabolites in rat plasma by a sensitive ultra‐high‐performance liquid chromatography with tandem mass spectrometry method and its application in a pharmacokinetic study

A sensitive and reliable ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of l ‐tetrahydropalmatine (l ‐THP) and its active metabolites l ‐isocorypalmine (l ‐ICP) and L ‐corydalmine (l ‐CD) in rat plasma. The analytes were extracted by liquid–liquid extraction and separated on a Bonshell ASB C₁₈ column (2.1 × 100 mm; 2.7 μm; Agela) using acetonitrile–formic acid aqueous as mobile phase at a flow rate of 0.2 mL/min in gradient mode. The method was validated over the concentration range of 4.00–2500 ng/mL for l ‐THP, 0.400–250 ng/mL for l ‐ICP and 1.00–625 ng/mL for l ‐CD. Intra‐ and inter‐day accuracy and precision were within the acceptable limits of <15% at all concentrations. Correlation coefficients (r ) for the calibration curves were >0.99 for all analytes. The quantitative method was successfully applied for simultaneous determination of l ‐THP and its active metabolites in a pharmacokinetic study after oral administration with l ‐THP at a dose of 15 mg/kg to rats.     

Simultaneous determination of tilianin and its metabolites in mice using ultra‐high‐performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

Tilianin is an active flavonoid glycoside found in many medical plants. Data are lacking regarding its pharmacokinetics and disposition in vivo. The objective of this study was to develop a sensitive, reliable and validated ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method to simultaneously quantify tilianin and its main metabolites and to determine its pharmacokinetics in wild‐type and breast cancer resistance protein knockout (Bcrp1−/−) FVB mice. Chromatographic separation was accomplished on a C₁₈ column by utilizing acetonitrile and 0.5 mm ammonium acetate as the mobile phase. Mass spectrometric detection was performed using electrospray ionization in both positive and negative modes. The results showed that the precision, accuracy and recovery, as well as the stability of tilianin and its metabolites in mouse plasma, were all within acceptable limits. Acacetin‐7‐glucuronide and acacetin‐7‐sulfate were the major metabolites of tilianin in mouse plasma. Moreover, systemic exposure of acacetin‐7‐sulfate was significantly higher in Bcrp1 (−/−) FVB mice compared with wild‐type FVB mice. In conclusion, the fully validated UHPLC–MS/MS method was sensitive, reliable, and was successfully applied to assess the pharmacokinetics of tilianin in wild‐type and Bcrp1 (−/−) FVB mice. Breast cancer resistance protein had a significant impact on the elimination of the sulfated metabolite of tilianin in vivo.     

Ultra‐high pressure liquid chromatography–tandem mass spectrometry method for the determination of omarigliptin in rat plasma and its application to a pharmacokinetic study in rats

Omarigliptin is a novel long‐acting dipeptidyl peptidase‐4 inhibitor used for the treatment of type 2 diabetes. In this work, a sensitive and selective ultra‐high pressure liquid chromatography tandem mass spectrometry method was developed and validated for the determination of omarigliptin in rat plasma. Sample preparation was performed by protein precipitation with acetonitrile. Chromatographic separation of analytes was achieved on an RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm), using gradient mobile phase (0.1% formic acid–acetonitrile) at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode, with target fragment ions m/z 399.1 → 152.9 for omarigliptin and m/z 237.1 → 194 for the internal standard. The total run time was 4 min. Retention time of omarigliptin and internal standard was 1.25 and 2.12 min, respectively. Relative standard deviation (%) of the intra‐ and inter‐day precision was below 10.0%, and accuracy was between 97.9% and 105.3%. Calibration curve was established over the range 2–5000 ng/mL with good linearity. The lower limit of quantification and limit of detection of omarigliptin were 2 and 0.25 ng/mL, respectively. Mean recoveries were in the range 87.3–95.1% for omarigliptin. No matrix effect was observed in this method. This novel method has been successfully applied to a pharmacokinetic study of omarigliptin in rats. The absolute bioavailability of omarigliptin was identified as high as 87.31%.     

An improved ultra‐high‐performance liquid chromatography–tandem mass spectrometric method for the quantitation of dexmedetomidine in small volume of pediatric plasma

Dexmedetomidine (Dex), a highly selective α₂‐adrenergic agonist, is used primarily for the sedation and anxiolysis of adults and children in the intensive care setting. A sensitive and selective assay for Dex in pediatric plasma was developed by employing ultra‐high‐performance liquid chromatography–tandem mass spectrometry with d4‐Dex as an internal standard. Dex was extracted from 0.1 mL of plasma by micro‐elution solid‐phase extraction. Separation was achieved with a Waters XBridge C₁₈ column with a flow rate of 0.3 mL/min using a mobile phase comprising 5 mm ammonium acetate buffer with 0.03% formic acid in water and methanol–acetonitrile (50:50, v/v). The intra‐day precision (coefficient of variation) and accuracy for quality control samples ranged from 1.32 to 8.91% and from 92.8 to 108%, respectively. The inter‐day precision and accuracy ranged from 2.13 to 8.45% and from 97.0 to 104%, respectively. The analytical method showed excellent sensitivity using a small sample volume (0.1 mL) with a lower limit of quantitation of 5 pg/mL. This method is robust and has been successfully employed in a pharmacokinetic study of Dex in neonates and infants postoperative from cardiac surgery.     

A fast and sensitive UHPLC–MS/MS method for the determination of N‐butylscopolamine in human plasma: application in a bioequivalence study

We have developed and validated a fast and sensitive ultra high‐performance liquid chromatography with positive ion electrospray ionization tandem mass spectrometry method for determining N‐ butylscopolamine levels in human plasma using propranolol as an internal standard. The acquisition was set up in the multiple reaction monitoring mode with the transitions m /z 360.3 → 138.0 for N‐ butylscopolamine and m /z 260.2 → 116.1 for IS. This method uses a liquid–liquid extraction process with dichloromethane. The analyte and IS were chromatographed on a C₁₈, 50 × 2.1 mm, 1.7 μm column through isocratic elution with acetonitrile–5 mm ammonium acetate (adjusted to pH 3.0 with formic acid). The method was linear in the 1–1000 pg/mL range for N‐ butylscopolamine and was selective, precise, accurate and robust. The validated method was successfully applied to perform a bioequivalence study of the reference (Buscopan^®, from Boehringer Ingelheim) and the test sample coated‐tablet formulations (from Foundation for Popular Remedy), both containing 10 mg of N‐ butylscopolamine bromide administered as a single dose. Using 58 healthy volunteers and accounting for the high intra‐individual variability confirmed by statistical calculations (38%), the two formulations were considered bioequivalent because the rate and extent of absorption (within 80–125% interval), satisfying international requirements.     

Metabolic profile of rosmarinic acid from Java tea (Orthosiphon stamineus) by ultra‐high‐performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry with a three‐step data mining strategy

Rosmarinic acid (RA) is a caffeic acid derivative and one of the most abundant and bioactive constituents in Java tea (Orthosiphon stamineus), which has significant biological activities. However, relatively few studies have been conducted to describe this compound's metabolites in vivo. Therefore, an ultra‐high‐performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry (UHPLC–QTOF–MS/MS) analysis with a three‐step data mining strategy was established for the metabolic profile of RA. Firstly, the exogenously sourced ions were filtered out by the MarkerView software and incorporated with Microsoft Office Excel software. Secondly, a novel modified mass detects filter strategy based on the predicted metabolites was developed for screening the target ions with narrow, well‐defined mass detection ranges. Thirdly, the diagnostic product ions and neutral loss filtering strategy were applied for the rapid identification of the metabolites. Finally, a total of 16 metabolites were reasonably identified in urine, bile and feces, while metabolites were barely found in plasma. The metabolites of RA could also be distributed rapidly in liver and kidney. Glucuronidation, methylation and sulfation were the primary metabolic pathways of RA. The present findings might provide the theoretical basis for evaluating the biological activities of RA and its future application.     

A rapid and sensitive UHPLC–MS/MS method for the determination of celosin A in rat plasma with application to pharmacokinetic study

Celosin A (CA), a natural compound isolated from Celosia argentea L., has been shown significant hepatoprotective effect on AHNP‐induced liver injury. This study described a rapid and sensitive ultra‐high‐pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) assay for determination of CA in rat plasma. Methanol‐mediated precipitation was used for sample pretreatment. Chromatographic separation was achieved on a T3 column with gradient elution using water and acetonitrile as mobile phase. Determination was obtained using an electrospray ionization source in negative selected reaction monitoring mode at the transitions of m/z 793.3 → m/z 661.2 and m/z 955.6 → m/z 793.2 for CA and IS, respectively. The assay was linear over the concentration range 0.25–2500 ng/mL (r > 0.995) with a lowest limit of quantification (LLOQ) of 0.25 ng/mL. The intra‐ and inter‐day precisions (RSD) were 1.65–9.84 and 2.46–13.49%, respectively, while accuracy (RR) ranged from 96.21 to 99.45%, respectively. The recovery ranged from 95.09 to 102.22% and the matrix effect from 98.29 to 100.13%. The analyte was stable under the tested storage conditions. The method has been successfully applied to a preclinical pharmacokinetic study in rats after a single intravenous (2 mg/kg) or oral (50 mg/kg) administration. The oral bioavailability of CA was ~1.94%; in addition, there was no difference between male and female rats. This is the first time of the use of an UHPLC–MS/MS method for determination of CA concentration in rat plasma and for evaluation of its pharmacokinetic behavior.     

Simultaneous quantification of oxybutynin and its active metabolite N‐desethyl oxybutynin in rat plasma by ultra‐high‐performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study of oxybutynin transdermal patch

A rapid, selective and sensitive ultra‐high‐performance liquid chromatography–tandem mass spectrometry method was developed to simultaneously determine oxybutynin and its active metabolite N‐desethyl oxybutynin in rat plasma. A 0.1 mL sample of plasma was extracted with n‐hexane. Chromatographic separation was performed on a UPLC BEH C₁₈ column (2.1 × 100 mm i.d.,1.7 μm) with mobile phase of methanol–water (containing 2 mmol/L ammonium acetate and 0.1% formic acid; 90:10, v/v). The detection was performed in positive selected reaction monitoring mode. Each plasma sample was chromatographed within 3 min. The linear calibration curves were obtained in the concentration range of 0.0944–189 ng/mL (r ≥ 0.99) for oxybutynin and 0.226–18.0 ng/mL (r ≥ 0.99) for N‐desethyl oxybutynin. The intra‐ and inter‐day precision (relative standard deviation) values were not more than 14% and the accuracy (relative error) was within ±7.6%. The method described was superior to previous methods for the quantitation of oxybutynin with three product ions and was successfully applied to a pharmacokinetic study of oxybutynin and its active metabolite N‐desethyl oxybutynin in rat plasma after transdermal administration.     

A UPLC–MS/MS approach for simultaneous determination of eight flavonoids in rat plasma,and its application to pharmacokinetic studies of Fu‐Zhu‐Jiang‐Tang tablet in rats

The purpose of this study is to establish and validate a UPLC–MS/MS approach to determine eight flavonoids in biological samples and apply the method to pharmacokinetic study of Fu‐Zhu‐Jiang‐Tang tablet. A Waters BEH C₁₈ UPLC column was employed with methanol/0.1% formic acid–water as mobile phases. The mass analysis was carried out in a triple quadrupole mass spectrometer using multiple reaction monitoring with negative scan mode. A one‐step protein precipitation by methanol was used to extract the analytes from blood. Eight major flavonoids were selected as markers. Our results showed that calibration curves for 3′‐hydroxypuerarin, mirificin, puerarin, 3′‐methoxypuerarin, daidzin, rutin, astragalin and daidzein displayed good linear regression (r ² > 0.9986). The intra‐day and inter‐day precisions (RSD) of the eight flavonoids at high, medium and low levels were <8.03% and the bias of the accuracies ranged from −5.20 to 6.75%.The extraction recoveries of the eight flavonoids were from 91.4 to 100.5% and the matrix effects ranged from 89.8 to 103.8%. The validated approach was successfully applied to a pharmacokinetic study in Sprague–Dawley rats after oral administration of FZJT tablet. Double peaks were emerged in curves of mean plasma concentration for 3′‐methoxypuerarin, which was reported for the first time.     

UHPLC–MS/MS analysis of sphingosine 1‐phosphate in joint cavity dialysate and hemodialysis solution of adjuvant arthritis rats: Application to geniposide pharmacodynamic study

Geniposide (GE) is an iridoid glycoside compound with anti‐inflammatory effect. The potential of sphingosine 1‐phosphate (S1P) as a plasma marker in human diseases was suggested recently in the literature, which demonstrated that, in patients with inflammatory diseases, plasma S1P was elevated. It follows that the obstructive coronary artery disease can be predicted with serum S1P. Therefore, S1P can also be potentially used as a pharmacodynamic marker to study adjuvant arthritis (AA) rats. In the current study, a UHPLC–MS/MS method combined with the microdialysis sampling technique (using FTY720 phosphate as an internal standard) was adopted and validated to measure S1P levels in the hemodialysis fluid and joint cavity dialysates of AA rats after oral administration of GE. A S1P concentration–time curve in the dialysate was established in this study. It was demonstrated that GE exerted an anti‐inflammatory effect by reducing AA‐induced elevated S1P levels. It is showed that changes in S1P concentrations over time can be used to monitor the pharmacodynamic effects of GE in treating AA rats in pharmacodynamic studies.     

Strategy for rapid screening of antioxidant and anti‐inflammatory active ingredients in Gynura procumbens (Lour.) Merr. based on UHPLC–Q‐TOF–MS/MS and characteristic ion filtration

Gynura procumbens (Lour.) Merr. is traditionally used as a raw material for making dumplings or steamed stuffed buns, and its fresh leaves are boiled with water for tea. Herein, we established an ultra‐high–performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC–Q‐TOF–MS/MS) combined with characteristic ion filtration (CIF) strategy to rapidly screen active ingredients with antioxidant and anti‐inflammatory properties in G. procumbens. This strategy involved screening the active part of G. procumbens using antioxidation and anti‐inflammatory activity assays; discovering the active compounds by speculating on the active site's chemical composition by UHPLC–Q‐TOF–MS/MS plus CIF; and verifying the active compounds' activities. The ethyl acetate extract (EEAF) of G. procumbens was the major active site. Eighty‐one compounds were identified from the EEAF using UHPLC–Q‐TOF–MS/MS plus CIF. Furthermore, polyphenols such as cynarine, isochlorogenic acids A and isochlorogenic acids C have excellent antioxidizing and anti‐inflammatory activities. This study provides a practical strategy for rapid in vitro screening of the antioxidizing and anti‐inflammatory activities of traditional vegetables and herbs and identification of active ingredients.     

A validated UHPLC–MS/MS method for the measurement of riluzole in plasma and myocardial tissue samples

Through blocking the cardiac persistent sodium current, riluzole has the potential to prevent myocardial damage post cardiac bypass surgery. A sensitive UHPLC–MS/MS method was developed and validated for quantitation of riluzole and 5‐methoxypsoralen in human plasma and myocardial tissue homogenate using a liquid–liquid extraction with dichloromethane. The chromatographic separation was achieved using Shimadzu Shim‐pack XR‐ODS III, 2.0 × 50 mm, 1.6 μm column with a gradient mobile phase comprising methanol and ammonium acetate buffer pH 3.6 in purified water. The analyte and internal standard were separated within 3.5 min. Riluzole quantitation was achieved using the mass transitions of 235–138 for riluzole and 217–156 for 5‐methoxypsoralen. The method was linear for riluzole plasma concentrations from 0.2 to 500 ng/mL and myocardial tissue homogenate concentrations from 0.2 to 100 ng/mL. The method developed was successfully applied to a clinical study for patients receiving riluzole while undergoing cardiac bypass surgery.     

Pharmacokinetic and tissue distribution studies of cassane diterpenoids,in rats through an ultra‐high‐performance liquid chromatography–Q exactive hybrid quadrupole–Orbitrap high‐resolution accurate mass spectrometry

Cassane diterpenoids (CA) are considered as the main active constituents of medicinal plants belonging to the Caesalpinia genus. Three cassane derivatives, bonducellpin G (BG), 7‐O‐acetyl‐bonducellpin C (7‐O‐AC) and caesalmin E (CE), isolated from Caesalpinia minax Hance seeds, showed strong anti‐inflammatory activity. In this paper, pharmacokinetics (BG, 7‐O‐AC, CE) and tissue distribution (7‐O‐AC, CE) properties were studied for the first time using a reliable, sensitive and rapid UHPLC–Q‐Orbitrap HR‐MS to develop new anti‐inflammatory agents. A novel quantitative method with full scan in positive ion mode was used to determine the contents of compounds. They were separated using acetonitrile–water (0.1% formic acid) as gradient mobile phase. The calibration curve displayed good linearity and the lower limit of quantitation was 0.005–0.02 μg/mL for all analytes. Meanwhile, the absorption, distribution, metabolism, excretion (ADME) property was predicted using PreADMET web. The pharmacokinetic parameters indicated that they were absorbed quickly, eliminated rapidly together with high blood concentration. The results of tissue distribution demonstrated that CE was distributed rapidly and widely among tissues, and stomach was the main tissue site of CE and 7‐O‐AC, followed by small intestine/liver. This study indicates that the structures and dosages of active CA should be modified to help improve the absorption rate and residence time, and the findings are helpful for the pharmaceutical design of CA derivatives.     

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.     

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.     

A simple and selective UHPLC–MS/MS method for quantification of plantagoguanidinic acid in rat plasma and its application to a pharmacokinetic study

A simple, sensitive and specific UHPLC–MS/MS method for quantification of plantagoguanidinic acid (PGA) in rat plasma was applied to investigate the pharmacokinetic behavior in vivo , using protopine as internal standard. The chromatography was separated on a Phenomenex® Luna‐C₁₈ column (2.1 × 150 mm, 3.0 μm) within 7.0 min using a mobile phase consisting of acetonitrile–0.1% formic acid solution under gradient elution at a flow rate of 0.4 mL/min. Prepared samples were monitored by multiple reaction monitoring mode, with the target fragmentions m/z 226.2 → 84.2 for PGA and m/z 354.2 → 188.9 for IS in positive electrospray ionization. The calibration curve of PGA was linear throughout the range 1–1000 ng/mL (r = 0.9962). The lower limit of quantitation in plasma for PGA was 0.1 ng/mL, and the recovery was >88.6%. Intra‐ and interday accuracy ranged from −8.6 to 4.9%. Furthermore, this validated method was successfully used for a pre‐clinical pharmacokinetic study of PGA at a single dose of 20 and 5 mg/kg in rats via oral and intravenous administration. The study showed that PGA was absorpted rapidly and eliminated gradually with a greater absolute oral bioavailability of 70.1% in rats.     

Stability‐indicating validation of acitretin and isoacitretin in human plasma by LC‐ESI‐MS/MS bioanalytical method and its application to pharmacokinetic analysis

LC‐ ESI‐ MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin‐d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid–liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin‐d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis‐RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025–753.217 ng/mL for acitretin and 0.394–289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra‐day and inter‐day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples. Copyright © 2010 John Wiley & Sons, Ltd.