Development and validation of LC–MS/MS method for quantification of bictegravir in human plasma and its application to an intracellular uptake study

A sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of bictegravir in human plasma. A small volume of only 50 μL aliquot of plasma was precipitated with acetonitrile containing an internal standard (IS). Chromatographic separation was performed on a Kinetex EVO C₁₈ column, 50 × 3.0 mm, 5 μm using an isocratic mobile phase containing 80:20 acetonitrile–water with 0.1% formic acid. A mass spectrometer was operated in ESI positive multiple reaction monitoring mode using the m/z 450.1/289.1 for bictegravir and 420.1/277.1 for IS. The dynamic range of the method was 1–10,000 ng/mL with a correlation coefficient of ≥0.9991. The precision results of calibration standards were in the range of 0.05–4.57% and accuracies were 95.07–104.70%. The mean extraction recovery was 98.64% with a precision of 2.91%. The method was validated as per US Food and Drug Administration guidelines and was found to be accurate and precise. The method was successfully applied to in vitro cellular uptake study.     

High‐throughput LC–MS/MS method with 96‐well plate precipitation for the determination of arotinolol and amlodipine in a small volume of rat plasma: Application to a pharmacokinetic interaction study

A rapid, sensitive, and selective liquid chromatography with tandem mass spectrometry method was developed and fully validated for the simultaneous quantification of arotinolol and amlodipine in rat plasma. Two internal standards were introduced with metoprolol as the internal standard of arotinolol and (S)‐amlodipine‐d4 as the internal standard of amlodipine. The analytes were isolated from 50.0 μL plasma samples by a simple protein precipitation using acetonitrile. The chromatographic separation was achieved in 5 min on a C18 column. The mobile phase consisted of phase A 5% methanol and phase B 95% methanol (both containing 0.5% formic acid and 5 mM ammonium acetate) and was delivered in gradient elution at 0.300 mL/min. Quantification was performed in multiple reaction monitoring mode with the transition m/z 372.1 → 316.1 for arotinolol, m/z 268.2 → 116.2 for metoprolol, m/z 409.1 → 238.1 for amlodipine and m/z 413.1 → 238.1 for (S)‐amlodipine‐d4. Linearity was obtained over the range of 0.200–40.0 ng/mL for arotinolol (r² = 0.9988) and 0.500–100 ng/mL for amlodipine (r² = 0.9985) in rat plasma. The validated data have met the acceptance criteria in FDA guideline. This method was successfully applied to a pharmacokinetic interaction study in rats, and the results indicated that there was no significant drug–drug interaction between arotinolol and amlodipine.     

Development and validation of a highly sensitive LC‐MS/MS method for simultaneous quantitation of nortriptyline and 10‐hydroxynortriptyline in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10‐hydroxynortriptyline (OH‐NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract NTP, OH‐NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH‐NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C₁₈ column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH‐NTP. A linear response function was established for the range of concentrations 1.09–30.0 ng/mL (r > 0.998) for both NTP and OH‐NTP. The intra‐ and inter‐day precision values for NTP and OH‐NTP met the acceptance as per FDA guidelines. NTP and OH‐NTP were stable in a battery of stability studies, i.e. bench‐top, auto‐sampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

The development and validation of a high‐throughput LC–MS/MS method for the analysis of endogenous β‐hydroxy‐β‐methylbutyrate in human plasma

A high‐throughput, sensitive, and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid quantitation of β ‐hydroxy‐β ‐methylbutyrate (HMB) in human plasma has been developed and validated for routine use. The method uses 100 μL of plasma sample and employs protein precipitation with 0.1% formic acid in methanol for the extraction of HMB from plasma. Sample extracts were analyzed using LC–MS/MS technique under negative mode electrospray ionization conditions. A ¹³C–labeled stable isotope internal standard was used to achieve accurate quantitation. Multiday validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2×), extraction recovery, freeze–thaw sample stability (three cycles), benchtop sample stability (6 h and 50 min), autosampler stability (27 h) and frozen storage sample stability (146 days). Linearity was demonstrated between 10 and 500 ng/mL. Inter‐day accuracies and coefficients of variation (CV) were 91.2–98.1 and 3.7–7.8%, respectively. The validated method was proven to be rugged for routine use to quantify endogenous levels of HMB in human plasma obtained from healthy volunteers.     

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.     

Identification of short‐chain poly‐3‐hydroxybutyrates in Saiga horn extracts using LC–MS/MS

Saiga horn extracts were analyzed with the goal of obtaining new information about compounds present in it. The purpose of this study is to find synthetic alternatives to Saiga horn extract, which is used in traditional Chinese medicine, by identifying potentially biologically active compounds in the extracts. Using high‐performance liquid chromatography coupled with high‐resolution mass spectrometry, we have been able to identify a series of short‐chain polyhydroxybutyrates in alcoholic extracts of Saiga horn. Optimized high‐performance liquid chromatography coupled with tandem mass spectrometry methods for analysis of short‐chain poly‐3‐hydroxybutyrates were developed and subsequently applied to investigate Saiga horn extract for the presence of these compounds, which might explain its biological actions, particularly for its antipyretic and procoagulant properties.     

Development of rapid and high‐throughput LC–MS/MS method for quantification of olprinone in rabbit plasma

A simple, highly sensitive and rapid method for quantification of olprinone (phosphodiesterase 3 inhibitor) in rabbit plasma using liquid chromatography–tandem mass spectrometry with electrospray was developed. An aliquot of 50 μL of plasma sample was cleaned up and extracted using Ostro? 96‐well plate followed by dilution. Chromatographic separation of olprinone and olprinone‐d3 was carried out on a CORTECS^® T3 column within 3 min. Detection was achieved using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive ion multiple reaction monitoring mode using the transitions m/z 251.07 → m/z 155.06 and m/z 254.21 → m/z 158.10 for olprinone and olprinone‐d3, respectively. The method was validated according to US Food and Drug Administration guideline for bioanalytical methods, and showed excellent linearity in the range 10.0–2000.0 ng/mL with coefficient of determination >0.99. The intra‐ and inter‐day precisions (CV) were <5.1% and the accuracies were within the range 99.7–103.2% at all quality control concentrations. Furthermore, olprinone was stable under various stability conditions. The developed method was used for quantification of olprinone in rabbit plasma after its intravenous administration at the dose of 1 mg/kg in order to better understand the metabolism of olprinone in a rabbit model of lung injury.     

Development and validation of an analytical method for multiresidue determination of pesticides in lettuce using QuEChERS–UHPLC–MS/MS

A new analytical method for multiresidue determination of 16 multiclass pesticides in lettuce was developed using ultra‐high performance liquid chromatography with tandem mass spectrometry with a triple quadrupole mass analyzer and positive mode electrospray ionization, using a previously optimized quick, easy, cheap, effective, rugged, and safe method for sample preparation. Validation studies, according to document SANTE/11945/2015, demonstrated that the developed method is selective, accurate, and precise, providing recoveries of 70–120%, relative standard deviations ≤20% and quantification limits from 3 μg/kg. The method was compared with one based on high‐performance liquid chromatography with tandem mass spectrometry, in terms of chromatographic performance, detectability and matrix effect for five varieties of lettuce. The new method provided a reduction in the time for the chromatographic analysis of 50%, from 30 to 15 min, using a lower mobile phase flow rate (0.147 mL/min), which reduced the consumption of mobile phase by 25%, and injection of smaller amounts of sample (1.7 μL). Lower limits of quantification were obtained for almost all pesticides studied for green‐leaf lettuce. However, in relation to the matrix effect, four of the five types of lettuce studied presented higher matrix effects.     

Development of a single‐run analytical method for the detection of ten multiclass emerging contaminants in agricultural soil using an acetate‐buffered QuEChERS method coupled with LC–MS/MS

This study was undertaken to develop and validate a single multiresidue method for the monitoring of ten multiclass emerging contaminants, viz. ceftiofur, clopidol, florfenicol, monensin, salinomycin, sulfamethazine, sulfathiazole, sulfamethoxazole, tiamulin, and tylosin in agricultural soil. Samples were extracted using an acetate‐buffered, modified quick, easy, cheap, effective, rugged, and safe method followed by liquid chromatography with tandem mass spectrometric analysis in positive ion mode. Separation on an Eclipse Plus C₁₈ column was conducted in gradient elution mode using a mobile phase of methanol (A) and distilled water (B), each containing 0.1% formic acid and 5 mM ammonium formate. The linearity of the matrix‐matched calibrations, expressed as determination coefficients, was good, with R ² ≥ 0.9908. The limits of quantification were in the range 0.05–10 μg/kg. Blank soil samples spiked with 4 × and 20 × the limit of quantification provided recovery rates of 60.2–120.3% (except sulfamethoxazole spiked at 4 × the limit of quantification, which gave 131.9%) with a relative standard deviation < 13% (except clopidol spiked at 20 × the limit of quantification, which gave 25.2%). This method was successfully applied to the monitoring of 51 field‐incurred agricultural loamy‐sand soil samples collected from 17 provincial areas throughout the Korean Peninsula. The detected and quantified drugs were clopidol (≤ 4.8 μg/kg), sulfathiazole (≤ 7.7 μg/kg), sulfamethazine (≤ 6.6 μg/kg), tiamulin (≤ 10.0 μg/kg), and tylosin (≤ 5.3 μg/kg). The developed method is simple and versatile, and can be used to monitor various classes of veterinary drugs in soil.     

Development and validation of a highly sensitive LC‐MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study

A sensitive, specific and simple LC‐MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under multiple‐reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C₁₈ 100 × 2.1 mm column with a mobile phase of water–methanol–0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple‐reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20–10,000 µg/L (r > 0.998) for bivalirudin. The intra‐ and inter‐day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench‐top, freeze–thaw cycles and long‐term stability. The developed assay method was applied to an intravenous administration study in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of tigecycline in human plasma and cerebrospinal fluid and its application to a pharmacokinetic study

Tigecycline (TGC) is an important antibiotic in treating various drug‐resistant bacteria. The dosage regimen for cerebral intraventricular TGC is still unknown. The aim of the study was to develop and validate liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methods for the determination of TGC in human plasma and cerebrospinal fluid (CSF) to obtain an applicable regimen. The ion transitions under ESI positive model were performed at m/z 586.3 > 513.2 and m/z 595.3 > 514.3 for TGC and d9‐TGC internal standard (IS). For plasma and CSF samples, the calibration curve of TGC was linear within the ranges 25–2000 and 250–100,000 ng/mL; the IS normalized matrix effect was within the ranges 96.46–101.06% and 101.13–103.58%, respectively, for all. TGC was stable under all tested conditions. The patient received 1 mg intraventricular and 49 mg intravenous administration of TGC. The AUC_0–12 in plasma and CSF calculated according to our noncompartment model were 4713 and 23,0238 h ng/mL, respectively. Given our findings cerebral intraventricular TGC may be a choice for clinicians to treat drug‐resistant Gram‐negative bacterial‐induced meningitis and the safety and efficacy of this administration route warrants further study.     

An LC–MS/MS method for quantitation of cyanidin‐3‐O‐glucoside in rat plasma: Application to a comparative pharmacokinetic study in normal and streptozotocin‐induced diabetic rats

A sensitive and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to determine cyanidin‐3‐O‐glucoside (Cy‐3G) in normal and streptozotocin‐induced diabetic rat plasma. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ (50 × 4.6 mm, 5 μm) column and mass spectrometric analysis was performed using a Thermo Finnigan TSQ Quantum Ultra triple‐quadrupole mass spectrometer coupled with an ESI source in the negative ion mode. Selected reaction monitoring mode was applied for quantification using target fragment ions m/z 447.3 → 285.2 for Cy‐3G and m/z 463.0 → 300.1 for quercetin‐3‐O‐glucoside (internal standard). The calibration curve was linear over the range 3.00–2700 ng/mL (r² ≥ 0.99) with the lower limit of quantitation at 3.00 ng/mL. Intra‐ and inter‐day precision was <14.5% and mean accuracy was from −11.5 to 13.6%. Stability testing showed that Cy‐3G remained stable during the whole analytical procedure. After validation, the assay was successfully used to support a preclinical pharmacokinetic comparison of Cy‐3G between normal and diabetic rats. Results indicated that diabetes mellitus significantly altered the in vivo pharmacokinetic characteristics of Cy‐3G after oral administration in rats.     

Development and validation of an LC‐MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU‐48 in rat plasma and its application to a pharmacokinetic study

A specific, sensitive and stable high‐performance liquid chromatographic–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantitative determination of methyl 3‐amino‐6‐methoxythieno [2,3‐b]quinoline‐2‐carboxylate (PU‐48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU‐48 was achieved with a reversed‐phase C₁₈ column (100 × 2.1 mm, 3 μm) at 35°C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3 mL/min. Samples were detected with the triple‐quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2 min for PU‐48 and 7.2 min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass‐to‐charge ratio (m/z) 289.1 → 229.2 for PU‐48 and m/z 385.3 → 267.1 for the internal standard. The calibration curve for PU‐48 was linear over the concentration range of 0.1–1000 ng/mL (r² > 0.99), and the lower limit of quantitation was 0.1 ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU‐48 in rats.     

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.     

Bioanalytical method development and validation of moxidectin in plasma by LC–MS/MS: Application to in vitro metabolism

Moxidectin (MOX) has recently been approved by the US Food and Drug Administration for the treatment of river blindness in select populations. It is also being evaluated as an alternative for the use of ivermectin, widespread resistance to which is becoming a global health issue. Moreover, MOX is becoming increasingly used as a prophylactic antiparasitic in the cattle industry. In this study, we developed and validated an LC–MS/MS method of MOX in human, monkey and mouse plasma. The separation was achieved on an ACE C₁₈ (50 × 3.0 mm, 3 μm) column with isocratic elution using 0.1% acetic acid and methanol–acetonitrile (1:1, v/v) as mobile phase. MOX was quantitated using MS/MS with an electrospray ionization source operating in negative multiple reaction monitoring mode. The multiple reaction monitoring precursor ion → product ion transitions for MOX and abamectin (IS) were m/z 638.40 → 236.30 and m/z 871.50 → 565.35 respectively. The MS/MS response was linear over the concentration range 0.1–1000 ng/mL in plasma with a correlation coefficient (r²) of 0.997 or better. The within‐ and between‐day precision (relative standard deviation, RSD) and accuracy were within the acceptable limits per US Food and Drug Administration guidelines. The method was successfully applied to an in vitro metabolic stability study of MOX.     

LC–MS/MS method development for quantification of doxorubicin and its metabolite 13‐hydroxy doxorubicin in mice biological matrices: Application to a pharmaco‐delivery study

This study describes the development of simple, rapid and sensitive liquid chromatography tandem mass spectrometry method for the simultaneous analysis of doxorubicin and its major metabolite, doxorubicinol, in mouse plasma, urine and tissues. The calibration curves were linear over the range 5–250 ng/mL for doxorubicin and 1.25–25 ng/mL for doxorubicinol in plasma and tumor, over the range 25–500 ng/mL for doxorubicin and 1.25–25 ng/mL for doxorubicinol in liver and kidney, and over the range 25–1000 ng/mL for doxorubicin and doxorubicinol in urine. The study was validated, using quality control samples prepared in all different matrices, for accuracy, precision, linearity, selectivity, lower limit of quantification and recovery in accordance with the US Food & Drug Administration guidelines. The method was successfully applied in determining the pharmaco‐distribution of doxorubicin and doxorubicinol after intravenously administration in tumor‐bearing mice of drug, free or nano‐formulated in ferritin nanoparticles or in liposomes. Obtained results demonstrate an effective different distribution and doxorubicin protection against metabolism linked to nano‐formulation. This method, thanks to its validation in plasma and urine, could be a powerful tool for pharmaceutical research and therapeutic drug monitoring, which is a clinical approach currently used in the optimization of oncologic treatments.     

Simultaneous quantitation of metformin and dapagliflozin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A single LC–MS/MS assay has been developed and validated for the simultaneous determination of metformin and dapagliflozin in human plasma using ion‐pair solid‐phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed‐phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile–15 mm ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using a polarity switching approach. Ion transitions studied using multiple reaction monitoring mode were m/z 130.1 [M + H]⁺/60.1 for metformin and m/z 467.1 [M + CH₃COO]^?/329.1 for dapagliflozin in the positive and negative modes, respectively. The linear calibration range of the assay was established from 1.00 to 2000 ng/mL for metformin and from 0.10 to 200 ng/mL for dapagliflozin to achieve a better assessment of the pharmacokinetics of the drugs. The limit of detection and limit of quantitation for the analytes were 0.39 and 1.0 ng/mL for metformin and 0.03 and 0.1 ng/mL for dapagliflozin, respectively. There was no interference of plasma matrix obtained from different sources, including hemolyzed and lipemic plasma. The method was successfully applied to study the effect of food on the pharmacokinetics of metformin and dapagliflozin in healthy subjects.     

Multiplexed therapeutic drug monitoring of antipsychotics in dried plasma spots by LC‐MS/MS

In this work, a convenient method for the therapeutic monitoring of seven common antipsychotic drugs in “dried plasma spot” samples has been developed. It is based on the liquid chromatography tandem mass spectrometry technique, operating in multiple reaction monitoring mode, and a straightforward procedure for the simultaneous extraction of all antipsychotics in a single step, with high extraction yield. The method was fully validated with proper accuracy, precision, selectivity and sensitivity, for all the drugs. Limits of quantification were 0.12, 1.09, 1.46, 1.47, 5.70, 1.32, 1.33 µg/L for haloperidol, aripiprazole, olanzapine, quetiapine, clozapine, risperidone, and paliperidone, respectively. Accuracy, intra‐ and interday precision values were <10% for all drugs at all concentration levels examined. The method was tested in the analysis of 30 plasma samples from real patients for each drug. The proposed analytical approach, by combining practical and logistical advantages of microsampling with liquid chromatography tandem mass spectrometry analytical performance, could offer an ideal strategy for accurate and timely therapeutic drug monitoring of antipsychotic drugs in most clinical settings, even in remote centers and/or in out‐patient settings, bringing so many potential improvements in psychiatric patient care.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of 106 pesticides in nuts by LC–MS/MS using freeze‐out combined with dispersive solid‐phase extraction purification

As a result of the low water content and high fat matrices in nuts, it is very difficult to simultaneously determine multi‐pesticides in trace levels. Here, a sample pretreatment method was developed in which, microwave‐assisted solvent extraction was firstly used to extract pesticides, and then a two‐step cleanup method was conducted combining freeze‐out with dispersive solid‐phase extraction to remove the lipidic matrix. By this way, 106 pesticides were simultaneously determined in the complicated nut sample by using an ultra‐high pressure liquid chromatography coupled with a tandem mass spectrometer. Average recoveries were 75.3–119.3% with relative standard deviations < 14% at three concentration levels. The limits of detection and quantification were in the ranges of 0.3–3.0 and 1.0–10.0 μg/kg, respectively. Furthermore, the method was successfully applied to the determination of pesticides in 180 commercial nut samples.