Validation and application of a liquid chromatography‐tandem mass spectrometric method for the determination of GDC‐0152 in human plasma using solid‐phase extraction

A liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0152 in human plasma to support clinical development. The method consisted of a solid‐phase extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₇‐GDC‐0152 was used as the internal standard. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 0.02–10.0 ng/mL for GDC‐0152. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 99.3% with a precision (%CV) of 13.9%. For quality control samples at 0.0600, 2.00 and 8.00 ng/mL, the between‐run %CV was ≤8.64. Between‐run percentage accuracy ranged from 98.2 to 99.6%. GDC‐0152 was stable in human plasma for 363 days at ?20°C and for 659 days at ?70°C storage. GDC‐0152 was stable in human plasma at room temperature for up to 25 h and through three freeze–thaw cycles. In whole blood, GDC‐0152 was stable for 12 h at 4°C and at ambient temperature. This validated LC‐MS/MS method for determination of GDC‐0152 was used to support clinical studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Highly sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by solid‐phase extraction and liquid chromatography‐tandem mass spectrometry

Using bamboo‐activated charcoal as SPE adsorbent, a novel SPE method was developed for the sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by rapid‐resolution LC‐ESI‐MS/MS. Important parameters influencing extraction efficiency, including type of eluent, eluent volume, sample pH, volume and flow rate, were investigated and optimized. Under the optimal extraction conditions (eluent: 8 mL methanol, pH: 7; flow rate: 4 mL/min; sample volume: 100 mL), low LODs (0.01–0.02 ng/mL), good repeatability (6.2–8.3%) and wide linearity range (0.10–10 ng/mL) were obtained. Satisfied results were achieved when the proposed method was applied to determine the two target compounds in real‐world environmental water samples with spiked recoveries over the range of 80.5–119.8%. All these facts indicate that trace determination of tetrabromobisphenol A and bisphenol A in real‐world environmental water samples can be realized by bamboo‐activated charcoal SPE‐rapid resolution‐LC‐ESI‐MS/MS.     

A sensitive and selective method for the quantitative analysis of miglitol in rat plasma using unique solid‐phase extraction coupled with liquid chromatography–tandem mass spectrometry

A sensitive, selective and robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of miglitol in rat plasma. The sample preparation procedures involved protein precipitation and unique solid‐phase extraction, which efficiently removed sources of ion suppression and column degradation interference present in the plasma. Chromatographic separation was achieved on an amide column using 10 mmol/L CH₃COONH₄ and CH₃CN:CH₃OH (90:10, v/v) as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in positive ion mode.The selected reaction monitoring transitions for miglitol and a stable isotope‐labeled internal standard were m/z 208 → m/z 146 and m/z 212 → m/z 176, respectively. The correlation coefficients of the calibration curves ranged from 0.9984 to 0.9993 over a concentration range of 0.5–100 ng/mL plasma. The quantification limit of the proposed method was more than 10 times lower than those of previously reported LC‐MS/MS methods. The novel method was successfully validated and applied to a pharmacokinetic study in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification and pharmacokinetics of crizotinib in rats by liquid chromatography–tandem mass spectrometry

Crizotinib is a small molecule inhibitor of anaplastic lymphoma kinase (ALK) and can be used to treat ALK‐positive nonsmall‐cell lung cancer. A rapid and simple high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of crizotinib in rat plasma using a chemical synthetic compound buspirone as the internal standard (IS). The plasma samples were pretreated by a simple protein precipitation with methanol–acetonitrile (1:1, v/v). Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C₁₈ column (2.1 × 50 mm, 3.5 µm). The gradient elution system was composed of 0.1% formic acid aqueous solution and 0.1% formic acid in methanol solution. The flow rate was set at 0.50 mL/min. The multiple reaction monitoring was based on the transitions of m/z = 450.3 → 177.1 for crizotinib and 386.2 → 122.2 for buspirone (IS). The assay was successfully validated to demonstrate the selectivity, matrix effect, linearity, lower limit of quantification, accuracy, precision, recovery and stability according to the international guidelines. The lower limit of quantification was 1.00 ng/mL in 50 μL of rat plasma. This LC‐MS/MS assay was successfully applied to the quantification and pharmacokinetic study of crizotinib in rats after intravenous and oral administration of crizotinib. The oral absolute bioavailability of crizotinib in rats was 68.6 ± 9.63%. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a liquid chromatography–tandem mass spectrometry method for the determination of Grayanotoxins in rat blood and its application to toxicokinetic study

A sensitive and specific high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid‐phase extraction using PEP solid‐phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C₁₈ (100 × 2.1 mm, 2.6 µm) reversed‐phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1–100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra‐day and inter‐day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of astragaloside III in rat plasma by liquid chromatography–tandem mass spectrometry and its application to a rat pharmacokinetic study

Astragaloside III (AST III), a naturally occurring saponin compound isolated from Radix Astragali, has been demonstrated to have anti‐gastric ulcer, immunomodulatory and antitumor effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high‐performance liquid chromatography–tandem mass spectrometric (HPLC‐MS/MS) method has been developed and validated for the quantification of astragaloside III in rat plasma. Samples were pretreated using a simple protein precipitation with methanol–acetonitrile (50:50, v/v) and the chromatographic separation was performed on a C₁₈ column by a gradient elution using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Astragaloside III and the internal standard (buspirone) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range of 5.00–5000 ng/mL together with satisfactory intra‐ and inter‐day precision, accuracy and recovery. Stability testing showed that astragaloside III spiked into rat plasma was stable for 24 h at 20°C temperature, for up to 30 days at ?80°C, and during three freeze–thaw cycles. The method was successfully used to investigate the pharmacokinetic profile of AST III after oral (10 mg/kg) and intravenous (1.0 mg/kg) administration in rats. The oral absolute bioavailability of AST III was calculated to be 4.15 ± 0.67% with an elimination half‐life value of 2.13 ± 0.11 h, suggesting its poor absorption and/or strong metabolism in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of polygalasaponin F in rat plasma using liquid chromatography–tandem mass spectrometry and its pharmacokinetics application

A rapid and highly selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of polygalasaponin F (PF) in rat plasma was developed and validated. The chromatographic separation was achieved on a reverse‐phase Zorbax SB‐C₁₈ column (150 × 4.6 mm, 5 µm), using 2 mm ammonium acetate (pH adjusted to 6.0 with acetic acid) and acetonitrile (25:75, v/v) as a mobile phase at 30 °C. MS/MS detection was performed using an electrospray ionization operating in positive ion multiple reaction monitoring mode by monitoring the ion transitions from m/z 1091.5 → 471.2 (PF) and m/z 700.4 → 235.4 (internal standard), respectively. The calibration curve showed a good linearity in the concentration range 0.0544–13.6 µg/mL, with a limit of quantification of 0.0544 µg/mL. The intra‐ and inter‐day precisions were <9.7% in rat plasma. The method was validated as per US Food and Drug Administration guidelines and successfully applied to pharmacokinetic study of PF in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of peimine and peiminine in rat plasma by LC‐ESI‐MS employing solid‐phase extraction

A simple and reliable LC‐ESI‐MS method for the determination of peimine and peiminine in rat plasma was developed for the first time. The method was proven to be specific and sensitive by carrying out validation. The analytes were extracted from rat plasma via solid‐phase extraction on Waters Oasis MCX cartridges. Chromatography separation was achieved on a C₁₈ column using 10 mM ammonium acetate (adjusted to pH 3.0 with glacial acetic acid)–acetonitrile (85:15, v/v) as mobile phase. The linear range was 1–100 ng/mL for peimine and peiminine. Intra‐ and inter‐day precisiond were less than 10%. Accuracies were within 85–115% of their nominal concentrations. The limit of quantification was 1 ng/mL for both analytes. The developed assay was successfully applied to pharmacokinetic study of peimine and peiminine in rats orally administered the alkaloids extracts from Bulbus Fritillariae, demonstrating a possible broader spectrum of applications of this method. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of corilagin in rat plasma using a liquid chromatography–electrospray ionization tandem mass spectrometric method

A sensitive and simple liquid chromatography–tandem mass spectrometric (HPLC‐MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C₈ column eluted with mobile phase consisting of methanol–0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5–1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra‐ and inter‐day relative standard deviationa across three validation runs for four concentration levels were both <9.8%. The relative error was within ±6.0%. This assay offers advantages in terms of expediency and suitability for the analysis of corilagin in rat plasma. The practical utility of this new HPLC‐MS/MS method was confirmed in pilot plasma concentration studies in rats following oral administration. Copyright © 2015 John Wiley & Sons, Ltd.     

Salting‐out‐assisted liquid–liquid extraction with acetonitrile for the determination of trimetazidine in rat plasma using liquid chromatography–mass spectrometry

A high‐throughout bioanalytical method based on salting‐out‐assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry‐compatible salts followed by LC‐MS/MS analysis of trimetazidine in rat plasma is presented. It required only 50 μL of plasma and allows the use of minimal volumes of organic solvents. The seamless interface of SALLE and LC‐MS eliminated the drying‐down step and the extract was diluted and injected into an LC‐MS/MS system with a cycle time of 2.5 min/sample. The retention times of trimetazidine and IS were approximately 1.1 and 1.7 min, respectively. Calibration curves were linear over the concentration range of 0.1–100 ng/mL, which can be extended to 500 ng/mL by dilution. The intra‐ and inter‐batch precision, accuracy and the relative standard deviation were all <15%. This method was successfully applied to determine trimetazidine concentrations in rat plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Qualification and application of a liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the determination of trastuzumab in rat plasma

An liquid chromatography–quadrupole time‐of‐flight (QqTOF) mass spectrometric method was developed for the determination of humanized or human monoclonal antibodies in rat plasma at the early drug discovery stage. Trastuzumab was used as a model monoclonal antibody. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC‐TOF‐MS/MS analysis of specific signature peptides in the positive ion mode using electrospray ionization for analysis. A stable isotope‐labeled signature peptide was also used as internal standard. A quadratic regression (weighted 1/concentration²), with an equation y = ax² + bx + c, was used to fit calibration curves over the concentration range of 0.500–100 µg/mL for trastuzumab. Samples from a pharmacokinetic study in rat were analyzed by this qualified LC‐TOF‐MS/MS method and concentrations were compared with those generated by enzyme linked immunosorbent assays method. The LC‐TOF‐MS/MS method was accurate and precise, with quantitative results comparable with those of ELISA. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. Within‐run accuracy ranged from 1.53 to 9.20% with precision values ≤10.29%. This LC‐TOF‐MS/MS method approach could be used as a complementary method for humanized or human monoclonal antibodies at the early drug discovery stage. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometric assay for the determination of vaccarin in rat plasma: application to a pharmacokinetic study

Vaccarin, a flavonoid glycoside, is considered one of the major active constituents of Vaccaria segetalis. A simple and specific liquid chromatography–tandem mass spectrometric method was developed and validated for quantifying vaccarin in rat plasma following intravenous dosing. Plasma samples were precipitated with methanol and separated on a Venusil‐C₁₈ analytical column (2.1 × 50 mm, 5 µm particles) with gradient elution consisting of methanol and 0.1% (v/v) formic acid as the mobile phase. The detection was performed on an Agilent Triple Quad LC/MS with electrospray ionization inlet in the positive multiple reaction monitoring mode. Good linearity was achieved over the concentration range of 12.5–25,000 ng/mL (r² > 0.99). Intra‐ and inter‐day precisions were <9.1%, and accuracy ranged from ?2.8 to 8.7%. The lower limit of quantification for vaccarin was 12.5 ng/mL, and the analyte was stable under various storage conditions. This validated method was successfully applied to the preliminary pharmacokinetic studies of vaccarin following intravenous administrations of 1.21, 2.41, and 4.82 mg/kg vaccarin in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

A simple,rapid and sensitive liquid chromatography–tandem mass spectrometry method for the determination of dienogest in human plasma and its pharmacokinetic applications under fasting

A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K₂EDTA plasma using levonorgestrel d₆ as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid–liquid extraction. Chromatographic separation was achieved on a Zorbax XDB‐Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile–5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003–200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra‐ and inter‐day precision (coefficient of variation) <3.97 and 6.10%, respectively, and accuracy within ±4.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administration of a single oral dose of 2.0 mg dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of ginsenosides and bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill for pharmacokinetic study by liquid chromatography tandem mass spectrometry following solid‐phase extraction

A sensitive and reliable bioanalytical method was established for quantitative and pharmacokinetic investigation of nine ginsenosides and seven bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill by liquid chromatography–electrospray ionization tandem mass spectrometry, using tinidazole and digoxin as internal standards (ISTDs). All of the analytes and ISTDs obtained satisfactory recoveries by solid‐phase extraction using an Oasis HLB μElution Plate, which was eluted with methanol and ethyl acetate successively, and chromatographic separation was achieved on a Shim‐pack XR‐ODSIIcolumn (75 × 2.0 mm, 2.2 μm) with gradient elution using a mixture of acetonitrile–0.1% formic acid solution (v /v) as the mobile phase at a flow rate of 0.3 mL/min. Detection was carried out by a triple‐quadrupole tandem mass spectrometry with positive/negative ion switching multiple reaction monitoring mode. All analytes showed good linearity over a wide concentration range (r ² > 0.99). The lower limit of quantification was in the range 0.625–12.5 ng/mL for bufadienolides and 2–5.5 ng/mL for ginsenosides, and the mean recoveries of all analytes were in the range 78.29–99.35%. The intra‐ and inter‐day precisions (RSD) were in the range 0.08–12.38% with the accuracies between 86.09 and 99.40%. The validated method was then successfully applied to pharmacokinetic study of the above 16 compounds in rat plasma. Pharmacokinetic results indicated that the developed extraction and analytical method could be employed as a rapid, effective technique for pharmacokinetic study of multiple components, especially various polarity that are difficult to extract simultaneously.     

Liquid chromatography‐tandem mass spectrometric method for determination of E121 in mouse plasma and its application to pharmacokinetics

A rapid LC‐MS/MS method for quantification of an enaminone analog, E121 in mouse plasma using E118 as an internal standard (IS) has been developed and validated. The analyte was analyzed on C₁₈ column using a mobile phase of acetonitrile/methanol/ammonium acetate/formic acid (60:20:20:0.025, v/v/v/v) at a flow rate of 0.25 mL/min. Quantitation was achieved using ESI+ interface, employing MRM mode at m/z 308>262 and 222>194 for E121 and IS, respectively. The calibration standards were linear over a range of 0.10–20 μg/mL (r²>0.99) with an LLOQ of 0.1 μg/mL (RSD%; 11.4% and bias%; 9.5%). Intra‐ and inter‐run precision of E121 assay ranged from 3.7 to 10.9% with accuracy (bias) that varied between ?10.0 and 12.0%, demonstrating good precision and accuracy. Recoveries of E121 and the IS from plasma were above 80%. Stability of E121 in plasma showed that the analyte was stable under various conditions. The matrix effect study showed a lack of effect. The applicability of the developed method was demonstrated by measuring E121 in mouse plasma samples following intraperitoneal administration of various doses ranging from 10 to 100 mg/kg and this study demonstrates that E121 exhibits linear kinetics in the dose range studied.     

A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody,antibody‐conjugated drug and free payload of antibody–drug conjugates

A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65–613.00 ng/mL with an equation y = ax² + bx + c for the antibody‐conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with an equation y = ax² + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01–2200 ng/mL with an equation y = ax² + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐captured‐LC‐TOF‐MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine–citrulline linker at the early drug discovery stage.     

Rapid and efficient method for the quantification of lychnopholide in rat plasma by liquid chromatography–tandem mass spectrometry for pharmacokinetic application

Lychnopholide is a sesquiterpene lactone usually obtained from Lychnophora and Eremanthus species and has pharmacological activities that include anti‐inflammatory and anti‐tumor. Lychnopholide isolated from Eremanthus matogrossenssis was analyzed in this study. The aims of this study were to develop and validate an analytical methodology by LC‐MS/MS and to quantify lychnopholide in rat plasma. Chromatographic separation was achieved on a C₁₈ column using isocratic elution with the mobile phase consisting of methanol and water (containing 0.1% formic acid) at a flow rate of 0.4 mL/min. The detection was performed in multiple‐reaction monitoring mode using electrospray ionization in positive mode. The method validation was performed in accordance with regulatory guidelines and the results met the acceptance criteria. The linear range of detection was 10–200 ng/mL (r > 0.9961). The intra‐ and inter‐day assay variability were <6.2 and <11.7%, respectively. The extraction recovery was approximately 63% using liquid–liquid extraction with chloroform. Lychnopholide was detected in plasma up to 60 min after intravenous administration in rats. This rapid and sensitive method for the analysis of the sesquiterpene lactone lychnopholide in rat plasma can be applied to pharmacokinetic studies of this compound. Copyright © 2016 John Wiley & Sons, Ltd.     

A combination of solid‐phase extraction and dispersive solid‐phase extraction effectively reduces the matrix interference in liquid chromatography–ultraviolet detection during pyraclostrobin analysis in perilla leaves

Perilla leaves contain many interfering substances; thus, it is difficult to protect the analytes during identification and integration. Furthermore, increasing the amount of sample to lower the detection limit worsens the situation. To overcome this problem, we established a new method using a combination of solid‐phase extraction and dispersive solid‐phase extraction to analyze pyraclostrobin in perilla leaves by liquid chromatography with ultraviolet absorbance detection. The target compound was quantitated by external calibration with a good determination coefficient (R² = 0.997). The method was validated (in triplicate) with three fortification levels, and 79.06– 89.10% of the target compound was recovered with a relative standard deviation <4. The limits of detection and quantification were 0.0033 and 0.01 mg/kg, respectively. The method was successfully applied to field samples collected from two different areas at Gwangju and Muan. The decline in the resiudue concentrations was best ascribed to a first‐order kinetic model with half‐lives of 5.7 and 4.6 days. The variation between the patterns was attributed to humidity. Copyright © 2015 John Wiley & Sons, Ltd.     

Proteome analysis of altered proteins in streptozotocin‐induced diabetic rat kidney using the fluorogenic derivatization–liquid chromatography–tandem mass spectrometry method

To find new molecular markers for early diagnosis of diabetic nephropathy, we applied fluorogenic derivatization–liquid chromatography–tandem mass spectrometry to identify the differentially expressed proteins in the kidney of control and streptozotocin‐induced diabetic rats. The Sprague–Dawley rats were injected with the sodium citrate buffer or streptozotocin and then killed after 1, 4, 12 and 24 weeks. The results showed that seven proteins were significantly changed after 1 week of injection. Only one protein had significantly changed after 4 weeks of injection. However, after 12 weeks of injection, the number of altered proteins rose to 10. After 24 weeks of injection, 18 proteins had altered significantly. Five common proteins were significantly altered at week 12 and 24 after injection, respectively. Importantly, these proteins appeared prior to microalbuminuria and may serve as new biomarkers that are able to improve early detection of and new drug development for diabetic‐related nephropathy. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of drugs of abuse in bovine dentin using liquid chromatography–electrospray ionization tandem mass spectrometry

Drugs deposited in human teeth are well preserved; the spectrum of toxicological investigations may therefore be supplemented by an analysis method for drugs in teeth. A liquid chromatography–electrospray ionization tandem mass spectrometry assay for the detection and quantification of basic drugs of abuse in bovine dentin samples was developed and validated. The drugs and metabolites amphetamine, methamphetamine, methylenedioxymethylamphetamine, methylenedioxyethylamphetamine, codeine, morphine, cocaine and benzoylecgonine were extracted from 50 mg ground dentin powder by ultrasonication for 60 min in methanol 3 times. The extracts were analyzed on a triple‐quadrupole mass‐spectrometer in multiple reaction monitoring mode. The method was validated and proved to be accurate, precise, selective, specific and stable with good linearity within the calibration range and a lower limit of quantification of 10 to 20 pg/mg. To artificially load bovine dentin samples with drugs, the natural process of de‐ and remineralization in the oral cavity was mimicked by a pH‐cycling experiment. The artificially drug‐loaded dentin samples showed drug concentrations of 20 to 80 pg/mg. The method can be applied in further in vitro experiments as well as in post‐mortem cases, especially where limited sample tissue is available. Copyright © 2014 John Wiley & Sons, Ltd.