Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a rapid method for the determination of glimepiride in human plasma using liquid-liquid extraction based on 96-well format micro-tubes and liquid chromatography/tandem mass spectrometry

A semi-automated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of glimepiride in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Glimepiride and the internal standard (IS) glibenclamide were extracted from human plasma by LLE, using a mixture of ethyl acetate/diethyl ether 50:50 (v/v) as the organic solvent. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analyte and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by reversed-phase LC/MS/MS with positive ion electrospray ionization, using multiple reaction monitoring. The method proved to be sensitive and specific for both drugs, and statistical evaluation revealed excellent linearity for the range of concentrations 2.0-500.0 ng/mL with very good accuracy and inter- and intra-day precisions. The proposed method enabled the rapid and reliable determination of glimepiride in pharmacokinetic or bioequivalence studies after per os administration of a 3 or 4 mg tablet of glimepiride.     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

Efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions as demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Presence of matrix ions could negatively affect the sensitivity and selectivity of liquid chromatography‐tandem mass spectrometer (LC‐MS/MS). In this study, the efficiency of a miniaturized silica monolithic cartridge in reducing matrix ions was demonstrated in the simultaneous extraction of morphine and codeine from urine samples for quantification with LC‐MS. The miniaturized silica monolith with hydroxyl groups present on the largely exposed surface area function as a weak cation exchanger for solid phase extraction (SPE). The miniaturized silica cartridge in 1 cm diameter and 0.5 cm length was housed in a 2‐ml syringe fixed over a SPE vacuum manifold for extraction. The cleaning effectiveness of the cartridge was confirmed by osmometer, atomic absorption spectrometer, LC‐MS and GC‐TOFMS. The drugs were efficiently extracted from urine samples with recoveries ranging from 86% to 114%. The extracted analytes, after concentration and reconstitution, were quantified using LC‐MS/MS. The limits of detection for morphine and codeine were 2 ng/ml and 1 ng/mL, respectively. The relative standard deviations of measurements ranged from 3% to 12%. The monolithic sorbent offered good linearity with correlation coefficients > 0.99, over a concentration range of 50–500 ng/ml. The silica monolithic cartridge was found to be more robust than the particle‐based packed sorbent and also the commercial cartridge with regards to its recyclability and repeated usage with minimal loss in efficiency. Our study demonstrated the efficiency of the miniaturized silica monolith for removal of matrix ions and extraction of drugs of abuse in urinary screening. Copyright © 2011 John Wiley & Sons, Ltd.     

Study of tanshinone IIA tissue distribution in rat by liquid chromatography-tandem mass spectrometry method

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for determining tanshinone IIA in rat tissues. After a single step liquid-liquid extraction with diethyl ether, tanshinone IIA and loratadine (internal standard) was subjected to LC/MS/MS analysis using positive electro-spray ionization under selected reaction monitoring mode. Chromatographic separation of tanshinone IIA and loratadine was achieved on a Hypersil BDS C(18) column (i.d. 2.1 x 50 mm, 5 microm) with a mobile phase consisting of methanol-1% formic acid (90:10, v/v) at a flow rate of 300 microL/min. The intra-day and inter-day precision of the method were less than 10.2 and 12.4%, respectively. The intra-day and inter-day accuracies ranged from 99.7 to 109.7%. The lowest limit of quantification for tanshinone IIA was 1 ng/mL. The method was applied to a tanshinone IIA tissue distribution study after an oral dose of 60 mg/kg to rats. Tanshinone IIA tissue concentrations decreased in the order of stomach > small intestine > lung > liver > fat > muscle > kidneys > spleen > heart > plasma > brain > testes. Tanshinone IIA still could be detected in most of the tissues at 20 h post-dosing. These results indicate that the LC/MS/MS method was rapid and sensitive to quantify tanshinone IIA in different rat tissues.     

Development and validation of UPLC‐MS/MS method for determination of domperidone in human plasma and its pharmacokinetic application

A sensitive, rapid and selective ultra‐performance liquid chromatography–tandem mass spectrometric (UPLC‐MS/MS) method was developed for the determination and pharmacokinetic study of domperidone in human plasma. Diphenhydramine was used as the internal standard. Plasma sample pretreatment involved a one‐step liquid–liquid extraction with a mixture of diethyl ether–dichloromethane (3:2, v/v). The analysis was carried out on an Acquity UPLC^TM BEH C₁₈ column. The mobile phase consisted of methanol–water containing 10 mmol/L ammonium acetate and 0.5% (v/v) formic acid (60:40, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionizationsource with positive mode. Each plasma sample was chromatographed within 2.1 min. The standard curves for domperidone were linear (r² ≥ 0.99) over the concentration range of 0.030–31.5 ng/mL with a lower limit of quantification of 0.030 ng/mL. The intra‐ and inter‐day precision (relative standard deviation) values were not higher than 13% and accuracy (relative error) was from ?7.6 to 1.2% at three quality control levels. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of domperidone in healthy Chinese volunteers after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxymethamphetamine in urine by online solid-phase extraction and ion-pairing liquid chromatography with detection by electrospray tandem mass spectrometry

A method using an online solid-phase extraction (SPE) and ion-pairing liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MS/MS) was developed for determination of amphetamine (Amp), methamphetamine (mAmp), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. A SPE cartridge column with both hydrophilic and lipophilic functions was utilized for online extraction. A reversed-phase C18 LC column was employed for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. This method was fully automated and the extraction and analysis procedures were controlled by a six-port switch valve. Recoveries ranging from 85-101% were measured. Good linear ranges (10-500 ng/mL) for Amp and mAmp were determined. For MDA, MDMA and MDEA, dual linear ranges were obtained from 5-100 and 100-500 ng/mL, respectively. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, ranged from 1-3 ng/mL. The applicability of this newly developed method was examined by analyzing several urine samples from drug users. Good agreement was obtained between the results from this method and a literature GC/MS method.     

Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.     

Identification of the aromatase inhibitors anastrozole and exemestane in human urine using liquid chromatography/tandem mass spectrometry

Anastrozole (2,2'-[5-(1H-1,2,4-triazol-1-ylmethyl)-1.3-phenylene]bis(2-methylpropionitrile)) and exemestane (6-methylenandrostan-1,4-diene-3,17-dione) are therapeutically used to treat hormone-sensitive breast cancer in postmenopausal women. For doping purposes they may be used to counteract adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase plasma testosterone concentrations. Excretion study urine samples and spot urine samples from women suffering from metastatic breast cancer, being treated with anastrozole or exemestane, were collected and analyzed to develop/optimize a detection system for anastrozole and exemestane to allow the identification of athletes who do not comply with the internationally prohibited use of these cancer drugs. The assay was based on liquid-liquid extraction after enzymatic hydrolysis following liquid chromatography/tandem mass spectrometry (LC/MS/MS). Anastrozole, exemestane and its main metabolite (17-dihydroexemestane) were identified in urine by comparison of mass spectra and retention times with respective reference substances. An assay validation for the analysis of anastrozole and exemestane was performed regarding lower limits of detection (anastrozole: 0.02 ng/mL; exemestane: 3.1 ng/mL; dihydroexemestane: 0.5 ng/mL), interday precisions (6.6-11.1%, 4.9-9.1% and 5.6-8.3% for low [10 ng/mL], medium [50 ng/mL] and high [100 ng/mL] concentration) and recoveries (ranged from 85-97%).     

Quantitative detection of salmeterol after inhalation in equine urine by liquid chromatography/tandem mass spectrometry

A sensitive, accurate and precise liquid chromatography/tandem mass spectrometry (LC/MS(2)) method was developed for the quantification of salmeterol in the urine of horses. The method consists of a liquid-liquid extraction with tert-butylmethyl ether and isopropanol at pH 12 after enzymatic hydrolysis. The extracts are analysed using an LC/MS system equipped with an electrospray ionisation (ESI) probe. Method validation showed excellent linearity, specificity, accuracy, precision and intra-laboratory repeatability and reproducibility. The limit of quantitative detection was 0.25 ng/mL and the limit of detection was 0.125 ng/mL. The excretion profile was determined after administration of 500 microg salmeterol (Serevent) to four standard-bred mares via a metered dose inhaler (MDI) with an Equinehaler adapter. Salmeterol was detected from 1 h until 12 h post-administration. Maximum urinary concentrations varied between 2.3 and 14.9 ng/mL.     

液相色谱-串联质谱法测定尿液中的内源性类固醇激素

建立了液相色谱-串联质谱(LC-MS/MS)测定尿液中的内源性类固醇激素的方法。尿样经葡萄糖醛酸甙酶酶解后进行液-液提取,以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68:32)为流动相,采用Cosmosil C18色谱柱分离,并以三重四极杆串联质谱多反应监测扫描方式对尿样中的脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素进行检测。方法的最低检出限为0.01～10 ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可替代气相色谱-质谱法用于体液中内源性类固醇激素兴奋剂的常规分析。     

Determination of fluorotelomer alcohols by liquid chromatography/tandem mass spectrometry in water

Fluorotelomer alcohols (FTOHs) are important polyfluorinated raw materials that belong to the general category of perfluoroalkyl substances (PFAS). PFAS, including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates, have recently attracted considerable attention because they are persistent and found globally in the environment. FTOHs are precursors that may degrade in the environment to PFCAs. The development of analytical methods for determination FTOHs in environmental samples is necessary to determine the environmental presence of FTOHs. This work presents the development and validation of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of FTOHs (6-2, 8-2, 10-2) in aqueous samples. Chromatographic conditions were optimized in order to obtain focused FTOH chromatographic peaks. The mobile phase and mass spectrometric conditions were optimized to enable formation of deprotonated FTOH molecules in the negative ion electrospray mode. Two extraction methods were investigated using acetonitrile and methyl tert-butyl ether (MTBE). These methods were validated for a range of environmental water samples fortified with FTOHs at three different levels. Both extraction methods resulted in recoveries from 70 to 120%. Detection limits of FTOHs were estimated to be approximately 0.09 ng/mL for LC/MS/MS detection. An LC/MS method was also developed for FTOH determination with an estimated 1.2 ng/mL limit of detection. Various sample storage scenarios were investigated. It was determined that the aqueous samples of FTOHs are best preserved by storing them frozen in sealed vials with aluminum foil lined septa.     

Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.     

Determination of ecgonine and seven other cocaine metabolites in human urine and whole blood by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry

Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 μL of sample by solid-phase extraction with a 96-well μElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1 % or less. The accuracy was in the range from 80.0 to 122.9 %, process efficiencies were in the range from 8.6 to 177.4 %, matrix effects were in the range from 28.7 to 171.0 %, and extraction recoveries were in the range from 41.0 to 114.3 %, except for ecgonine (12.8 % and 9.3 % at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion.

Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor-buprenorphine in urine, blood and hair samples

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the analysis of buprenorphine (BUP) and nor-buprenorphine (NBUP) in biological fluids. Analytes are isolated from urine and blood, after addition of d4-buprenorphine (d4-BUP) as internal standard, by solid-phase extraction. Preparation of hair involves external decontamination, mechanical pulverization, overnight incubation in acidic medium, and neutralization prior to extraction. Enzymatic hydrolysis with beta-glucuronidase may be performed to distinguish between free and total BUP. Chromatographic separation is accomplished by gradient elution on a cyanopropyl 2.1 x 150 mm column. Positive ion ESI and MS analyses are carried out in an ion trap mass spectrometer. The use of this mass analyzer allows effective collisional experiments to be performed on ESI-generated MH+ species. Abundant product ions are produced, which can be monitored together with precursor ions without losing sensitivity. Thus, assay selectivity is definitely increased with respect to LC/ESI-MS/MS methods in which only precursor ions are monitored. The method has good linearity (calibration curves were linear in the range 0.1-10 ng/mL in urine and blood, in the range 10-160 pg/mg in hair) and limits of detection of 0.05 ng/mL for both BUP and NBUP in blood and urine samples, of 4 pg/mg for both analytes in hair. Both intra- and inter-assay precision and accuracy were satisfactory at three concentrations studied: relative standard deviations were <13.7% in urine, <17.3% in blood, <17.8% in hair; percent deviation of the mean from the true value was always <10.5% in urine and blood, <16.1% in hair. The method can be used to determine both analytes in the urine and hair of drug addicts on replacement therapy, and in post-mortem blood specimens when there is suspicion of drug-related death.     

Quantification of cyproheptadine in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid‐liquid extraction using a diethyl‐ether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile/water (50/50 v/v) + 0.1% of acetic acid. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC‐MS/MS). Chromatography was performed isocratically using an Alltech Prevail C18 5 µm analytical column, (150 mm x 4.6 mm I.D.). The method had a chromatographic run time of 4 min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r2 > 0.99). The limit of quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two cyproheptadine + cobamamide (4 mg + 1 mg) tablet formulations (Cobactin® [cyproheptadine + cobamamide] test formulation supplied from Zambon Laboratórios Farmacêuticos Ltda. and Cobavital® from Solvay Farma (standard reference formulation)). A single 4 mg + 1 mg [cyproheptadine + cobamamide] dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two‐period crossover design with a 1‐week washout interval. Since the 90% CI for Cmax and AUCs ratios were all within the 80‐125% bioequivalence limit proposed by the US Food and Drug Administration, it was concluded that the cyproheptadine test formulation (Cobactin®) is bioequivalent to the Cobavital® formulation for both the rate and the extent of absorption of cyproheptadine. Copyright © 2011 John Wiley & Sons, Ltd.     

Screening for anabolic steroids in doping analysis by liquid chromatography/electrospray ion trap mass spectrometry

A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human urine has been developed and validated. Liquid-liquid extraction with diethyl ether was applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds. The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10 ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-hydroxystanozolol, were assessed using the described method.     

A direct injection high-throughput liquid chromatography tandem mass spectrometry method for the determination of a new orally active alphavbeta3 antagonist in human urine and dialysate

A generic high-throughput liquid chromatography (HTLC) tandem mass spectrometry (MS/MS) assay for the determination of compound I in human urine and dialysate (hemodialysis) was developed and validated. By using the HTLC on-line extraction technique, sample pretreatment was not necessary. The sample was directly injected onto a narrow bore large particle size extraction column (50 x 1.0 mm, 60 microm) where the sample matrix was rapidly washed away using a high flow rate (5 mL/min) aqueous mobile phase while analytes were retained. The analytes were subsequently eluted from the extraction column onto an analytical column using an organic-enriched mobile phase prior to mass spectrometric detection. The analytes were then eluted from the analytical column to the mass spectrometer for the determination. The linear dynamic range was 2.0-6000 ng/mL for the urine assay and 0.1-300 ng/mL for the dialysate assay. Intraday accuracy and precision were evaluated by analyzing five replicates of calibration standards at all concentrations used to construct the standard curve. For the urine assay, the precision (RSD%, n=5) ranged from 1.9 to 8.0% and the accuracy ranged from 87.8 to 105.2% of nominal value. For the dialysate assay, the precision (RSD%, n=5) ranged from 1.1 to 10.0% and the accuracy from 94.5 to 105.2% of nominal value. In-source fragmentation of the acyl glucuronide metabolite (compound III) did not interfere with the determination of parent compound I. The developed HTLC/MS/MS methodology was specific for compound I in the presence of compound III. Column life-time is increased and sample analysis time is decreased over traditional reversed-phase methods when direct injection assays for urine and dialysate are coupled with the technology of HTLC.     

Simultaneous LC‐MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self‐microemulsifying drug‐delivery systems

In this study, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid–liquid extraction using methyl tert‐butyl ether–dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB‐C₁₈ column (100 × 2.1 mm, 3 μm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide‐d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra‐ and inter‐day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel‐loaded self‐microemulsifying drug‐delivery systems.     

Simultaneous determination of nalbuphine and its prodrug sebacoly dinalbuphine ester in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic study in humans

A rapid, simple, sensitive and selective ultraperformance liquid chromatography–tandem spectrometry (UPLC‐MS/MS) method for the determination of nalbuphine and its prodrug sebacoly dinalbuphine ester (SDE) was developed and validated in human plasma. The sample pretreatment involves basification and iterative liquid–liquid extraction with ethyl‐ether–dichloromethane (7:3, v/v) solution, followed by LC separation and positive electrospray ionization (ESI) API‐3000 mass spectrometry detection. The chromatography was on a Waters Acquity UPLC BEH HILIC column (2.1 × 100 mm, 1.7 µm). The mobile phase was composed of acetonitrile and water (83:17, v/v) that contained 0.2% formic acid and 4 mm ammonium formate at a flow rate of 0.25 mL/min. Ethylmorphine and naloxine were selected as the SDE and nalbuphine internal standard (IS), respectively. The calibration curve for both was linear over the range from 0.05 to 20 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantification was set at 0.05 ng/mL. The intra‐ and inter‐day precision values for nalbuphine and SDE were acceptable as per FDA guidelines. The method was applied successfully to determine nalbuphine concentration in human plasma samples obtained from four Taiwanese volunteers receiving intramuscularly administration of sebacoyl dinalbuphine ester. The method is sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2013 John Wiley & Sons, Ltd.