Simple methodology for the therapeutic drug monitoring of the tyrosine kinase inhibitors dasatinib and imatinib

A simple HPLC method has been developed to measure imatinib and N‐desmethylimatinib (norimatinib) in plasma or serum at concentrations attained during therapy. Adaptation of this method to LC‐MS/MS also allows dasatinib assay. A small sample volume (100 μL HPLC‐UV, 50 μL LC‐MS/MS) is required and analysis time is <5 min in each case. Detection was by UV (270 nm) or selective reaction monitoring (two transitions per analyte) tandem mass spectrometry. Assay calibration was linear (0.05–10 mg/L imatinib, 0.01–2.0 mg/L norimatinib and 1–200 µg/L dasatinib), with acceptable accuracy (86–114%) and precision (<14% RSD) for both methods. A comparison between whole blood and plasma confirmed that plasma is the preferred sample for imatinib and norimatinib assay. For dasatinib, although whole blood concentrations were slightly higher, plasma is still the preferred sample. Despite considerable variation in the (median, range) plasma imatinib and norimatinib concentrations in patient samples [1.66 (0.02–4.96) and 0.32 (0.01–0.99) mg/L, respectively, N = 104], plasma imatinib was >1 mg/L (suggested target for response) in all but one sample from patients achieving complete molecular response. As to dasatinib, the median (range) plasma dasatinib concentration was 13 (2‐143) µg/L (N = 33). More observations are needed to properly assess the potential role of therapeutic drug monitoring in guiding treatment with dasatinib. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of selected‐ion flow‐tube mass spectrometry for the measurement of ethanol,methanol and isopropanol in physiological fluids: effect of osmolality and sample volume

Selected‐ion flow‐tube mass spectrometry (SIFT‐MS) is particularly suited for the analysis of volatile low molecular weight compounds. We have evaluated this technique for the assay of different alcohols in aqueous solutions, including blood plasma, and in particular whether the osmolality or sample volume affected vapourisation. Solutions of three different alcohols (methanol, ethanol and isopropanol) ranging from 0.005 to 50 mmol/L were prepared in deionised water (0 milliosmol), phosphate‐buffered saline (690 mOsm), isotonic saline (294 mOsm) and plasma (296 mOsm). The vapour above the sample (50 to 1000 µL) contained in air‐tight tubes at 37°C was aspirated into the instrument. The outputs for ethanol, methanol and isopropanol were linear over the concentration range and independent of the sample volume and relatively independent of the osmolar concentration. SIFT‐MS can reliably and accurately measure common alcohols in the headspace above aqueous solutions, including serum/plasma. This novel application of SIFT‐MS is easy to follow, requires no sample preparation and the wide dynamic range will facilitate measurement of alcohols present from normal metabolism as well as when taken in excess or in accidental poisoning. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on‐line post‐column derivatization

In the present study, we propose the first HPLC method coupled to postcolumn derivatization for the determination of rimantadine in human urine samples. The analyte and amantadine (internal standard) were isocratically separated using an RP monolithic stationary phase (100 × 4.6 mm id) with a mobile phase consisting of CH₃OH/phosphate buffer (25 mmol/L, pH 3.0) at a volume ratio of 50:50. Postcolumn derivatization involved on‐line reaction with o‐phthalaldehyde (20 mmol/L) and N‐acetyl‐cysteine (5 mmol/L) at alkaline medium (100 mmol/L borate pH 11.0). Spectrofluorimetric detection at λ_ex/λ_em = 340/455 nm enabled the selective and sensitive determination of rimantadine in urine samples at a range of 50–500 ng/mL with an LOD of 5 ng/mL. Human urine samples were analyzed successfully after SPE using hydrophilic‐lipophilic balanced RP cartridges (30 mg/mL, Oasis HLB). Recoveries ranged between 89.7 and 102.7%.     

A New Sensor for Simultaneous Determination of Tyrosine and Dopamine Using Iron(III) Doped Zeolite Modified Carbon Paste Electrode

A new chemically modified electrode is constructed based on iron(III) doped zeolite modified carbon paste electrode (Fe³⁺Y/ZMCPE). The new sensor could be used for the simultaneous determination of the biologically important compounds dopamine (DA) and tyrosine (Tyr). The measurements are carried out using differential pulse voltammetric (DPV) method. The prepared modified electrode shows voltammetric responses of high sensitivity, selectivity and stability for DA and Tyr under optimal conditions, which makes it a suitable sensor for simultaneous trace detection of DA and Tyr in solution. Application of the DPV method demonstrates that in the Briton Robinson buffer solutions (pH=5) containing 50 µmol/L Tyr, there is a linear relationship between the oxidation peaks and the concentrations of DA over the range of 0.1–200 µmol/L, with a detection limit of 0.05 µmol/L (S/N=3). For Tyr a linear correlation between oxidation peak current and concentration of Tyr over the range of 0.5–200 µmol/L (containing 50 µmol/L DA), with a detection limit of 0.08 µmol/L is obtained. The analytical performances of this sensor are evaluated for the detection of DA and Tyr in human serum and a medicine.     

Upregulated plasma and urinary levels of nucleosides as biological markers in the diagnosis of primary gallbladder cancer

We first detected aberrant nucleoside levels in the plasma, urine, bile, and tissues from cases and controls to explore them as biomarkers in the diagnosis of gallbladder cancer. Reversed‐phase high‐performance liquid chromatography was used to assess the levels of ten nucleosides in these samples from gallbladder cancer patients, gallstone patients, and healthy controls. Plasma and urine samples were collected from patients with gallbladder cancer (n = 202), patients with gallstones (n = 203), and healthy controls (n = 205); bile and tissue samples were collected from 91 gallbladder cancer patients, 93 gallstone patients; and 90 were donated after cardiac death. Of the ten nucleosides analyzed, eight urinary nucleosides, five plasma nucleosides, three bile nucleosides, and one tissue nucleoside were significantly upregulated in the gallbladder cancer patients compared to control groups (p < 0.05). Among these upregulated nucleosides, the sensitivity, specificity, and accuracy of urinary nucleosides in the diagnosis of gallbladder cancer patients were 89.4, 97.1, and 95.7%, respectively, those of plasma nucleosides were 91.2, 95.6, and 94.2%, respectively, those of bile nucleosides were 95.3, 96.4, and 95.1%, respectively, and those of tissue nucleosides were 86.2, 93.8, and 92.6%, respectively. These results suggest that nucleosides may be as useful as biological markers for gallbladder cancer.     

Liquid‐phase microextraction combined with liquid chromatography‐mass spectrometry. Extraction from small volumes of biological samples

Liquid‐phase microextraction (LPME) is a sample preparation technique based on disposable polypropylene hollow fibres, which results in efficient sample clean‐up and high preconcentration. The present paper describes the combination of LPME with LC‐MS utilising electrospray ionisation for high sensitivity. Nine antidepressant drugs were extracted from 50 or 500 μL of plasma or whole blood samples, through a thin layer of dodecyl acetate immobilised in the pores of the hollow fibre, and into 15 μL of 200 mM formic acid as acceptor solution inside the hollow fibre. Analyte recoveries in the range 12–68% and 9–52% were obtained from 50 μL of plasma and whole blood respectively. The acceptor solution (15 μL) was diluted with 60 μL of 5 mM ammonium formate pH = 2.7 prior to injection into the LC‐MS system. The system was qualitatively investigated for matrix effects utilising a post‐column infusion system. Whole blood from 5 different persons was cleaned‐up by LPME and injected onto the analytical column while a solution of the 9 model compounds was continuously infused post‐column. No signs of ion suppression were seen for any of the model compounds. Limits of quantification (S/N = 10) were in the low ng/mL range for 6 of the 9 model compounds utilising a whole blood sample volume of only 50 μL. The repeatability of the extractions was investigated utilising paroxetine as internal standard. Acceptable RSDs (%) were obtained (< 20%) for 5 of the antidepressants. By increasing the sample volume from 50 to 500 μL of whole blood RSDs below 20% (3–16%) were observed for all 8 antidepressants.     

Study of normal and modified nucleosides in serum by RP-HPLC

Summary Modified nucleosides derived predominantly from transfer ribonucleic acid (tRNA) have been studied as possible tumor markers. In this paper a reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been applied to study 15 normal and modified nucleosides in serum. The nucleoside levels in normal human serum were established, and the concentrations of 15 nucleosides in serum from 19 cancer patients were determined. It was found that the concentrations of modified nucleosides were significantly higher in patient sera. Based on 15 nucleoside concentrations in serum, factor analysis could classify correctly 90% of cancer patients from the normal humans. Further work showed that urine was slightly better than serum when determining nucleosides as biological marker candidates. More work is ongoing to determine the clinical usefulness of modified nucleosides as tumor markers.     

Monosegmented flow-analysis of slow enzymatic reactions: Determination of triglycerides in serum

 A monosegmented flow system (MCFA) is proposed to achieve slow enzymatic spectrophotometric determinations, here applied to the determination of triglycerides in blood serum. The sample (4.5 μL), enzymatic reagent (150 μL) and an air plug (100 μL) are simultaneously inserted into a carrier stream buffered to pH 7.9 (Tris ⋅ HCl). In order to avoid the cumbersome step of air removal, a relocating detector was used. This system handles about 60 samples/h, yielding precise results (r.s.d. usually<2.5%). Sensitivity is 56 mAU ⋅ L/mmol up to 6 mmol/L triglycerides. Accuracy was assessed by running 50 samples already analysed by a conventional procedure yielding the equation C_MCFA(mmol/L)=1.00(±0.04) C_Ref(mmol/L)−0.03(±0.08); r=0.990. Received: 22 January 1997/Revised: 12 March 1997/Accepted: 28 March 1997     

A Rapid,Amplification‐Free,and Sensitive Diagnostic Assay for Single‐Step Multiplexed Fluorescence Detection of MicroRNA

The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short‐sequence nucleic acids next‐generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single‐step, single‐temperature, and amplification‐free assay provides a unique combination of performance parameters compared to state‐of‐the‐art miRNA detection technologies. Precise multiplexed quantification of miRNA‐20a, ‐20b, and ‐21 at concentrations between 0.05 and 0.5 nM in a single 150 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high‐throughput and point‐of‐care clinical diagnostics.     

Reversed-Phase High Performance Liquid Chromatography (HPLC) of Nucleosides in Cell Extracts with Special Reference to Deoxythymidine

Abstract

A reversed-phase high performance liquid chromatography (HPLC) system was designed to assay the nucleoside concentrations, especially deoxythymidine, of tissue cultured cells. Concentration and purification of the acid extracted cell samples were achieved by utilizing a Sep-Pak C₁₈ cartridge. After adsorption of the sample, the cartridge was washed with 0.5 ml of water followed by 2 × 0.5 ml of 2.5% methanol. The compounds of interest were subsequently eluted in 2 × 0.5 ml of methanol. The cartridge procedure was found to be fast, inexpensive and showed good recoveries for most tested nucleosides. The nucleosides uridine, deoxythymidine and adenosine could be detected in green monkey kidney cells. Ribonucleotides and deoxyribonucleotides could be separated from each other with the HPLC system used.     

胶束电动力学毛细管色谱法测定尿中核苷

用胶束电动力学毛细管色谱法测定尿中核苷.通过苯基硼酸亲和色谱柱对尿中核苷进行纯化,以十二烷基磺酸钠(SDS)-十水合四硼酸钠-磷酸二氢钠为缓冲液(pH=6.96),在未涂层石英毛细管上,于7kV恒压,29℃下进行电泳分离,检测波长为254nm.此方法用于28个正常人尿中14种核苷的测定,确定了正常成年人(汉族)尿中核苷的排放范围.     

Application of ISO 13528 robust statistical method for external quality assessment of blood glucose measurements in China

External quality assessment (EQA) including extensive participants often result in abnormal distribution. Robust statistical methods are insensitive to slight deviation for a given probability model and can estimate the population parameters utilizing robust algorithm. The aim of this study was to evaluate the blood glucose testing performance of EQA in China using ISO 13528 robust statistical method. A total of 4571 participants measured two lots of materials at different concentrations and submitted the results through the Clinet-EQA reporting system V1.5. Data were collected and analyzed by Clinet-EQA evaluating system and SPSS 13.0. Participants were classified by method principles they adopted. Acceptability of relative differences between each result and robust mean of the group was calculated using biological variation desirable quality specification for allowable total error. The kernel density plots and Youden Plot were also applied to show the distribution of z-scores. The robust means of the two lots were 6.31 mmol/L and 15.71 mmol/L, and the robust standard deviations were 0.23 mmol/L and 0.5 mmol/L, respectively. Acceptability of relative differences was more than 90 % among all groups. About 91.5% and 89.7 % of the participants out of the two lots fulfilled the condition of |z| ≤ 2. In addition, 87.2 % participants fell inside the confidence ellipse for probability levels of 95 % in Youden Plot. According to the statistical results, we can conclude that most participants are qualified for performing blood glucose test and ISO 13528 robust statistical method is appropriate for EQA result analysis.     

Application of solid-phase microextraction and gas chromatography-mass spectrometry for the determination of chlorophenols in leather

This paper proposes a new analytical procedure based on the headspace solid‐phase microextraction (HS‐SPME) technique and gas chromatography‐selected ion monitoring‐mass spectrometry (GC‐SIM‐MS) for the determination of 16 phenols extracted from leather samples. The optimized conditions for the HS‐SPME were obtained through two experimental designs – a two‐level fractional factorial design followed by a central composite design – using the commercial SPME fiber polyacrylate 85 μm (PA). The best extraction conditions were as follows: 200 μL of derivatizing agent (acetic anhydride), 20 mL of saturated aqueous NaCl solution and extraction time and temperature of 50 min and 75°C, respectively. All optimized conditions were obtained with fixed leather sample mass (250 mg), vial volume (40 mL) and phosphate buffer pH (12) and concentration (50 mmol/L). Detection limits ranging from 0.03 to 0.20 ng/g, and relative standard deviation (RSD) lower than 10.23% (n=6) for a concentration of 800 ng/g (chlorophenols) and 1325 ng/g (2‐phenylphenol) in the splitless mode were obtained. The recovery was studied at three concentration levels by adding different amounts of phenols to the leather sample and excellent recoveries ranging from 90.0 to 107.2% were obtained. The validated method was shown to be suitable for the quantification of phenols in leather samples, as it is simple, relatively fast and sensitive.     

Screening of seized cocaine samples using electrophoresis microchips with integrated contactless conductivity detection

This study describes the development of a new analytical method for the separation and detection of cocaine (COC) and its adulterants, or cutting agents, using microchip electrophoresis (ME) devices coupled with capacitively coupled contactless conductivity detection (C⁴D). All the experiments were carried out using a glass commercial ME device containing two pairs of integrated sensing electrodes. The running buffer composed of 20 mmol/L amino‐2‐(hydroxymethyl) propane‐1,3‐diol and 10 mmol/L 3,4‐dimethoxycinnamic acid provided the best separation conditions for COC and its adulterants with baseline resolution (R > 1.6), separation efficiencies ranging from (2.9 ± 0.1) to (3.2 ± 0.2) × 10⁵ plates/m, and estimated LOD values between 40 and 150 μmol/L. The quantification of COC was successfully performed in four samples seized by the Brazilian Federal Police Department and all predicted values agree with values estimated by the reference method. Some other interfering species were detected in the seized samples during the screening procedure on ME–C⁴D devices. While lidocaine was detected in sample 3, the presence of levamisole was observed in samples 2 and 4. However, their concentrations were estimated to be below the LOQ. ME–C⁴D devices have proved to be quite efficient for the identification and quantification of COC with errors lower than 10% when compared to the data obtained by a reference method. The approach herein reported offers great potential to be used for on‐site COC screening in seized samples.     

毛细管区带电泳-间接紫外检测法快速测定食品中乳糖、蔗糖、葡萄糖和果糖

建立了毛细管区带电泳-间接紫外检测快速测定食品中乳糖、蔗糖、葡萄糖和果糖的方法。以水或5 mmol/L醋酸为样品提取液,未涂层熔融石英毛细管(30.2 cm(有效长度20 cm)×50 μm)为分离柱,4 mmol/L山梨酸钾+10 mmol/L磷酸钠+30 mmol/L NaOH(pH 12.56)+0.5 mmol/L十六烷基三甲基溴化铵(CTAB)为分离缓冲液,在-8 kV下分离,于254 nm波长下检测,10 min内实现了食品中上述4种糖的同时分离与测定。乳糖、蔗糖、葡萄糖和果糖的检出限(S/N=3)分别为50、75、25和25 mg/L,定量限(S/N=10)分别为150、225、75和75 mg/L,回收率在87.0%~107.0%之间,相对标准偏差在1.2%~4.7%之间。整个实验过程未使用有机溶剂。用该法测定了9种食品样品及1个质控样品,结果表明该法简单、快速、准确,适用于食品中乳糖、蔗糖、葡萄糖和果糖的日常测定。     

Development of a Carbon Paste Electrode Modified with Reduced Graphene Oxide and an Imidazole Derivative for Simultaneous Determination of Biological Species of N‐acetyl‐L‐cysteine,Uric Acid and Dopamine

In this work, the modified carbon paste electrode (CPE) with an imidazole derivative 2‐(2,3 dihydroxy phenyl) 4‐methyl benzimidazole (DHPMB) and reduced graphene oxide (RGO) was used as an electrochemical sensor for electrocatalytic oxidation of N‐acetyl‐L‐cysteine (NAC). The electrocatalytic oxidation of N‐acetyl‐L‐cysteine on the modified electrode surface was then investigated, indicating a reduction in oxidative over voltage and an intensive increase in the current of analyte. The scan rate potential, the percentages of DHPMB and RGO, and the pH solution were optimized. Under the optimum conditions, some parameters such as the electron transfer coefficient (α) between electrode and modifier, and the electron transfer rate constant) k_s) in a 0.1 M phosphate buffer solution (pH=7.0) were obtained by cyclic voltammetry method. The diffusion coefficient of species (D) 3.96×10^?5 cm² s^?1 was calculated by chronoamperometeric technique and the Tafel plot was used to calculate α (0.46) for N‐ acetyl‐L‐cysteine. Also, by using differential pulse voltammetric (DPV) technique, two linear dynamic ranges of 2–18 µM and 18–1000 µM with the detection limit of 61.0 nM for N‐acetyl‐L‐cysteine (NAC) were achieved. In the co‐existence system of N‐acetyl‐L‐cysteine (NAC), uric acid (UA) and dopamine (DA), the linear response ranges for NAC, UA, and DA are 6.0–400.0 µM, 5.0–50.0 µM and 2.0–20.0 µM, respectively and the detection limits based on (C=3s_b/m) are 0.067 µM, 0.246 µM and 0.136 µM, respectively. The obtained results indicated that DHPMB/RGO/CPE is applicable to separate NAC, uric acid (UA) and dopamine (DA) oxidative peaks, simultaneously. For analytic performance, the mentioned modified electrode was used for determination of NAC in the drug samples with acceptable results, and the simultaneous determination of NAC, UA and DA oxidative peaks was investigated in the serum solutions, too.     

Affinity capillary electrophoresis coupling with partial filling technique and field‐amplified sample injection for enantioseparation and determination of DL‐tetrahydropalmatine

A novel, simple and sensitive method for the enantioseparation and determination of DL ‐tetrahydropalmatine (DL ‐THP) was developed using ACE in combination with partial filling technique and field‐amplified sample injection. A chiral selector, i.e. BSA, was used for the enantioseparation of DL ‐THP in ACE. Effects of BSA concentration, pH and separation voltage on the effectiveness of the enantiomer separation were evaluated. In an optimal condition, D ‐ and L ‐THP were completely enantio‐separated in less than 9 min by partially filling an electrophoretic capillary with 50 μmol/L BSA (50 mbar, 100 s) and carrying out an electrophoresis with 20 mmol/L phosphate buffer (pH 7.4) at 15 kV. The sensitivity was further improved by making use of field‐amplified sample injection to lower the LOD (defined as S/N=3) down to 6 ng/mL. Real samples were also tested and promising results for the determination of DL ‐THP enantiomers were obtained.     

Monitoring disopyramide, lidocaine, and quinidine by micellar liquid chromatography

A micellar liquid chromatography (MLC) method using a C18 column was developed to determine three antiarrhythmic drugs--disopyramide, lidocaine, and quinidine--that are most usually monitored in serum samples. After the application of an interpretative strategy for optimization of sodium dodecyl sulfate (SDS) and modifier concentrations in order to ensure the minimum analysis time, maximum sensitivity, and good resolution, the optimum chromatographic conditions for the determination of the three antiarrhythmics were flow rate, 1 mL/min; injection volume, 20 microL; separation temperature, 25 degrees C; mobile phase, 150 mmol/L SDS-7% (v/v) butanol-phosphate buffer, 10 mmol/L, pH 7-0.9% (w/v) NaCl; and detection at 214 nm. The calibration curves for the drugs were linear (r2 > 0.999). The intraday and interday precisions were lower than 3.9% (CV). Recoveries were 100 +/- 0.6% when the method was applied to both serum samples spiked with the antiarrhythmics (n = 10) and real serum samples. In all cases, the results were similar to those obtained using the reference method (fluorescence polarization immunoassay) usually used in the Spanish hospital. The proposed method is useful for hospital monitoring of the antiarrhythmics by direct injection into the chromatograph.     

Nanosensor for dopamine and glutathione based on the quenching and recovery of the fluorescence of silica-coated quantum dots

We have constructed a fluorescent nanosensor for dopamine (DA) and glutathione (GSH) in physiologically relevant concentrations. CdTe quantum dots (QDs) were coated with silica, and dopamine-quinone (formed by oxidation of DA) is captured on the surface of silica via dual interactions (hydrogen bonding and electrostatic interaction) and quenches the photoluminescence of the modified QDs by an electron transfer process. GSH, in being a strong reducing agent, can chemically reduce the dopamine-quinone on the QDs, and this results in recovered photoluminescence. There are linear relationships between the concentrations of dopamine and GSH respectively and the intensity of the photoluminescence intensity of the QDs both in the quenched and regenerated form, the ranges being 0.0005 to 0.1 mmol?L^?1 for dopamine, and 0.1 to 10 mmol?L^?1 for GSH. The method was applied to the determination of dopamine and GSH in human serum samples with satisfactory results.

Determination of inorganic cations in biological fluids using a hybrid capillary electrophoresis device coupled with contactless conductivity detection

We describe the assembly of a hybrid electrophoresis device that contains fused silica capillaries interconnected to a microfabricated interface in a cross format for the determination of inorganic cations in biological samples. The sample transport in the proposed hybrid device was performed under gated injection mode and the separations were monitored with a capacitively coupled contactless conductivity detector. The capillary extremities were inserted into polypropylene tubes to create solution reservoirs. Sensing electrodes were produced using stainless‐steel hypodermic needles previously cut with 2.0 mm length. The running composition and injection time were optimized and the best results were found using 50 mmol/L lactic acid, 20 mmol/L histidine and 3 mmol/L 18‐crown‐6 ether, and an electrokinetic injection time of 15 s. The separation of six inorganic cations was achieved with baseline resolution, and efficiencies were between 9.1 × 10³ and 5.4 × 10⁴ plates/m. The proposed hybrid device was explored for determining the concentration levels of inorganic cations in urine, saliva, and tear samples, employing Li⁺ as an internal standard. The achieved results were in good agreement with the data reported in the literature. The reliability of the proposed method ranged from 93 to 98%, thus suggesting satisfactory accuracy for bioanalytical applications.