High-performance liquid chromatography coupled with tandem mass spectrometry for the detection of amyloid Beta peptide related with Alzheimer's disease.

Recent studies show that quantitative and qualitative differences in amyloid beta (Abeta ) peptides may be implicated in the development of Alzheimer's disease. New evidence seems to support the existence of a dynamic equilibrium between Abeta peptide in the brain and peripheral blood circulation. The quantitation of Abeta in the blood may allow the development of the potential value of Abeta peptides as a biomarker in the development of Alzheimer's disease. In this communication, quantitation of Abeta peptides using high-performance liquid chromatography coupled with tandem mass spectrometry in a linear ion trap mode is presented. RP-HPLC was performed using a Waters Xterra MS C8 column (3.0 mm x 150 mm). Abeta(1-40) peptide was eluted using a gradient elution program. Eluate from the RP-HPLC column was split to both the UV detector and electrospray ionization MS source. The product ion scan was performed in a linear ion trap mode utilizing the transition of a multiply charged molecular ion of Abeta(1-40) to a singly charged product ion. The detection limit of 31.25 ng in column load using a 3.0-mm-diameter conventional C8 column was achieved. The Abeta(1-40) standard calibration curves show excellent linearity from 34 ng to 2500 ng Abeta(1-40) of column sample load. The product ion scan enhances sensitivity 10 times compared with the best previously achieved by a single-quadrupole instrument in the selective ion monitoring mode. Moreover, the product ion scan of Abeta(1-40) provides superior selectivity and specificity, which is very important in the quantitation of Abeta(1-40) in a complex biological matrix.     

Field gas chromatography-mass spectrometry for fast analysis

The objective of this presentation is to demonstrate the original device and procedure for fast gas chromatography-mass spectrometry (GC-MS) analysis of gaseous and liquid samples and to discuss its features and capabilities. The concept was developed in order to expand the range of compounds suitable for GC separation and to reduce the time of analysis. Field GC-MS, consisting of original "concentrator-thermodesorber" (CTD) unit, multiple module GC system and compact magnetic mass spectrometer with powerful two-stage vacuum system and multicollector ion detector, is represented. The whole weight of the device is 90 kg. Power consumption is 250 W. The device and analytical procedures allow high speed screening of toxic substances in air and extracts within 100 s per sample. The examples of applications are described, including fast screening of tributyl phosphate (TBP) in air at low ppt level at the rate 1 sample/min.     

Analyzing the performance of ArcCHECK diode array detector for VMAT plan

Aim

The aim of this study is to evaluate performance of ArcCHECK diode array detector for the volumetric modulated arc therapy (VMAT) patient specific quality assurance (QA). VMAT patient specific QA results were correlated with ion chamber measurement. Dose response of the ArcCHECK detector was studied.

Background

VMAT delivery technique improves the dose distribution. It is complex in nature and requires proper QA before its clinical implementation. ArcCHECK is a novel three dimensional dosimetry system.

Materials and methods

Twelve retrospective VMAT plans were calculated on ArcCHECK phantom. Point dose and dose map were measured simultaneously with ion chamber (IC-15) and ArcCHECK diode array detector, respectively. These measurements were compared with their respective TPS calculated values.

Results

The ion chamber measurements are in good agreement with TPS calculated doses. Mean difference between them is 0.50% with standard deviation of 0.51%. Concordance correlation coefficient (CCC) obtained for ion chamber measurements is 0.9996. These results demonstrate a strong correlation between the absolute dose predicted by our TPS and the measured dose. The CCC between ArcCHECK doses and TPS predictions on the CAX was found to be 0.9978. In gamma analysis of dose map, the mean passing rate was 98.53% for 3% dose difference and 3 mm distance to agreement.

Conclusions

The VMAT patient specific QA with an ion chamber and ArcCHECK phantom are consistent with the TPS calculated dose. Statistically good agreement was observed between ArcCHECK measured and TPS calculated. Hence, it can be used for routine VMAT QA.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection

A liquid chromatographic method with on-line activity determination for microperoxidases has been developed. After enzymatic digestion of a cytochrome, possibly under formation of microperoxidases, the product mixture is separated by reversed-phase liquid chromatography. The products first pass a diode-array detector, and are then subjected to a reaction with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) and hydrogen peroxide. In a reaction coil, microperoxidases catalyze the reaction under formation of the fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA). Quantification of the microperoxidases is performed using a fluorescence detector at an excitation wavelength of 470 nm and an emission wavelength of 545 nm, respectively. For this LC-based detection system, limits of detection are 3 x 10(-8) mol/L, limits of quantification are 9 x 10(-8) mol/L, and a linear range from 9 x 10(-8) mol/L to 3 x 10(-6) mol/L is obtained for the microperoxidases MP-9 and MP-11. A highly active microperoxidase MP-6 was found in the reaction of cytochrome c from bovine heart with protease from streptomyces griseus.     

A task-based evaluation of PEM detector element size

Positron Emission Mammography (PEM) is a planar imaging method that utilizes arrays of discrete detector elements for the detection of radiotracer-avid breast cancer. In this investigation we have systematically studied, through computer simulations, the effect of detector element size (width and length) on breast lesion detection and localization tasks. The contrast-to-noise ratios of the spheres simulating breast lesions were calculated as a function of detector element dimension to gauge detectability. System resolution (fwhm) across the field-of-view was used as the metric for the localization task. For both tasks, individual detector elements of lyso with cross sectional dimensions of 2×2 mm (96×72 element arrays, step 2.1mm) and 3×3mm (65×49 element arrays, step 3.1 mm), and lengths of 10,15 and 20 mm were simulated. The results revealed that narrower pixel dimensions reduced the partial volume effect, while the thicker pixels increased pixel sensitivity, thus reducing noise per pixel and increasing the contrast-to-noise ratio.     

Flow injection amperometric detection of OP nerve agents based on an organophosphorus-hydrolase biosensor detector

A flow-injection system with an organophosphorus-hydrolase (OPH)-biosensor detector has been developed and characterized for the rapid detection of organophosphorus (OP) nerve agents. The enzyme was immobilized onto a thin-film gold detector through a cystamine-glutaraldehyde coupling. Factors influencing the performance were optimized. The resulting flow system offered a fast, sensitive, selective, and stable response. The peak current increased linearly with the concentration of paraoxon and methyl parathion over the 1-10 microM range (sensitivity, 2.29 and 1.04 nA/microM, respectively). The OPH-biosensor flow injection systems offered low detection limits (e.g. 0.1 microM paraoxon), along with a good precision (R.S.D. of 3.6% for 20 successive injections of a 1.0 microM paraoxon solution). The OPH-biosensor flow detector offers great promise for rapid field screening of OP pesticides and nerve agents.     

High-resolution magnetoencephalography--studies with a small-volume phantom]

To investigate the spatiotemporal organisation of neuronal processes in an animal model using magnetoencephalography (MEG), a high temporal resolution (ms) and an appropriate spatial resolution of about 1 mm is necessary. With the aim of determining the localization error and the resolution power of high-resolution MEG systems, we developed a phantom capable of simulating the characteristics of animal models. The phantom enables us to variably position at least two magnetic field sources to within 0.1 mm. For source localization on the basis of the magnetic field data, a spatial filtering algorithm was used. The investigation of a 16-channel micro SQUID-MEG system with a current dipole orientated tangentially to the phantom surface produced the following localization data (min ... max, x, y--horizontal plane, z--depth); systematic localization error e(x) = 1.16 ... 1.67 mm, e(y) = -1.01 ... -1.28 mm, e(z) = -5.22 ... -7.64 mm, standard deviation of the individual measurements perpendicular to the dipole axis s(perp) = 0.05 ... 0.22 mm, along this axis s(long) = 0.20 ... 1.73 mm, in the depths sz = 0.17 ... 3.17 mm. The "goodness of fit" was > 95%. Separation of two dipoles was still possible for parallel dipoles at a distance apart of d(parallel) = 0.03 mm and for those oriented perpendicularly to each other at a distance apart of d(perp) = 0.10 mm. On the basis of these results we conclude that the MEG system can achieve a resolution sufficient to permit the investigation of neuronal microstructures. The spatial errors detected were related to sensor position in the cryostatic vessel as well as to external low-frequency noise.     

Determination of aesculin in rat plasma by high performance liquid chromatography method and its application to pharmacokinetics studies

A sensitive and reproducible high performance liquid chromatography method with UV detection was described for the determination of aesculin in rat plasma. After deproteinization by methanol using metronidazole as internal standard (I.S.), solutes were evaporated to dryness at 40 degrees C under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of mobile phase and a volume of 20 microl was injected into the HPLC for analysis. Solutes were separated on a Diamonsil C18 column (250 mm x 4.6 mm i.d., 5 microm particle size, Dikma) protected by a ODS guard column (10 mm x 4.0 mm i.d., 5 microm particle size), using acetonitrile-0.1% triethylamine solution (adjusted to pH 3.0 using phosphoric acid) (10:90, v/v) as mobile phase (flow-rate 1.0 ml/min), and wavelength of the UV detector was set at 338 nm. No interference from any endogenous substances was observed during the elution of aesculin and internal standard (I.S., metronidazole). The retention times for I.S and aesculin were 10.4 and 12.4 min, respectively. The limit of quantification was evaluated to be 57.4 ng/ml and the limit of detection was 24.0 ng/ml. The method was used in the study of pharmacokinetics of aesculin after intraperitoneal injection (i.p.) administration in rats.     

Assay of tramadol in urine by capillary electrophoresis using laser-induced native fluorescence detection

Capillary electrophoresis (CE) with UV laser-induced native fluorescence detection was developed as a sensitive and selective assay for the direct determination of tramadol in human urine without extraction or preconcentration. The main problem in CE is the small inner diameter of the capillary which causes a low sensitivity with instruments equipped with a UV detector. Laser-induced native fluorescence with a frequency doubled argon ion laser at an excitation wavelength of 257 nm was used for the direct assay of tramadol in urine to enhance the limit of detection about 1000-fold compared to UV absorption detection. The detection system consists of an imaging spectrograph and an intensified CCD camera, which views an illuminated 1.5 mm section of the capillary. This set-up is able to record the whole emission spectra of the analytes to achieve additionally wavelength-resolved electropherograms. In the concentration range of 20 ng/ml–5 μg/ml in human urine coefficients of correlation were better than 0.998. Within-day variation determined on four different concentrations showed accuracies ranging from 90.2 to 108.4%. The relative standard deviation (RSD) was determined to be less than 10%. Day-to-day variation presented accuracies ranging from 90.9 to 103.1% with an RSD less than 8%.     

Determination of echinacoside in rat serum by reversed-phase high-performance liquid chromatography with ultraviolet detection and its application to pharmacokinetics and bioavailability

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of echinacoside (ECH) in rat serum. After protein precipitation of serum sample with trichloroacetic acid, the supernatant was directly injected and analyzed on a C(18) CapcellACR analytical column (150 mm x 4.6mm I.D. 5 microm) with a mobile phase consisting of acetonitrile-0.5% acetic acid (15.5:84.5, v/v). The UV detector was set at 330 nm. The lower limit of detection and quantification were 9 and 29.2 ng/mL, respectively, and the calibration curves were linear over the concentration range of 29.2-18250 ng/mL. The assay method was successfully applied to the study of the pharmacokinetics and bioavailability of ECH in rat.     

Identification and quantitative analysis of cytokinins from shoot apices of Mercurialis ambigua by gas chromatography—Mass spectrometry computer system

This paper describes the identification and quantitative analysis of cytokinins from natural sources (150–500 g fresh weight) at the submicrogram level. It summarizes an improved purification procedure with high resolution power that permits the detection of Trimethylsilylderivatives by gas chromatography-mass spectrometry. A comparison of the intensity of a characteristic ion in the mass spectrum of suitable standard (5 g) and theintensity of the same ion in the mass spectrum of the extraction product permitted precise quantitative analysis. The method has been used to determine zeatin, trans- and cis-ribosylzeatin, and ²-isopentenyladenosine concentrations in extracts from female and monoecious Mercurialis ambigua apices. It has been proved that differences appear in the endogenous cytokinin pools of monoecious and female individuals.Abbreviations IPA isopentenyladenosine - Z Zeatin - RZ Ribosylzeatin - GC gas chromatography - TMS Trimethylsilyl     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Determination of L-cysteine in amino acid mixture and human urine by flow-injection analysis with a biamperometric detector.

Based on the electrocatalytic oxidation of cysteine at pretreated platinum electrode and the flow-injection biamperometry for irreversible couple, a novel electrochemical detector is proposed for the selective determination of cysteine in amino acid mixtures and human urine samples. A thin-layer flow through cell was used to achieve large electrode surface area to volume ratio. Two identical pretreated platinum electrodes were mounted in the cell with an applied potential difference of 10 mV. By coupling two independent and irreversible electrode processes, namely, the oxidation of cysteine and the reduction of platinum oxide, the biamperometric detection scheme has been established. The resulting current is linear to cysteine over the range 4 x 10(-7)-4 x 10(-5) M with the detection limit 1 x 10(-7) M (15 pmol). The selectivity of the detector is tested by 55 foreign species including 26 ions, 11 amino acids, 6 vitamins, and 12 other compounds possibly found in urine. The detector performs well as a routine assay, showing high efficiency (180 samples/h) and good reproductivity shown by a RSD of 0.6% for eight repeated determinations of 2 x 10(-6) M cysteine. The urine samples are detected directly without the need of pretreatment or adding other reagents.     

Determination of lapatinib (GW572016) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of lapatinib (GW572016) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Plasma samples (100 microL) were prepared using solid phase extraction (SPE) columns, and 6.0 microL of the reconstituted eluate was injected onto a Phenomenex CuroSil-PFP 3 mu analytical column (50 mm x 2.0mm) with an isocratic mobile phase. Analytes were detected with a PE SCIEX API-365 LC-MS/MS system at unit (Q1) and low (Q3) resolution in positive multiple reaction monitoring mode (m/z 581 (precursor ion) to m/z 364 (product ion) for lapatinib). The mean recovery for lapatinib was 75% with a lower limit of quantification of 15 ng/mL (S/N=11.3, CV< or =14%). This method was validated over a linear range of 100-10,000 ng/mL, and results from a 5-day validation study demonstrated good within-day and between-day precision and accuracy. This method has been used to measure plasma lapatinib concentrations in a Phase I study in children with cancer.     

A solid-state,portable instrument for measurement of chlorophyll luminescence induction in plants

A newly developed compact instrument is described for the measurement of chlorophyll luminescence induction in plants. The instrument operates with a pulsed light emitting diode (LED) as light source and a photodiode as luminescence detector. A special emitter-detector geometry provides for high irradiance of the sample and efficient collection of luminescence by the detector. With insertion of appropriate filters the same probe is also suited for measuring prompt chlorophyll fluorescence. The instrument shows considerable flexibility with respect to pulse frequency, relative lengths of light/dark intervals and luminescence sampling periods. Due to a selective amplifier system only that part of luminescence is processed which is induced by the individual excitation pulses. By this approach, the problem of slow phase accumulation, encountered with conventional phosphoroscopes, is eliminated. Some examples are given for system operation, demonstrating satisfactory performance in measurements with intact leaves and isolated chloroplasts.     

Rapid determination of valsartan in human plasma by protein precipitation and high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of valsartan in human plasma is reported. The assay is based on protein precipitation with methanol and reversed-phase chromatography with fluorimetric detection. The preparation of a batch of 24 samples takes 20 min. The liquid chromatography was performed on an octadecylsilica column (50 mm x 4 mm, 5 microm particles), the mobile phase consisted of acetonitrile -15 mM dihydrogenpotassium phosphate, pH 2.0 (45:55, v/v). The run time was 2.8 min. The fluorimetric detector was operated at 234/374 nm (excitation/emission wavelength). The limit of quantitation was 98 ng/ml using 0.2 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Human Intracranial High Frequency Oscillations (HFOs) Detected by Automatic Time-Frequency Analysis

Objectives

High frequency oscillations (HFOs) have been proposed as a new biomarker for epileptogenic tissue. The exact characteristics of clinically relevant HFOs and their detection are still to be defined.

Methods

We propose a new method for HFO detection, which we have applied to six patient iEEGs. In a first stage, events of interest (EoIs) in the iEEG were defined by thresholds of energy and duration. To recognize HFOs among the EoIs, in a second stage the iEEG was Stockwell-transformed into the time-frequency domain, and the instantaneous power spectrum was parameterized. The parameters were optimized for HFO detection in patient 1 and tested in patients 2–5. Channels were ranked by HFO rate and those with rate above half maximum constituted the HFO area. The seizure onset zone (SOZ) served as gold standard.

Results

The detector distinguished HFOs from artifacts and other EEG activity such as interictal epileptiform spikes. Computation took few minutes. We found HFOs with relevant power at frequencies also below the 80–500 Hz band, which is conventionally associated with HFOs. The HFO area overlapped with the SOZ with good specificity > 90% for five patients and one patient was re-operated. The performance of the detector was compared to two well-known detectors.

Conclusions

Compared to methods detecting energy changes in filtered signals, our second stage - analysis in the time-frequency domain - discards spurious detections caused by artifacts or sharp epileptic activity and improves the detection of HFOs. The fast computation and reasonable accuracy hold promise for the diagnostic value of the detector.     

Automated determination of amisulpride by liquid chromatography with column switching and spectrophotometric detection

A fully automated chromatographic method including on-line blood serum or plasma clean-up, isocratic high-performance liquid chromatography (HPLC) and spectrophotometric detection was developed for quantitative analysis of the new antipsychotic drug amisulpride. After injection of serum or plasma onto the HPLC system and clean-up on a pre-column (10x4.0 mm I.D.) filled with Silica CN 20 micrometer (pore size 10 nm) by an eluent consisting of 8% acetonitrile in deionized water, the chromatographic separation was performed on Lichrospher CN (5 micrometer; 250x4.6 mm I.D.) by an eluent consisting of 50% acetonitrile and 50% aqueous potassium phosphate buffer (0.008 M, pH 6.4). The UV detector was set at 254 nm. The limit of quantification was about 10 microgram/l. The method revealed linearity between 10 and 600 microgram/l (correlation coefficients R(2)>0.9996). The inter-assay reproducibility (coefficient of variation) of quality control samples was between 2.8 and 11.3%. Inaccuracy was between -0.6 and +9.1%. The performance of daily calibration standards revealed an imprecision always below 15% and maximum inaccuracy of 7.7%. The method can be applied to therapeutic drug monitoring as well as pharmacokinetic studies of amisulpride.     

Determination of four water-soluble compounds in Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to determine the four water-soluble components-Danshensu (I), protocatechuic acid (II), protocatechuic aldehyde (III) and salvianolic acid B (IV) in Chinese medicine plant Salvia miltiorrhiza Bunge using high-performance liquid chromatography with a coulometric electrode array detection (HPLC-CEAD) system. Heat reflux extraction was used to pretreat the sample. This analysis was carried on a column of Hypersil C18 (250 mm x 4.6 mm, 5 microm) with a mobile phase of sodium acetate (pH 2.5, 50 mM) and acetonitrile in gradient mode. An ESA electrochemical detector monitored the four compounds. Potentials of four electrodes in series were set at 100, 150, 200 and 250 mV, respectively. Optimization of the pH of mobile phase and the proportion of acetonitrile were also performed. Calibration curve showed good linearity with correlation coefficients (r) more than 0.9937. Average recoveries of the four compounds were more than 92% and relative standard deviations were less than 6.6%. This method appeared to be stable, sensitive and reproducible for determination of the four water-soluble compounds in Chinese medicine plant S. miltiorrhiza Bunge.