Coefficient Of Variation For Precision Research Articles

Pharmacokinetics of ebeiedinone in mouse blood by UPLC–MS/MS

An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was established to determine ebeiedinone in mouse blood, and the pharmacokinetics of ebeiedinone after intravenous (0.5 mg/kg) and oral (2, 4, and 8 mg/kg) administration was studied. Twenty-four mice were randomly divided into 4 groups, 1 group was for intravenous administration (0.5 mg/kg), and other 3 groups were for oral administration (2, 4, and 8 mg/kg), with 6 rats in each group. Yubeinine was used as an internal standard. Multiple reaction monitoring (MRM) mode was used to quantitatively analyzed ebeiedinone m/z 414.4 → 91.1 and the internal standard m/z 430.4 → 412.3 in the electrospray ionization (ESI) positive interface. In the concentration range of 1–2000 ng/mL, the ebeiedinone in the mouse blood was linear (r2 > 0.995), and the lower limit of quantification was 1.0 ng/mL. In the mouse blood, the intra-day precision coefficient of variation (CV) was less than 15%, and the inter-day precision CV was less than 15%. The accuracy ranged from 85.4% to 114.6%, and the average recovery was higher than 61.3%. The matrix effect was between 87.0% and 106.5%. These data met the pharmacokinetic study requirements of ebeiedinone. The UPLC–MS/MS method was sensitive, rapid, and selective and was successfully applied to the pharmacokinetic study of ebeiedinone in mice. The absolute bioavailability of ebeiedinone was 30.6%.

Analytical validation of fecal 3-bromotyrosine concentrations in healthy dogs and dogs with chronic enteropathy.

Studies that have used serum 3-bromotyrosine (3-BrY) to investigate eosinophil activation in dogs have found elevated 3-BrY levels in clinical patients with chronic enteropathy (CE). To our knowledge, a method to measure 3-BrY concentrations in feces has not been reported. We developed and analytically validated an electron ionization gas chromatography-mass spectrometry method to measure fecal 3-BrY concentrations in dogs. The mean and maximum fecal 3-BrY concentrations in healthy dogs ( n = 40) and dogs with CE ( n = 40) over 3 consecutive days were compared. Analytical validation had a limit of blank and a limit of detection of 2.5 and 3.7 mmol/g of feces, respectively. The mean coefficients of variation for precision and reproducibility for 3-BrY were 11.2% (range: 7.5-14.2%) and 10.1% (4.8-15.2%), respectively. The ranges of observed-to-expected ratios for linearity and accuracy were 81.3-125% and 85.4-120%, respectively. The reference intervals for mean and maximum fecal 3-BrY concentrations in 40 healthy dogs were 3.7-23.0 and 3.7-37.8 mmol/g of feces. Mean and maximum fecal 3-BrY concentrations in dogs with CE were significantly higher than those of healthy dogs ( p < 0.001). Further research is warranted to determine the clinical usefulness of fecal 3-BrY concentrations in dogs with CE.

Rapid, Validated UPLC-MS/MS Method for Determination of Glibenclamide in Rat Plasma.

Quick and specific bioanalytical methods are required for analyzing drugs in biological samples. A simple, quick, sensitive, and specific UPLC-MS/MS method was developed and validated for glibenclamide determination in plasma samples. The plasma samples were processed by protein precipitation technique. Glimepiride was used as internal standard (IS). Glibenclamide and glimepiride were eluted on C18 column (Acquity UPLC®BEH). Mobile phase consisting of acetonitrile (0.1% formic acid) and water (0.1% formic acid) was pumped in binary gradient mode at flow rate of 150 μL/min. Glibenclamide and IS elution time was about 1.0 min, and total run time was 2.0 min. The mass spectrometer (triple-quadrupole) was operated in positive electrospray ionization mode. Sodium adducts [M + Na]+ of glibenclamide and IS were monitored in MRM mode. A linear calibration curve was obtained in the range of 10-1280 ng/mL, with regression equation Y = 0.0076 X – 0.0165 and linear regression coefficient r2 = 0.999. Lower limit of quantitation was 10 ng/mL. Accuracy of the method at LQC, MQC, and HQC was 109.7% (± 6.7), 93.6% (± 0.4), and 99.3% (± 1.9), respectively. The coefficient of variation for precision at all QC concentrations was less than 6%. Recovery at LLQC, MQC, and HQC was 104.2% (± 4.9), 100.6% (± 0.9), and 102.9% (± 5.8), respectively. The method was successfully implemented for pharmacokinetic investigations (in-house data).

VALIDACIÓN DE UN MÉTODO ANALÍTICO PARA CUANTIFICAR ALUMINIO EN TEJIDOS DE RATAS SPRAGUE DAWLEY MEDIANTE EAA-FLAMA Y EAA-HORNO DE GRAFITO

The method was validated to quantify aluminum (Al) in tissues of Sprague Dawley rats by flame atomic absorption spectrophotometry (F-AAS) and graphite furnace (GF-AAS). The linearity was evaluated by the correlation coefficient (r) and the results were 1.000 and 0.9993 for F-AAS and GF-AAS, respectively. Accuracy was 100.62 and 97.75 %, while the precision coefficient of variation was of 0.61 and 1.30 % for F-AAS and GF-AAS, respectively. Precision was also evaluated under repeatability conditions; the result obtained indicates that the method is repeatable for the Al concentrations used and determined by F-AAS and GF-AAS. The results of limit of detection and limit of quantification of Al determined by F-AAS were 0.06 and 0.19 mg/L, and 0.05 to 0.18 µg/L of Al determined by GF-AAS. Furthermore, the accuracy of the analytical method was evaluated with oyster tissue as certified reference material (NIST-1566b) digested in microwave system and containing a concentration of 197.2 ± 6.0 mg of Al/kg of tissue. The results for accuracy through the percent recovery rate were 100.60 and 100.51 % for F-AAS and GF-AAS, respectively. Finally, the applicability of the method was evaluated by fortifying rats’ tissues and controls. The results for accuracy by the recovery rate ranged between 99.32 and 109.00 %.

Application of QuEChERS Coupled with HPLC-DAD-ESI-MS/MS for Determination of Heterocyclic Amines in Commercial Meat Products

This study aims to evaluate analysis of heterocyclic amines (HAs) in meat products by an established method using QuEChERS coupled with HPLD-DAD-ESI-MS/MS. A high extraction efficiency could be attained by QuEChERS method. Twenty HA standards plus one internal standard (4,7,8-TriMeIQx) could be separated within 27 min by using an Inspire C18 column and gradient mobile phase by HPLC-DAD, followed by identification and quantitation using selected reaction monitoring mode by HPLC-ESI-MS/MS. Limit of quantitation of HAs in meat samples ranged from 0.005 to 0.1 ng/g. Average recovery of 20 HAs in pork tenderloin, Pacific saury, and chicken breast ranged from 56.51 to 113.97%, 55.12 to 105.47%, and 59.06 to 103.29%, with coefficient of variation (CV) from 2.93 to 15.88%, 3.00 to 15.63%, and 4.84 to 16.60%, respectively. The CV for intra-day and inter-day precision ranged from 4.12 to 16.10% and 6.40 to 43.53% for pork tenderloin, 2.25 to 30.56% and 2.63 to 19.42% for Pacific saury, and 2.88 to 17.78% and 5.94 to 40.00% for chicken breast, respectively. Analysis of 30 commercial meat samples revealed harman and norharman to be the major HAs present at a level of 0.052–34.98 ng/g.

Method validation of flame atomic absorption spectrophotometry (FAAS) for determination of iron (FE) in multivitamin mineral capsule dosage form

Flame Atomic Absorption Spectrophotometry (FAAS) has been developed for determination of iron (Fe) in multivitamin mineral capsule dosage form. The method of sample preparation used combination of dry ashing (450 ± 20˚C for 2 hours or until it becomes ashes) and wet ashing with a mixture H2O2 30%, HCl 6N, and HNO3 65%. The method validation comprises of the study of selectivity, linearity, accuracy, and precision. The proposed method was linear over the concentration ranges of 2–8 μg/mL. The regression was y=0, 0435x+0, 0237 with the correlation coefficient (r) obtained for those regression is 0, 9996 and the relative process standard deviation value (Vxo) is 1, 39%. The accuracy of Fe was 100, 12%. The precision coefficients of variation in the range 0, 99% for repeatability and 0, 83% for intermediate precision. The method was successfully applied for determination of iron (Fe) in multivitamin mineral capsule dosage form. The conclusion of this study that the method of FAAS (Flame Atomic Absorption Spectrophotometry) meets the validation requirements in selectivity, linearity, accuracy, and precision.

Development of high performance thin layer chromatography for simultaneous analysis of lamivudine and tenofovir disoproxil fumarate

Background: The quality of antiretroviral medicines (ARVs) is vital in the management of HIV infection. Nevertheless, the effort for the management of HIV infection is impeded by the increasing problem of counterfeit ARVs.Objectives: The aim of this study was to avail a less expensive, accurate, and precise analytical method for the simultaneous determination of lamivudine and tenofovir disoproxil fumarate in fixed dosage combination.Methods: During the method development and validation, a high performance thin layer chromatography (HPTLC) system with WinCATS software was used. Freshly prepared solutions in methanol were spotted on HPTLC silica gel 60F254 plates. The mobile phase was composed by 20 mL of toluene and 10 mL of methanol, both analytical grades.Results: The retention factor (Rf) was from 0.35 to 0.38 and from 0.57 to 0.59, respectively for lamivudine and tenofovir disoproxil fumarate. The coefficients of variation (CV) for repeatability were 1.3% and 3.4%, respectively for tenofovir disoproxil fumarate and lamivudine. The CVs for intermediate precision were 3.9% and 3.8%, respectively for lamivudine and tenofovir disoproxil fumarate. The analytical range was 2.000-3.100 μg for both lamivudine and tenofovir disoproxil fumarate.Conclusions: This method is recommended in routine analysis of pharmaceutical products containing lamivudine and tenofovir disoproxil fumarate.Keywords: HPTLC, Lamivudine, Tenofovir disoproxil fumarate

Development of stable isotope dilution assays for the quantitation of intra- and extracellular folate patterns of Bifidobacterium adolescentis

Folate-producing bifidobacteria have been studied extensively but appropriate methods for detailed quantitation of intra- and extracellular pteroylmono- and pteroylpolyglutamate patterns are lacking. Therefore, B. adolescentis DSM 20083T was cultivated in folate-free medium (FFM) for 24h to develop and validate stable isotope dilution assays (SIDAs) coupled with LC–MS/MS for the determination of 5-formyltetrahydrofolic acid (5-HCO-H4folate), 10-formylfolic acid (10-HCO-PteGlu), tetrahydrofolic acid (H4folate), folic acid (PteGlu) and 5-methyltetrahydrofolic acid (5-CH3-H4folate) including its di-, tri-, and tetraglutamic vitamers (5-CH3-H4PteGlu2-4). The respective monoglutamylated isotopologues labelled with deuterium were used as internal standards for quantitation. Limits of detection and quantitation (LOD/LOQ) were sufficiently low to quantify 48.2nmol L−1 5-CH3-H4folate (5.7/17nmolL−1) and 71.0nmolL−1 5-HCO-H4folate (10/30nmolL−1) as major folate vitamers extracellularly and 124nmolL−1 5-CH3-H4folate (3.4/10nmolL−1), 213nmolL−1 5-HCO-H4folate (4.8/14nmolL−1), and 61.4nmolL−1 H4folate (2.3/7.0nmolL−1) intracellularly after deconjugation. The major portion of native 5-CH3-H4folate vitamer was ascribed to its tetraglutamate ( > 95%). Concentrations of mono-, di-, tri-, and pentaglutamylated folates were below LOD or LOQ. Intra-assay precision coefficients of variation (CVs) ranged from 7% (at a concentration of 53.9nmolL−1 for 5-CH3-H4PteGlu4), 15% (25.5nmolL−1 5-CH3-H4folate) to 18% (78.5nmolL−1 5-HCO-H4folate), extracellularly, and from 6% (60.7nmolL−1 5-CH3-H4PteGlu4), 7% (202nmolL−1 5-HCO-H4folate), 10% (67.1nmolL−1 H4folate) to 11% (127nmolL−1 5-CH3-H4folate), intracellularly. Inter-assay precision CVs ranged from 2% (54.7nmolL−1 5-CH3-H4PteGlu4), 3% (71nmolL−1 5-HCO-H4folate) to 11% (48.2nmolL−1 5-CH3-H4folate), extracellularly, and from 1% (61.4nmolL−1 H4folate), 5% (213nmolL−1 5-HCO-H4folate), 6% (63.5nmolL−1 5-CH3-H4PteGlu4) to 10% (124nmolL−1 5-CH3-H4folate), intracellularly, thus showing excellent reproducibility. Recoveries for all analytes under study ranged between 81 and 113%. These newly developed methods enable reproducible, precise and sensitive quantitation of eight bacterially synthesized folate vitamers in two totally different matrices, including both monoglutamates and polyglutamates. Furthermore, we here present the first assay using solely monoglutamylated [2H4]-5-CH3-H4folate to quantify native polyglutamate patterns of this vitamer in bacteria which might replace time-consuming determination of monoglutamates in the future.

Estandarización y validación en Colombia de una metodología basada en HPLC para la determinación de la concentración sérica de posaconazol

BackgroundColombia currently does not have a specialised service for measuring antifungal levels in serum, which is of prime importance for the proper treatment and correct management of invasive fungal infections. AimsTo standardise and validate a simple, sensitive, and specific protocol, based on high performance liquid chromatography, complying with the parameters recommended by the Food and Drug Administration, to detect, identify, and quantify serum concentrations of posaconazole. MethodsA high performance liquid chromatography Agilent series-1 200 equipment was used with ultraviolet diode array detector and analytical column-Eclipse XDB-C18. Posaconazole-SCH56592 (batch IRQ-PAZ-10-X-103) was used as the primary control and itraconazole (batch ZR051211PUC921) was used as an internal control. The validation was performed taking into account all criteria recommended by the Food and Drug Administration (selectivity, calibration curves, recovery, accuracy, precision, sensitivity, reproducibility, and stability of the sample). ResultsThe most suitable chromatographic conditions were the following: column temperature 25°C, ultraviolet detection at 261nm, 50μl injection volume, flow volume 0.8ml/min, 10min running time, mobile phase of acetonitrile:water (70:30), and final retention times of 3.4 and 7.2min for posaconazole and itraconazole, respectively, with a wide and reliable quantification range (0.125μg/ml to 16μg/ml). Using these parameters, the method was selective, R2 in the calibration curves was≥0.99, and the percentage recovery was 98.7%, with a coefficient of variation less than 10%. The relative error for accuracy and the coefficient of variation for precision were less than 15%, all meeting the acceptance criteria recommended by the Food and Drug Administration. ConclusionsThe selectivity and chromatographic purity of the obtained signal, as well as the standardised limits of detection and quantification, make this method an excellent tool for therapeutic monitoring of patients treated with posaconazole.

Evaluation of point-of-care testing for D-dimer in elderly patients

Objective To evaluate the clinical performance of AQT90 FLEX, a novel time-resolved fluorescence based point-of-care test (POCT) for quantification of D-dimer in elderly patients. Methods The method from Quantitative D-dimer assay (WS/T 477-2015) for testing equipment performance was used as a reference to evaluate the clinical performance of AQT90 FLEX. The correlation was compared between testing results of D-dimer using the AQT90 immune-assay analyzer and those using the ACL TOP coagulation analyzer. Results At high concentration of D-dimer, the within-run precision coefficient of variation (CV) was 2.619%, and at low concentration of D-dimer, the within-run precision CV was 2.767%. The pollution-carrying rate was 0.12%. The measured data from AQT90 and ACL TOP had a correlation coefficient of r=0.9491(P <0.01). The equation of the line of best fit for D-dimer with which all AQT90 results can be adapted to the ACL TOP was: AQT90=2.52 ACL TOP + 0.15. The number from the equation was slightly greater in female than that in male, and it was also increased in elderly. Conclusions The AQT90 FLEX had rational precision and linearity in determination of concentration. There was a high agreement between the testing results from AQT90 and those from ACL TOP. It was recommended to use a slope of 2.52 and an intercept of 0.15 to adjust the D-dimer values of the ACL TOP to the AQT90 FLEX assay systems. POCT for D-dimer by AQT90 FLEX raises feasibility for use in elderly patients. Key words: D-dimer; AQT90 FLEX immunoassay analyzer; ACL TOP Coagulation analyzer

Development and analytic validation of an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-bromotyrosine in canine serum.

The activation of eosinophils causes the release of eosinophil peroxidase and subsequent production of 3-bromotyrosine (3-BrY), a stable byproduct. In people, 3-BrY is used as a biomarker for eosinophil activation. The method for measuring 3-BrY concentrations in biologic samples from dogs has not previously been described. The objective of this study was to develop and analytically validate an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-BrY in canine serum samples. Pooled canine serum samples were utilized to validate the assay. Serum samples from healthy control dogs (n = 41) were used to evaluate 3-BrY concentrations and establish a reference interval. The analytic validation revealed that the limit of blank and limit of detection were 0.33 and 0.63 μmol/L, respectively. The coefficients of variation for precision and reproducibility for 3-BrY were < 13.9% and < 11.0%, respectively. The means ± SD of observed-to-expected ratios for linearity and accuracy were 109.6 ± 17.2% and 98.7 ± 11.3%, respectively. The reference interval was determined as ≤ 1.12 μmol/L (median [range]: ≤ 0.63 μmol/L [≤ 0.63-1.13]). The EI-GC/MS assay described here for the measurement of 3-BrY in canine serum samples was precise, reproducible, linear, and accurate. Further studies are underway to determine the diagnostic utilities in canine patients with eosinophilic diseases.

Validation of an isotope dilution gas chromatography–mass spectrometry method for combined analysis of oxysterols and oxyphytosterols in serum samples

We describe the validation of a method for the analysis of oxysterols, i.e. oxycholesterols and oxyphytosterols, in human serum using gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM). Concentrations of 7α- and 7β-hydroxy-, and 7oxo-cholesterol, -campesterol, and -sitosterol as well as 4β-hydroxycholesterol and side-chain oxygenated 24S-, 25-, and 27-hydroxycholesterol were determined by isotope dilution methodology.After saponification at room temperature the oxysterols were extracted, separated from their substrates, cholesterol, campesterol, and sitosterol, by solid phase extraction, and subsequently derivatised to their corresponding trimethylsilyl-ethers prior to GC–MS-SIM. In order to prevent artificial autoxidation butylated hydroxytoluene and ethylenediaminetetraacetic acid were added. The validation of the method was performed according to the International Conference on Harmonisation guidance, including limits of detection and quantification, ranges, recovery and precision. Due to improved instrumental settings and work-up procedure, limits of detection and quantification ranged between 8.0–202.0pg/mL and 28.0–674pg/mL, respectively. Recovery data in five calibration points varied between 91.9% and 116.8% and in serum samples between 93.1% and 118.1%. The mean coefficient of variation (CV) for the recovery of all compounds was <10%. Well satisfying CVs for within-day precision (2.1–10.8%) and for between-day precision (2.3–12.1%) were obtained. More than 20 samples could be processed in a single routine day and test series of about 300 samples can be realised without impairment of the validation parameters during a sequence. Comparison of oxysterol and oxyphytosterol content in serum and plasma revealed no difference.A fully validated isotope dilution methodology for the quantification of oxycholesterols and oxyphytosterols from human serum or plasma is presented.

High Performance Liquid Chromatography Fluorescence Method for the Determination of Seleno-Amino Acids in Ovine Blood Plasma

This study was performed to validate an effective high performance liquid chromatography-fluorescence detection method for the separation of seleno-amino acids and sulfur amino acids in biological samples using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Standard calibration and standard addition procedures were developed, but the quantification of seleno-amino acids was carried out by a second method. The coefficient of variation for precision was < 15% and accuracies were 96 to 98%. Recoveries in the extraction method were between 63 and 99%. This method was applied to the contribution of seleno-amino acids containing methylselenocysteine and selenomethionine in plasma tissue of lambs during infusion study. The method demonstrated good selectivity, sensitivity, precision, and accuracy.

Abstract P1-04-05: Subpopulation heterogeneity demonstrated in circulating tumor cells isolated from breast cancer patients using ApoStream™, an antibody-independent cancer cell recovery device

Abstract Background: Current established methods of circulating tumor cell (CTC) isolation and identification rely on antibodies against epithelial specific markers such as epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). The classical phenotypic definition of a CTC is a CK positive, CD45 negative, nucleated cell, yet several reports have shown that EpCAM and CK detect only a fraction of CTCs and are not sufficient to detect the heterogeneous subpopulations of CTCs. Moreover, subsets of primary tumor cells acquire features of invasiveness and transform into an aggressive phenotype. During this process, EpCAM and CK are down regulated or lost leaving a lethal population of CTCs undetectable and unstudied using antibody dependent CTC technologies. It is imperative to isolate CTCs in an unbiased, EpCAM independent manner and expand the phenotypic characterization of CTCs to elucidate the subpopulation heterogeneity. Here we used ApoStream™, a novel, antibody-independent device which exploits differences in the dielectric properties between cancer cells and normal blood cells to enrich CTCs from the blood of cancer patients. We demonstrate device performance and integration with additional methods to perform subsequent phenotyping and molecular marker analysis. Methods: The performance of ApoStream™ was assessed using SKOV3 (ovarian cancer) and MDA-MB-231 (breast cancer) cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. A side-by-side comparison of CellSearch® and ApoStream™ was performed on 10 metastatic breast cancer patients. A multiplexed immunofluorescent assay and laser scanning cytometry (LSC) analyses were applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI cells, expression of EpCAM and vimentin. Results: In system precision performance studies, the average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 million peripheral blood mononuclear cells obtained from 7.5 mL normal donor blood was 75.4 ± 3.1% (n = 12) and 71.2 ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation (CVs) of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4-2600 cells. ApoStream™ consistently recovered significantly higher numbers of CTCs compared to CellSearch® (p = 0.024). ApoStream™ recovered varying numbers of CK+/CD45−/DAPI+, CK+/CD45+/DAPI+, CK−/CD45−/DAPI+ cells from each cancer patient sample tested. ApoStream™ recovered both EpCAM+ and EpCAM− CTCs in 50% and 90% of patients, respectively. Vimentin+ CTCs were isolated from 90% of patients. Conclusions: The ApoStream™ technology circumvents dependence on expression of EpCAM and recovers CTCs in high percentage of patients. ApoStream™ coupled with LSC analysis is a sensitive method for phenotyping and detecting biomarker expression in CTCs. These results demonstrate the broad applicability of ApoStream™ for enrichment and molecular characterization of CTCs as a foundation for improved clinical applications of CTCs. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-05.

Abstract A079: Subpopulation heterogeneity demonstrated in circulating tumor cells isolated from breast cancer patients using ApoStream, an antibody-independent cancer cell recovery device

Abstract Background: Current established methods of circulating tumor cell (CTC) isolation and identification rely on antibodies against epithelial specific markers such as epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). The classical phenotypic definition of a CTC is a CK positive, CD45 negative, nucleated cell, yet several reports have shown that EpCAM and CK detect only a fraction of CTCs and are not sufficient to detect the heterogeneous subpopulations of CTCs. Moreover, subsets of primary tumor cells acquire features of invasiveness and transform into an aggressive phenotype. During this process, EpCAM and CK are down regulated or lost leaving a lethal population of CTCs undetectable and unstudied using antibody dependent CTC technologies. It is imperative to isolate CTCs in an unbiased, EpCAM independent manner and expand the phenotypic characterization of CTCs to elucidate the subpopulation heterogeneity. Here we used ApoStream™, a novel, antibody-independent device which exploits differences in the dielectric properties between cancer cells and normal blood cells to enrich CTCs from the blood of cancer patients. We demonstrate device performance and integration with additional methods to perform subsequent phenotyping and molecular marker analysis. Methods: The performance of ApoStream™ was assessed using SKOV3 (ovarian cancer) and MDA-MB-231 (breast cancer) cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. A side-by-side comparison of CellSearch® and ApoStream™ was performed on 10 metastatic breast cancer patients. A multiplexed immunofluorescent assay and laser scanning cytometry (LSC) analyses were applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI cells, expression of EpCAM and vimentin. Results: In system precision performance studies, the average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 million peripheral blood mononuclear cells obtained from 7.5 mL normal donor blood was 75.4 ± 3.1% (n=12) and 71.2 ± 1.6% (n=6), respectively. The intra-day and inter-day precision coefficients of variation (CVs) of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4-2600 cells. ApoStream™ consistently recovered significantly higher numbers of CTCs compared to CellSearch® (p=0.024). ApoStream™ recovered varying numbers of CK+/CD45–/DAPI+, CK+/CD45+/DAPI+, CK–/CD45–/DAPI+ cells from each cancer patient sample tested. ApoStream™ recovered both EpCAM+ and EpCAM– CTCs in 50% and 90% of patients, respectively. Vimentin+ CTCs were isolated from 90% of patients. Conclusions: The ApoStream™ technology circumvents dependence on expression of EpCAM and recovers CTCs in high percentage of patients. ApoStream™ coupled with LSC analysis is a sensitive method for phenotyping and detecting biomarker expression in CTCs. These results demonstrate the broad applicability of ApoStream™ for enrichment and molecular characterization of CTCs as a foundation for improved clinical applications of CTCs. Citation Format: Kenna L. Anderes, Vladislava O. Melnikova, Vishal Gupta, Dave K. Hasegawa, Darren W. Davis. Subpopulation heterogeneity demonstrated in circulating tumor cells isolated from breast cancer patients using ApoStream, an antibody-independent cancer cell recovery device. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A079.

Validation of a New LC-MS/MS Method for the Detection and Quantification of Phenolic Metabolites from Tomato Sauce in Biological Samples

Tomato is a good source of bioactive molecules such as vitamin C, carotenoids, and phenolic compounds. Up to now, only a few studies have evaluated the bioavailability of phenolic compounds from tomato. This paper presents the optimization of a method for the determination of phenolics in tomato and their metabolites in human urine and plasma after ingestion of tomato sauce. The sample preparation includes a SPE step to obtain cleaner extracts for injection in the LC-MS/MS system. The mean recovery of analytes ranged from 73 to 104% in plasma and from 65 to 106% in urine, the accuracy was between 90.3 and 115.0% in urine and between 85.7 and 115.0% in plasma, and the precision coefficient of variation was <15%. The method allowed detection and quantification limits of 0.5-29 and 2.0-90 ng mL⁻¹ in urine, respectively, and 0.5-30 and 2.0-105 ng mL⁻¹ in plasma, respectively, for the same phenolic compounds.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests: A study in 9 Swiss laboratories

IntroductionRivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting.Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. MethodsThe same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 – 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. ResultsRXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43μg/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. ConclusionsRXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

SIMULTANEOUS ESTIMATION OF GLIBENCLAMIDE, GLICLAZIDE, AND METFORMIN HYDROCHLORIDE FROM BULK AND COMMERCIAL PRODUCTS USING A VALIDATED ULTRA FAST LIQUID CHROMATOGRAPHY TECHNIQUE

A rapid, precise, sensitive, economical, and validated analytical method is reported for simultaneous separation and quantification of three anti-diabetic drugs, viz., glibenclamide (GLB), gliclazide (GLC), and metformin hydrochloride (MHC) using ultra fast liquid chromatography (UFLC). The separation of the three drugs was achieved using a XR-ODS C18 column (30°C) with a mobile phase comprised of acetonitrile-water-trifluoroacetic acid-triethylamine (54:46:0.1:0.1v/v) in isocratic elution mode at a flow rate of 0.38 mL/min and detected at 230 nm. System suitability tests essential for the assurance of quality performance of the method were performed. The method was validated for accuracy, precision, reproducibility, robustness, detection (LOD), and quantification (LOQ) limits according to FDA and ICH guidelines. MHC (Rt = 0.98 min), GLC (Rt = 4.10 min), and GLB (Rt = 6.40 min) separated with good resolution in a single chromatographic run of 7.5 min. Linear relationship (r2 > 0.999) was observed between the peak area and concentration for all the three compounds within the range of 5–50 µg/mL. Accuracy ranged from 98 to 103% and the coefficient of variation for precision was found to be less than 3%; in all cases. LOD and LOQ values were 10 ng/mL and 20 ng/mL, respectively, for GLC and GLB; whereas 25 ng/mL and 35 ng/mL, respectively, for MHC. The method was found to be robust with minor changes in injection volume and column temperature. Validation results indicated that the method shows satisfactory linearity, precision, accuracy, and ruggedness. The extremely low flow rate, short run time, and simple mobile phase composition makes the method cost effective, rapid, non-tedious, and can also be successfully employed for simultaneous analysis of the three anti-diabetic drugs from commercial products.

An acid hydrolysis method for quantification of plasma free and total carnitine by flow injection tandem mass spectrometry

Objectives To develop a tandem mass spectrometry (MS/MS) method for plasma free and total carnitine, incorporating acid hydrolysis for total carnitine measurement, for improved accuracy and sample throughput. Design and methods Limit of detection (LOD), limit of quantitation (LOQ), linearity, accuracy, precision coefficient of variation (CV), method comparison with a radioenzymatic assay (REA) and liquid chromatography tandem mass spectrometry assay (LC-MS/MS) were performed. The method was tested for interferences and for long-term performance. Results The method LOD and LOQ were 0.87 and 1.36 μmol/L for free carnitine and 1.79 and 2.54 μmol/L for total carnitine, respectively. No interferences were detected. Long-term measurement CVs for both free and total carnitine were < 4%. Conclusions The method shows acceptable performance characteristics with improved speed and low level accuracy compared to existing REA and MS/MS methods.

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL−1 in plasma and 0.32–20 μg mL−1 in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.