Day Coefficients Of Variation Research Articles

Close association between flowering time and aridity gradient for Sarcopoterium spinosum in Israel

The climate gradient in the southeast Mediterranean region offers an opportunity to associate local conditions with adaptive inter-population phenotypic variability. In a common garden experiment, we monitored flowering time and plant biomass in Sarcopoterium spinosum (Rosaceae) representing plant populations along an aridity gradient that in nature differ according to two environmental parameters: elevation and temperature regime. As a significant correlation was found between elevation and the average winter and summer temperatures at the natural sites, elevation was used in the analysis as a proxy for annual temperature regimes. A significant positive correlation was found between flowering day and annual rainfall and elevation, as well as between the aboveground plant biomass and annual rainfall. In addition, a significant negative correlation was found between the flowering day coefficient of variation and the two environmental parameters. Our results show inter-population differentiation in a perennial species, emphasizing the ecological importance of early flowering and trade-offs between growth and reproduction in arid vs. mesic environments.

Non‐Invasive 3‐Dimensional Facial Modeling for the Quantification of Facial Edema

BACKGROUNDWith the lengthening of spaceflight mission durations, many crewmembers have experienced in‐flight and/or post‐flight visual and anatomical changes including optic‐disc edema, globe flattening, choroidal folds, and hyperopic shifts. These changes define the visual impairment/intracranial pressure (VIIP) syndrome. Although the underlying mechanism behind VIIP is unknown at this time, it is suspected that the microgravity‐induced cephalad fluid shift plays a significant role. Therefore, quantification of the microgravity‐induced fluid shifts may provide important prognostic information. However, there are limited non‐invasive tools currently available to assess volume changes and facial edema. A standardized method for quantifying the fluid shift and establishing reliability across repeated trials also remains to be clarified. Accordingly, using a commercially available Structure Sensor 3‐dimensional (3D) scanner, our lab developed a protocol to acquire and analyze 3D facial images in seated and supine positions and assessed the validity and reliability of the method. We hypothesized that within day coefficient of variation and across day coefficient of variation would be low (<5%).METHODSVolume measurement validity was assessed by acquiring and analyzing 3D scans of an object with a known volume across three trials. Reliability was assessed in 10 subjects. Structure Sensor 3D scanner (Occipital Inc., San Francisco, CA) images were obtained in two positions: upright and supine on three separate days. Images were immediately viewed on an Apple IPad with ItSeezit3D (ItSeez3D Inc., San Francisco, CA) application to ensure complete scans were obtained. Participants were instructed to look at the same spot each time to ensure constancy across scans. Analysis was completed in Rhinoceros (Robert McNeel & Associates, Seattle, WA, version 5, 64‐bit) software with a standardized technique developed by our lab.RESULTSValidity assessments yielded a mean absolute difference of 1.13 in3 . Average intra‐trial and intra‐day variability (CV) was 2.79%. Upon initial analysis, a cephalic fluid shift was evident in participants in a seated position when compared to the supine position. The mean fluid shift across participants was approximately 600 mL.CONCLUSIONOur findings indicate that the Structure Sensor 3D scanner and accompanying analysis software are valid and reliable tools in the assessment of cephalic fluid shift across different positions. This technology may be useful in identifying inter‐individual differences in cephalic fluid shifts in crewmembers. Future studies should examine the potential applications of this technology in response to a microgravity environment.

Age and sex corrected normal reference values of T1, T2\xa0T2* and ECV in healthy subjects at 3T CMR

BackgroundMyocardial T1, T2 and T2* imaging techniques become increasingly used in clinical practice. While normal values for T1, T2 and T2* times are well established for 1.5 Tesla (T) cardiovascular magnetic resonance (CMR), data for 3T remain scarce. Therefore we sought to determine normal reference values relative to gender and age and day to day reproducibility for native T1, T2, T2* mapping and extracellular volume (ECV) at 3T in healthy subjects.MethodsAfter careful exclusion of cardiovascular abnormality, 75 healthy subjects aged 20 to 90 years old (mean 56 ± 19 years, 47% women) underwent left-ventricular T1 (3-(3)-3-(3)-5 MOLLI)), T2 (8 echo- spin echo-imaging) and T2 * (8 echo gradient echo imaging) mapping at 3T CMR (Philips Ingenia 3T and computation of extracellular volume after administration of 0.2 mmol/kg Gadovist). Inter- and intra-observer reproducibility was estimated by intraclass correlation coefficient (ICC). Day to day reproducibility was assessed in 10 other volunteers.ResultsMean myocardial T1 at 3T was 1122 ± 57 ms, T2 52 ± 6 ms, T2* 24 ± 5 ms and ECV 26.6 ± 3.2%. T1 (1139 ± 37 vs 1109 ± 73 ms, p < 0.05) and ECV (28 ± 3 vs 25 ± 2%, p < 0.001), but not T2 (53 ± 8 vs 51 ± 4, p = NS) were significantly greater in age matched women than in men. T1 (r = 0.40, p < 0.001) and ECV (r = 0.37, p = 0.001) increased, while T2 decreased significantly (r = −0.25, p < 0.05) with increasing age. T2* was not influenced by either gender or age. Intra and inter-observer reproducibility was high (ICC ranging between 0.81-0.99), and day to day coefficient of variation was low (6.2% for T1, 7% for T2, 11% for T2* and 11.5% for ECV).ConclusionsWe provide normal myocardial T2, T2*,T1 and ECV reference values for 3T CMR which are significantly different from those reported at 1.5 Tesla CMR. Myocardial T1 and ECV values are gender and age dependent. Measurement had high inter and intra-observer reproducibility and good day-to-day reproducibility.

Determination of Urinary Oxalate with Arylamine Glass-Bound Sorghum Oxalate Oxidase and Horseradish Peroxidase

We describe an enzymic colourimetric method for determination of oxalate level in urine using arylamine glass-bound sorghum leaf oxalate oxidase and horseradish peroxidase. The method is based on quantification of H2O2 generated from oxidation of urinary oxalate by immobilized oxalate oxidase, by a colour reaction consisting of 4-aminophnazone, phenol and immobilized peroxidase as chromogen. Minimum detection limit of the method was 0.05 mmol/l. Analytical recovery of added oxalate in urine was 96.8± 3.0% (mean ±S.D.). Within and between day coefficient of variation (CV) for urinary oxalate in urine were &lt; 3.5% and &lt;6.46 % respectively. The urinary oxalate values in apparently healthy and urinary stone formers as measured by the present method were correlated with those by modified Sigma Kit method (r= 0.929). The method has the advantages that it provides ca 200 times reuse of oxalate oxidase and peroxidase and free from interferences by Cl- and NO3- normally found in urine. Int J Appl Sci Biotechnol, Vol 4(3): 346-351

A simple and rapid RP-HPLC method for the simultaneous determination of piperacillin and tazobactam in human plasma

A rapid and sensitive reverse phase HPLC (RP-HPLC) method for the simultaneous quantitation of piperacillin and tazobactam in human plasma has been developed and validated. The method utilizes a novel, simple and rapid solid phase extraction step, which results in an improved extraction yield of analytes from human plasma, as well as significantly reduced interference from serum components at low UV wavelength detection compared to previously published liquid–liquid extraction methods. Chromatographic separation was carried out on a Hypersil ODS C18, 3μm column using an acetonitrile-trifluoroacetic acid-water gradient elution with dual wavelength quantitation at 254nm for piperacillin and 218nm for tazobactam. Linear relationships between peak area and drug concentration were obtained in the range of 1.0–200μg/mL for piperacillin and 0.78–50μg/mL for tazobactam, with r2=0.9997 and 0.9994 respectively. The assay proved to be sensitive (with a lower limit of quantitation of 1μg/mL for piperacillin and 0.78μg/mL for tazobactam), specific (no interference from plasma components at either 218nm or 254nm), and reproducible (both intra- and inter- day coefficients of variation were ≤6%). With a total process/assay time of less than 30min, the method provides a simple, precise and reproducible assay for monitoring piperacillin and tazobactam plasma levels that can be readily adapted for routine clinical use.

Determination of low-molecular-weight organic acids in non-small cell lung cancer with a new liquid chromatography–tandem mass spectrometry method

As compared to other classes of metabolites, determination of organic acids is an underrepresented field in cancer research and till now there has been a lack of appropriate analytical procedure for determination of serum levels of organic acids potentially associated with cancer development. The aim of the study was to develop a new rapid liquid chromatography–tandem mass spectrometry method for the quantification of six low-molecular-weight organic acids in human serum and to apply this method in an analysis of samples collected from non-small cell lung cancer (NSCLC) patients and a matched control group. The samples were prepared by solid phase extraction (Clean-up CUQAX, UCT). Chromatography was conducted on a Synergi Hydro–RP column (Phenomenex) and a gradient run of 15min. Detection was performed using a negative multiple reaction monitoring mode. The calibration ranges were as follows: 0.24–38.42μmol/L for 2-hydroxybutyric acid, 0.09–17.23μmol/L for fumaric acid, 0.08–15.13μmol/L for glutaric acid, 0.11–2.22mmol/L for lactic acid, 0.39–30.98μmol/L for pyroglutamic acid, and 0.08–16.93μmol/L for succinic acid. Mean relative recovery range was 85.99–114.42% and the determined intra- and inter day coefficients of variation were ≤14%. Among the studied acids, pyroglutamic acid showed the best discriminating potential and enabled to identify accurately NSCLC patients and control subjects regardless of the cancer stage. Further investigations of serum organic acids may allow us to better understand the underlying mechanisms involved in NSCLC and develop novel means of its detection and treatment. The developed method may be also a valuable tool to study metabolic changes associated with other types of cancer.

Simultaneous Determination of Six Benzodiazepines in Spiked Soft Drinks by High Performance Liquid Chromatography with Ultra Violet Detection (HPLC-UV).

A high performance liquid chromatographic method with ultra violet detection for simultaneous analysis of six benzodiazepines (BZDs) (chlordiazepoxide, diazepam, clonazepam, midazolam , flurazpam, and lorazepam) has been developed for forensic screening of adulterated non-alcoholic drinks. Samples were analyzed after a simple procedure for preparation using pH adjustment and filtering. Isocratic elution on a C18 column (250mm × 4.6 mm, 5μm) in the temperature 45ºC with a mobile phase consisting of 15mM phosphate buffer: methanol (50:50 v/v) at a flow rate 1.4 mL/min has been done. The column eluent was monitored with a UV detector at 245 nm. This allowed a rapid detection and identification as well as quantization of the eluting peaks. Calibration curves for all drugs in the range of 0.5- 10 µg/ mL that all the linear regression and has more than 0.996. Recovery rates for the BZDs were in the range 93.7- 108.7%. The limits of detection were calculated between 0.01- 0.02 µg/ mL. Also, the limits of quantification were 0.03- 0.05 µg/mL. Within-day and between -day coefficient of variation for all BZDs at all concentrations in the range of 0.45 - 7.69 % was calculated. The procedure can provide a simple, sensitive and fast method for the screening of six BZDs in adulterated soft drinks in forensic analysis.

Pharmacokinetic comparison of two 4 mg tablet formulations of tizanidine

To assess the bioequivalence of two Tizanidine 4 mg tablet formulations (Tizanidine® of the Pharma International company, as test product, and Sirdalud® of Novartis as a reference product), and to investigate possible effects of smoking on pharmacokinetics of tizanidine. A single-blind, randomized, single dose, two treatment, two-period, two-sequence, crossover bioequivalence study with 1 week washout period in 36 healthy volunteers. The drug was administered with 240 ml of water after 10-hour overnight fasting. After dosing, serial blood samples were collected for a period of 14 hours. Plasma harvested from blood was analyzed for tizanidine by a newly developed method using HPLC coupled with an MS/MS detector. The limit of quantitation of tizanidine was 0.080 ng/ml. Matrix-based calibration curves were linear over the range 0.080 - 8.00 ng/ml for tizanidine. The between- day coefficient of variation for quality control samples was less than 10%. The average bioavailability measures and pharmacokinetic parameters of the two tizanidine tablets were as follows: peak plasma concentration, Cmax, was 1.21 ± 0.84 ng/ml and 1.28 ± 1.11 ng/ml for Tizanidine PIC® and Sirdalud®, respectively. The time to peak plasma concentrations tmax were 0.83 ± 0.43 and 1.01 ± 0.5 hours, while the plasma half-life (t1/2) values were 1.20 ± 0.84 and 1.29 ± 0.57 hours. The area under the plasma concentration-time profiles AUC0→last were 2.53 ± 2.10 ng×h/ml and 2.46 ± 2.23 ng×h/ ml, whereas the AUC0→∞were 2.81 ± 2.27 ng×h/ml and 2.75 ± 2.37 ng×h/ml for Tizanidine PIC® and Sirdalud®, respectively. The mean residence time (MRT) values were 2.16 ± 0.693 hours and 2.33 ± 0.65 hours. The 90% confidence intervals for test/reference ratio of Cmax, AUC0→last and AUC0→∞ were found within the acceptable limits of 0.00 -125.00%, consequently no significant difference was found between the test and reference. Based on the pharmacokinetic and statistical results, it was concluded that; Tizanidine PIC® 4 mg tablets is bioequivalent to Sirdalud® 4 mg tablets of Novartis and smoking decreases Cmax and AUC of tizanidine.

Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum Samples.

The present study reports the development and validation of a sensitive and rapid extraction method beside high performance liquid chromatographic method for the determination of crocetin in human serum. The HPLC method was carried out by using a C18 reversed-phase column and a mobile phase composed of methanol/water/acetic acid (85:14.5:0.5 v/v/v) at the flow rate of 0.8 ml/min. The UV detector was set at 423 nm and 13-cis retinoic acid was used as the internal standard. Serum samples were pretreated with solid-phase extraction using Bond Elut C18 (200mg) cartridges or with direct precipitation using acetonitrile. The calibration curves were linear over the range of 0.05-1.25 µg/ml for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction. The mean recoveries of crocetin over a concentration range of 0.05-5 µg/ml serum for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction were above 70 % and 60 %, respectively. The intraday coefficients of variation were 0.37- 2.6% for direct precipitation method and 0.64 - 5.43% for solid-phase extraction. The inter day coefficients of variation were 1.69 - 6.03% for direct precipitation method and 5.13-12.74% for solid-phase extraction, respectively. The lower limit of quantification for crocetin was 0.05 µg/ml for direct precipitation method and 0.5 µg/ml for solid-phase extraction. The validated direct precipitation method for HPLC satisfied all of the criteria that were necessary for a bioanalytical method and could reliably quantitate crocetin in human serum for future clinical pharmacokinetic study.

Development and clinical application of a LC-MS/MS method for simultaneous determination of various tyrosine kinase inhibitors in human plasma

BackgroundIncreasing numbers of tyrosine kinase inhibitors (TKIs) were studied and approved for therapy of malignancies and other diseases. The aim of this study was to develop and validate a specific, simple and rapid quantification method for various TKIs in human plasma. MethodsA simultaneous test for six TKIs (erlotinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib) was developed using liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. After protein precipitation the specimens were applied to the HPLC system and separated using a gradient of acetonitrile containing 1% formic acid with 10mM ammoniumformiate on an analytic RP-C18 column. ResultsThe calibration range was 10–1000ng/mL for sunitinib and 50–5000ng/mL for the other TKIs with coefficients of determination ≥0.99 for all analytes. The intra- and inter day coefficients of variation were ≤15% and the chromatographic run time was 12min. Plasma specimens were stable for measurement for at least 1week at 4°C. Clinical applications of the assay are exemplarily discussed. ConclusionsThis novel high-throughput method is suitable for specific simultaneous determination of different TKIs in routine clinical practice.

Reproducibility of spirometry during cystic fibrosis pulmonary exacerbations

To compare the within day variation of spirometry between hospital admission, discharge, and outpatient follow up among children with cystic fibrosis (CF) hospitalized for a pulmonary exacerbation. Within day variation of spirometry will be greater at hospital admission than at hospital discharge or outpatient follow up. We performed a retrospective review of spirometry data for all patients with CF >or=6 years old admitted to our pediatric CF center for a pulmonary exacerbation in 2004 or 2005. For patients who had previously performed spirometry successfully, measurements were used from one admission only during 2004-2005 if the spirometry occurred within 3 days of hospital admission, 3 days of discharge, or at a follow up clinic visit when well. We compared the within day coefficients of variation (CV) for FVC, FEV(1), and FEF(25-75) between time points using the Wilcoxon signed rank-test. We also determined the change in spirometry that is likely to be beyond measurement variability during inpatient treatment of a pulmonary exacerbation. Spirometry data were available from 40 subjects at admission and follow up and 35 at hospital discharge. There was no significant difference in CV at admission, discharge, and follow up for FVC, FEV(1), or FEF(25-75). The mean (SD) CV was 3.1% (2.7) for FVC, 3.2% (2.1) for FEV(1), and 9.7% (7.0) for FEF(25-75) at admission, 2.8% (2.2) for FVC, 3.1% (2.1) for FEV(1), and 8.1% (6.7) for FEF(25-75) at discharge, and 2.7% (1.7) for FVC, 2.8% (2.0) for FEV(1), and 8.4% (7.8) for FEF(25-75) at follow up. These are similar to previous reports of outpatients with CF. The improvement in spirometry that exceeded measurement variability for our cohort was 80 ml for FVC, 70 ml for FEV(1), and 220 ml/sec for FEF(25-75). The presence of an acute pulmonary exacerbation in children and adolescents with CF does not substantially contribute to the within day variation in spirometry. Within day variation of spirometry for children with CF during pulmonary exacerbations is similar to previously reported values from clinically stable CF patients.

New RP‐HPLC Method with UV‐Detection for the Determination of Carvedilol in Human Serum

Carvedilol has been used as an antihypertensive drug and also possesses antioxidant and antiproliferative effects. A simple and sensitive analytical method for carvedilol in human serum by using high performance liquid chromatography (HPLC) was developed. The method employs a liquid‐liquid extraction for isolation and sample concentration followed by reversed phase liquid chromatography (RPLC) analysis using ultraviolet (UV) detection at 238 nm. Analytes were extracted from serum samples, that were previously mixed with 300 µL of 0.1 N sodium hydroxide solution into an n‐hexane, dichloromethane (7∶3) solvent system. The mobile phase was made of acetonitrile, 15 mM orthophosphoric acid (37∶63), and 0.25% v/v of triethylamine, with a flow rate of 1 mL/min. Serum samples containing the carvedilol and internal standard, amitriptyline were eluted through a C8, Kromasil KR 100 5C8 column. Retention times of carvedilol and amitriptyline were 6.10 min and 8.44 min, respectively. The intra day and inter day coefficient of variation and percent error values of the assay method were less than 5%. The calibration curve was linear over a concentration range of 5–500 ng/mL. The extraction recovery of carvedilol is more than 75%. The validated method was applied to a pharmacokinetic study of carvedilol in human serum following the administration of a single carvedilol tablet (6.25 mg). Such a method would be ideally suitable for estimation of the drug for pharmacokinetic studies in human volunteers after oral administration of a single or multiple dosage(s) of carvedilol.

Rapid Quantitation of Digoxin in Human Plasma and Urine Using Isotope Dilution Liquid Chromatography‐Tandem Mass Spectrometry

A rapid LC/MS/MS assay to quantitate digoxin in human plasma and urine has been developed and validated using a Packard liquid handling systems‐Multiprobe® II AMP8E1. Following automatic solid phase extraction on Waters Oasis HLB 30 mg 96 plates, digoxin and its internal standard (Digoxin D3) were analyzed by reverse phase chromatography (LUNA C18, 5 µm, 150×2 mm), and introduced into the mass spectrometer (Sciex ‐ API 3000) via the turboIonspray ion source operating in positive mode. The assay was validated for human plasma and human urine over a concentration range of 0.2–20 ng/mL and 1–100 ng/mL, respectively, using 0.5 mL of sample. The between day and within day coefficients of variation for all matrices were <17% at the concentrations of lower limit of quantification (LLOQ), and <13% at other quality control concentrations. The average recovery was 80.3% from plasma and 74.3% from urine. No matrix effect was observed. Freeze‐thaw stability, stability of digoxin in matrix, and stability of extracted samples were also evaluated. The mean concentration after three freeze‐thaw cycles was within±3.2% of the nominal value in plasma and within±11.4% of the nominal value in urine. After storage for 4 hours at room temperature, greater than 97.2% of digoxin remained in plasma and 96% remained in urine. Digoxin in extract was stable in the autosampler at+5°C for 77 hours in plasma and 80 hours in urine.

Fully automated on-line quantification of quetiapine in human serum by solid phase extraction and liquid chromatography

A quantitative method for determination of quetiapine (QTP) in human serum is presented. The method is fully automated and based on high performance liquid chromatography (HPLC) with on-line solid phase extraction (SPE). The extraction procedure is based on a C2 cartridge, which is eluted with methanol. The eluate is injected onto a silica column with a mobile phase consisting of methanol:20 mM NH 4CH 3COO, pH 5.0 (99:1). Quetiapine is quantified by ultra-violet (UV) absorbance at 257 nM with trifluoperazine as the internal standard (I.S.). The extraction recoveries for quetiapine and trifluoperazine were 69 and 57%, respectively. The total inter day coefficient of variation was 11.1, 3.8 and 3.1% at 20, 500 and 1000 nM, respectively. The detection limit was 10.3 nM quetiapine. The method has been used in our therapeutic drug monitoring (TDM) laboratory where co-administered drugs often are observed. In an investigation of analytical interference from co-administered drugs, demethyl-mianserine was the only drug which interfered with the internal standard. There was no interference with quetiapine itself. The method showed good agreement with mass spectrometric quantification of quetiapine.

Co-immobilization of cholesterol esterase, cholesterol oxidase and peroxidase onto alkylamine glass beads for measurement of total cholesterol in serum

Commercially available cholesterol esterase, cholesterol oxidase and peroxidase have been co-immobilized onto alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling with a conjugation yield of 2.3 mg/g of support and 76% retention of initial activity. The co-immobilized enzyme system showed maximum activity at pH 7.0, when incubated at 37 °C for 12 min. A method was developed for total serum cholesterol determination employing co-immobilized enzymes. There was a linear relationship between A520 and cholesteryl acetate concentration ranging from 5 mg to 50 mg/dl reaction mixture. The minimum detection limit of the method is 50 mg/dl. Within day and between day coefficient of variation were <1.0% and <6%, respectively. A good correlation (r=0.83) was found between the total serum cholesterol obtained by the present method and commercial Enzo-kit method employing free enzymes. Among the various serum substances tested at their physiological concentrations; Testosterone, vit D and progesterone caused 59%, 41% and 39% inhibition, while NaCl, KCl, CuSO4, creatinine, NaHCO3, albumin and estrogen had practically no effect.

Quantification of plasma and intracellular levels of the HIV protease inhibitor ritonavir by competitive ELISA

The HIV protease inhibitor ritonavir (Norvir; ABT-578), currently used in combination with nucleoside analogs and other protease inhibitors in anti-HIV therapy, has previously been quantified by an HPLC procedure. Here, we report the first convenient one-step competitive ELISA for measuring plasma and intracellular ritonavir in HIV patients. Anti-ritonavir antibody was raised in rabbits using ritonavir–KLH conjugate as immunogen, and the enzymatic tracer was prepared by coupling the drug to acetylcholine esterase. Samples for analysis were first extracted with methanol. Bound/free separation was achieved in a microtiter plate previously coated with anti rabbit IgG monoclonal antibody. Fifty percent inhibition was observed at 1 ng/ml ritonavir and the method accurately and specifically detected as little as 3–4 ng/ml of plasma ritonavir as well as intracellular drug in the peripheral blood mononuclear cells of patients undergoing ritonavir therapy. Within-run and day to day coefficients of variation were below 10% and the drugs currently used in HIV therapy did not interfere with the test. The ELISA was applied to the measurement of plasma ritonavir and to the determination of the extracellular/intracellular drug level ratios in HIV patients receiving long-term multidrug therapy.

Simultaneous determination of artemether and its major metabolite dihydroartemisinin in plasma by gas chromatography–mass spectrometry-selected ion monitoring

A sensitive, selective, and reproducible GC–MS–SIM method was developed for determination of artemether (ARM) and dihydroartemisinin (DHA) in plasma using artemisinin (ART) as internal standard. Solid phase extraction was performed using C 18 Bond Elut cartridges. The analysis was carried out using a HP-5MS 5% phenylmethylsiloxane capillary column. The recoveries of ARM, DHA and ART were 94.9±1.6%, 92.2±4.1% and 81.3±1.2%, respectively. The limit of quantification in plasma was 5 ng/ml (C.V.≤17.4% for ARM and 15.2% for DHA). Calibration curves were linear with R 2≥0.988. Within day coefficients of variation were 3–10.4% for ARM and 7.7–14.5% for DHA. Between day coefficients of variations were 6.5–15.4% and 7.6–14.1% for ARM and DHA. The method is currently being used for pharmacokinetic studies. Preliminary data on pharmacokinetics showed C max of 245.2 and 35.6 ng/ml reached at 2 and 3 h and AUC 0–8h of 2463.6 and 111.8 ngh/ml for ARM and DHA, respectively.

Simpler technique for measuring oxidative susceptibility by heparin affinity column isolation of lipoproteins

Variations in the in vitro oxidative susceptibility or resistance of lipoproteins have been used to test the effect of ingested antioxidants and may prove to be a marker for coronary artery disease. Here we describe a simple technique for isolating and oxidizing β-lipoproteins that may have utility in the clinical laboratory. Electrophoretic profiles showed that β-lipoproteins were separated from α-lipoproteins and essentially from most other serum proteins using heparin affinity columns. Lipoproteins were normalized in the reaction mixture by measuring apo B in the β-lipoprotein eluate using an automated apo B method, which gave good recoveries of 106–112%. Copper mediated oxidation was monitored by measurement of conjugated dienes formation at 234 nm for 300 min. When the reaction mixture included β-lipoprotein eluate containing 0.03 g/L of apoB and 5 μmol/L copper sulfate, conditions were effective for obtaining complete oxidation while allowing reproducible measurements, with between day coefficients of variation of 2.35%, 14.6%, and 10.5% for lag, propagation and plateau phases, respectively. β-lipoproteins isolated from serum were more susceptible to oxidation than β-lipoproteins from plasma apparently due to inhibition of oxidation by fibrinogen which co-eluted with β-lipoprotein from plasma. For this reason, we recommend using serum preserved with EDTA for this assay.

Simple liquid chromatographic method for the determination of naltrexone in human plasma using amperometric detection

A simple liquid chromatographic method using amperometric detection was developed for the determination of naltrexone in human plasma. Prior to analysis, naltrexone and the internal standard (naloxone) were extracted from plasma samples using a 9:1 mixture of chloroform and isopropyl alcohol. The mobile phase comprised 0.1 M disodium hydrogen orthophosphate (pH 3.5) and acetonitrile (85.5:14.5, v/v). Analysis was run at a flow-rate of 0.8 ml/min with the detector operating under oxidative mode at an applied potential of +0.95 V. The method is specific and sensitive with a detection limit of approximately 1 ng/ml at a signal-to-noise ratio of 3:1. Mean recovery value of the extraction procedure was about 93%, while the within day and between day coefficient of variation and percent error values of the assay method were all less than 10%. The calibration curve was linear over a concentration range of 1.5–100 ng/ml.

Components of variance when assessing there producibility of body composition measurements using bio-impedance and the Hologic QDR-2000 DXA scanner

This study evaluated the reproducibility of measuring lean tissue mass (LTM), fat mass(FM DXA), bone mineral content (BMC) and density (BMD) with a Hologic QDR-2000 DXA scanner, and both fat free mass (FFM bio) and fat mass (FM bio) with an impedance meter. Furthermore, the study aimed to assess whether sheets and pillows significantly altered the DXA results. Fifty-one healthy volunteers aged 20–61 years were examined before and after repositioning ( n = 20), removal of the sheets ( n = 10), addition of two foam pillows ( n = 11) or a regular pillow ( n = 10). One to 7 days later a re-examination was performed ( n = 35). Eight surgical patients were also scanned twice 1–7 days apart. The day to day coefficients of variation were about 1% for most measurements, though more than 2%for FM DXA, and FM bio. For total tissue mass, LTM, and BMC there was a significant biological day to day variance whereas for the other measurements the day to day variation could be accounted for by just measurement variance. Pillows systematically changed the estimates of bone mineral and soft tissue, but sheets did not.