The gravimetric method for the determination of residual moisture in freeze-dried biological products

The gravimetric test for the determination of residual moisture in freeze-dried biological products performed in a humidity- and temperature-controlled room with the use of scrupulous gravimetric analytical technique can be used to accurately determine residual moisture in freeze-dried biological products such as antihemophilic factor (human) or honey bee venom allergenic extract. This method determines the first water of hydration of sodium tartrate dihydrate (7.93%) to within 1.3% of the calculated value with a relative standard deviation of 0.3% for 10 replicates. For this gravimetric procedure, freeze-dried samples containing from 1.12 to 4.4% residual moisture had relative standard deviations ranging from 3.6 to 9.1%. Samples containing less than 1.0% residual moisture by the gravimetric method such as intravenous immune globulin and antihemophilic factor (human) had relative standard deviations ranging from 16.7 to 47.0%. Relative standard deviations for residual moisture tests performed on comparable samples by the Karl Fischer and thermogravimetric methods showed similar variability.     

Estimates of measurement uncertainty from proficiency testing schemes, internal laboratory quality monitoring and during routine enforcement examination of foods

AIMS: To determine the levels of measurement uncertainty (MU) obtained in proficiency testing and routine microbiological analyses of foods and to compare these with estimates of MU obtained for results of analyses obtained in collaborative interlaboratory studies of microbiological methods. METHODS AND RESULTS: Raw data submitted by participants in the Food Examination Proficiency Assessment Scheme were obtained from the Central Science Laboratory (York). Internal quality monitoring data were obtained from Health Protection Agency (HPA) laboratories, together with data from routine food examinations undertaken in HPA laboratories. The data sets were analysed to determine the relative standard deviations of reproducibility (RSD(R)), based on log(10) colony count values, and thence the relative measures of expanded uncertainty. Analysis of proficiency test data showed extreme values of RSD(R) up to +/-30% depending upon the organism, the laboratory and the method of examination. RSD(R) values on routine samples averaged around +/-12% but ranged up to +/-41% in a few instances. Internal quality assessments for different organisms ranged up to +/-27%, depending upon the particular organism and examination procedure. The results show little difference in uncertainty for counts obtained using different plating systems (e.g. pour plates, spread plates or spiral plating) on the same dilutions of the same food samples. The data are compared with estimates of microbiological uncertainty derived in interlaboratory studies. CONCLUSIONS: The estimates of uncertainty ranged widely, both within and between laboratories, and appeared to bear little relationship to the foodstuff under examination. The extent of MU associated with many routine microbiological examinations is generally no worse than those produced in inter-laboratory trials, although notable exceptions were seen. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the levels of MU may have wide impact on the establishment of international standard methods for microbiological examination of foods and the ability to set realistic microbiological criteria.     

Residue analysis of organophosphorus and organochlorine pesticides in fatty matrices by gas chromatography coupled with electron-capture detection

A multiresidue method for the simultaneous determination of 22 organochlorine (OCs) and organophosphorus (Ops) pesticides (including isomers and metabolites), representing a wide range of physicochemical properties, was developed in fatty matrices extracted from meat. Pesticides were extracted from samples with acetonitrile/n-hexane (v:v, 1:1). The analytical screening was performed by gas chromatography coupled with electron-capture detection (ECD). The identification of compounds was based on their retention time and on comparison of the primary and secondary ions. The optimized method was validated by determining accuracy (recovery percentages), precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) from analyses of samples fortified at 38 to 300 ng/g levels. Correlation coefficients for the 22 extracted pesticide standard curves (linear regression analysis, n = 3) ranged from 0.998 to 1.000. Recovery studies from 2 g samples fortified at 3 levels demonstrated that the GC-ECD method provides 64.4-96.0% recovery for all pesticides except 2,4'-DDE (44.6-50.4%), 4,4'-DDE (51.1-57.5%) and 2,4'-DDT (50.0-51.2%). Both repeatability and reproducibility relative standard deviation values were < 20% for all residues. Detection limits ranged from 0.31 to 1.27 ng/g and quantification limits were between 1.04 and 4.25 ng/g. The proposed analytical method may be used as a simple procedure in routine determinations of OCs and Ops in meat. It can also be applied to the determination of pesticide multi-residues in other animal products such as butter and milk.     

Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid,isoascorbic acid and dehydroisoascorbic acid in food and biological samples

The total vitamin C amount in different food and plasma samples was determined by a dual detection system, after HPLC separation, with direct detection of ascorbic acid and indirect fluorimetric detection of dehydroascorbic acid after a post-column O-phenyldiamine derivatisation. The two active forms of vitamin C and their d-isomers were separated within 10 min. The repeatability was determined by measurement of several fruits and vegetables and ranged from 0.3 to 1.9% (relative standard deviation) for vitamin C. The reproducibility, based on double determinations, ranged from 1.9 to 3.6% for vitamin C, depending on the matrix. The reproducibility, based on several determinations of reference materials, ranged from 2.4 to 3.7% for ascorbic acid and from 4.3 to 5.8% for dehydroascorbic acid, again depending on the matrix.     

Evaluation of the Nova StatSensor? XpressTM Creatinine Point-Of-Care Handheld Analyzer

Creatinine is a parameter that is required to monitor renal function and is important to follow in patients under treatment with potentially toxic renal drugs, such as the anti-HIV drug Tenofovir. A point of care instrument to measure creatinine would be useful for patients monitoring in resource-limited settings, where more instruments that are sophisticated are not available. The StatSensor Xpress Creatinine (Nova Biomedical Cooperation, Waltham, MA, USA) point of care analyzer was evaluated for its diagnostic performance in indicating drug therapy change. Creatinine was measured in parallel using the Nova StatSensor Xpress Creatinine analyzer and the Vitros 5,1FS (Ortho Clinical Diagnostics, Inc, Rochester, USA), which served as reference standard. The precision (i.e., repeatability and reproducibility) and accuracy of the StatSensor Xpress Creatinine analyzer were calculated using a panel of specimens with normal, low pathological and high pathological values. Two different Nova StatSensor Xpress Creatinine analyzers were used for the assessment of accuracy using repeated measurements. The coefficient of variation of the StatSensor Xpress Creatinine analyzers ranged from 2.3 to 5.9% for repeatability and from 4.2 to 9.0% for between-run reproducibility. The concordance correlation agreement was good except for high values (>600 µmol/L). The Bland-Altman analysis in high pathological specimens suggests that the Nova StatSensor Xpress Creatinine test tends to underestimate high creatinine values (i.e., >600 µmol/L). The Nova StatSensor Xpress Creatinine analyzers showed acceptable to good results in terms of repeatability, inter-device reproducibility and between-run reproducibility over time using quality control reagents. The analyzer was found sufficiently accurate for detecting pathological values in patients (age >10 year) and can be used with a moderate risk of misclassification.     

Evaluation of accuracy and reliability of the plaque reduction neutralization test (micro-PRNT) in detection of yellow fever virus antibodies

Yellow fever is a disease caused by the prototype virus of the genus Flavivirus and remains endemic in tropical forest regions from Africa and South America, despite the availability of effective vaccines. These are capable of inducing a rapid specific immune response, with the formation of neutralizing antibodies that appear early, are protective and long lasting. The Plaque Reduction Neutralization Test is considered the most sensitive and specific test for quantification of neutralizing antibodies, and the reference method for assessing the protective immune response after vaccination. This study evaluated the reliability (repeatability and reproducibility) and accuracy (sensitivity, specificity and overall accuracy) of micro-PRNT50 and compared its performance with the micro-PRNT90. Although the micro-PRNT50 has showed satisfactory levels of reliability (ICCs ranged from 0.62 to 0.NorNormas e Manuais Técnicosas e Manuais Técnicos6 for repeatability and 0.72 for reproducibility) and accuracy (sensitivity of 91.1%, specificity of 72.9% and overall accuracy of 78%), the micro-PRNT90 showed higher performance, with ICCs for repeatability ranged from 0.78 to 0.79 and 0.81 for reproducibility, sensitivity of 100%, specificity of 94.7% and overall accuracy of 95%. Modifications in the test methodology and changes in the classification criteria in the readings of the results obtained will be important to improve the accuracy of micro-PRNT.     

Reproducibility of stable isotope-labeled tracer measures of VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics

To gain insight into the mechanisms regulating plasma lipid homeostasis, FFA, VLDL-triglyceride (TG), and VLDL-apolipoprotein B-100 (apoB-100) kinetics are commonly assessed using stable isotope-labeled tracer methods. The reproducibility of these measurements, which is critical for the experimental design, is unknown. Therefore, we investigated the repeatability of plasma FFA, VLDL-TG, and VLDL-apoB-100 kinetics in eight healthy men using stable isotope-labeled tracer techniques. There were no systematic differences in plasma FFA, VLDL-TG, and VLDL-apoB-100 concentrations and kinetics between the two studies. Intraindividual day-to-day variability for various outcome variables ranged from 15% to 25%, and almost all of this was of biological origin. The most robust outcome variables were FFA rate of appearance and hepatic VLDL-TG and VLDL-apoB-100 secretion rates; the least robust were VLDL-TG and VLDL-apoB-100 plasma clearance rates and mean residence times. Overall, physiologically meaningful differences in mean values (i.e., 25-30% in magnitude) can be obtained with a sample size of 6-10 subjects for paired studies and 12-20 subjects per group for cross-sectional studies, assuming a type I error rate of 0.05 and a type II error rate of 0.20 (i.e., 80% power). These findings will be useful for future studies investigating FFA, VLDL-TG, and VLDL-apoB-100 kinetics with the methods described.     

A novel support for laccase immobilization: cellulose acetate modified with ionic liquid and application in biosensor for methyldopa detection

A material based on cellulose acetate (CA) and the room temperature ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI·N(Tf)(2)) was developed and characterized by scanning electron microscopy, electron dispersive spectroscopy and infrared analysis. Laccase (Lac) from Aspergillus oryzae was immobilized in this material to investigate the behavior of methyldopa by square-wave voltammetry. Under optimized conditions, the Lac biosensor based on CA/BMI·N(Tf)(2) exhibited an excellent electrocatalytic performance: the analytical curve showed good linear range for methyldopa concentrations from 34.8 to 370.3 μM with a detection limit of 5.5 μM. This sensor demonstrated acceptable stability (ca. 60 days; at least 350 determinations), good repeatability and reproducibility (relative standard deviations of 1.5 and 4.3%, respectively). The recovery study of methyldopa in pharmaceutical formulations ranged from 94.1 to 105.9%. The determination of this substance using the biosensor compared favorably with that using a spectrophotometry procedure at the 95% confidence level, and indicated potential application to methyldopa determination in pharmaceutical samples.     

Determination of the enantiomeric purity of (−−) 2-(N-propyl-n-2-thienylethylamino)-5-hydroxytetralin (n-0923) by chiral stationary phase HPLC

The determination of the enantiomeric impurity, i.e., the percentage of (+) N?0437 (= N?0924) in several batches of (??) N-0437 (= N-0923) by chiral HPLC is described. Enantiomeric impurities were calculated based on the peak areas of the two baseline separated enantiomers in the chromatogram. The enantiomeric impurities found in different batches ranged from 0.02% to 0.11%. Calibration curves of the two isomers of N-0437 (Fig. 1,) were made twice to study the reproducibility and linearity of the method. The absorbance ratio, N-0923/N-0924, was found to be 1.02 with a relative standard deviation (RSD) of 9% over the whole concentration range used for the calibration curves.     

In vivo micro-CT scanning of a rabbit distal femur: repeatability and reproducibility

Before in vivo micro-CT scanning can be used to investigate femoral trabecular microarchitecture over time in rabbits, its repeatability and reproducibility must be demonstrated. To accomplish this, both distal femurs of two 6-month-old New Zealand white rabbits were scanned five times each in 1 day under different conditions (repeatability). Scanning was done at 28 microm isotropic voxel size to produce five image stacks of each femur. Three operators then followed a standard image processing protocol (reproducibility) to isolate two separate cubes from each anterior femoral condyle [total n = (8 cube sites)(5 scans)(3 operators) = 120]. Bone volume fraction (BV/TV) of the eight different cube sites (sample) ranged from 0.408 to 0.501 (mean: 0.453); trabecular thickness (Tb.Th) ranged from 158.1 to 185.5 microm (mean: 168.6 microm); and trabecular separation (Tb.Sp) ranged from 179.4 to 233.1 microm (mean: 204.7 microm). Using ANOVA and the variance component method, the total process variation was +/- 14.1% of the mean BV/TV of 0.453. The sample variation was +/- 13.9% (p < 0.001), the repeatability was +/- 2.1% (p < 0.001), and the reproducibility was +/- 0.1% (p > 0.05). Results were similar for Tb.Th and Tb.Sp. Though the contribution due to repeatability was statistically significant for each of the three indices, the natural sample differences were far greater than differences caused by repeated scanning under different conditions or by different operators processing the images. These findings suggest that in vivo micro-CT scanning of rabbit distal femurs was repeatable and reproducible and can be used with confidence to measure differences in trabecular bone microarchitecture at a single location in a longitudinal study design.     

Validation of an HPLC Analytical Method Coupled to a Multifunctional Clean-up Column for the Determination of Deoxynivalenol

To evaluate a method using a multifunctional clean-up column coupled with high performance liquid chromatography as an official analytical method for the determination of deoxynivalenol in wheat used as food or feed, an inter-laboratory study was performed in 12 laboratories using four naturally contaminated wheat samples and one spiked sample. The relative standard deviations for repeatability (RSD_r) and reproducibility (RSD_R) of naturally contaminated wheat were in the range 5.8–11.3% and 12.0–20.7%, respectively. The HORRAT was less than 1.0 in each sample. From the spiking test, the recovery rate, RSD_r, RSD_R and HORRAT value were 100.0%, 11.2%, 10.3% and 0.5, respectively. The limit of quantification is 0.10 mg/kg from the range obtained in a linear calibration. Thus, it should be useful as a sensitive and validated analytical method for the determination of deoxynivalenol in wheat intended for use in food and feed.     

Measurement of pyrethroid,organophosphorus, and carbamate insecticides in human plasma using isotope dilution gas chromatography–high resolution mass spectrometry

We have developed a gas chromatography–high resolution mass spectrometry method for measuring pyrethroid, organophosphorus, carbamate and fipronil pesticides and the synergist piperonyl butoxide in human plasma. Plasma samples were extracted using solid phase extraction and were then concentrated for injection and analysis using isotope dilution gas chromatography–high resolution mass spectrometry. The limits of detection ranged from 10 to 158 pg/mL with relative recoveries at concentrations near the LODs (e.g., 25 or 250 pg/mL) ranging from 87% to 156% (9 of the 16 compounds were within ±15% of 100%). The extraction recoveries ranged from 20% to 98% and the overall method relative standard deviations were typically less than 20% with some exceptions. Analytical characteristics were determined at 25, 250, and 1000 pg/mL.     

Statistical Evaluation of Diluents and Automatic Diluting and Pipetting Machines in Influenza Serology

The use of three diluents (i.e., 0.01 m phosphate-buffered saline, PBS; PBS with 0.2% gelatin, PBS/GEL; and PBS with 0.4% bovine plasma albumin) and three methods (i.e., the standard tube macro-procedure, TUBE; the manual microtechnique, MANUAL; and the semiautomatic microtechnique, AUTO) were statistically compared for their reproducibility and sensitivity in determining hemagglutinin (HA) and hemagglutination-inhibition (HI) antibody titers. In the HA test, analyses of between-cell variances of the different methods showed the AUTO microtiter procedure to be more reproducible than the standard TUBE method. The MANUAL microtiter procedure was the least reproducible. In the HI test, the TUBE method was the most reproducible. No significant difference in the reproducibility of the diluents was observed in either the HA or HI test. When a comparison of the sensitivity of test methods and diluents was made for determining HA titers, the AUTO microtiter procedure and PBS/GEL diluent appeared to be the method and diluent of choice. Evaluation of another instrument, the autopipetter, which standardizes the volume of diluent to be added in the microtechnique, suggests that the reproducibility of the AUTO microtiter procedure might be further increased.     

Liquid chromatography-tandem mass spectrometry determination of loperamide and its main metabolite desmethylloperamide in biological specimens and application to forensic cases

A liquid chromatographic mass spectrometric (LC/MS/MS) method has been developed for the determination of loperamide in whole blood and other biological specimens. The procedure involves liquid-liquid extraction of loperamide, desmethylloperamide and methadone-D3 (internal standard) with butyl acetate. Confirmation and quantification was done by positive electrospray ionisation with a triple quadrupole mass spectrometer operating in multiple reaction-monitoring (MRM) mode. Two MRM transitions of each compound were established and identification criteria were set up based on the ratio of the responses between the two MRM transitions of each compound. The standard curves were linear over a working range of 0.1-500 microg/kg for all transitions. The limit of quantification was 0.1 microg/kg in whole blood. The repeatability and reproducibility within the laboratory expressed by relative standard deviation were less than 5 and 11%, respectively, and the accuracy was better than 9%. The method was developed to examine a feces sample from a child whose mother was suspected of Münchausen syndrome by proxy and it proved to be suitable for forensic cases being simple, selective and reproducible. The method was also applied for a case investigation involving a overdose of loperamide.     

Quantitative determination of thalidomide in human serum with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and ultraviolet detection

A validated and precise reversed-phase high-performance liquid chromatographic method for the determination of thalidomide in serum, with phenacetin as an internal standard, is described. Protein precipitation, using trichloroacetic acid, was used for clean-up. The aliquot was chromatographed on a octadecyl column, using an eluent composed of 250 ml 0.01 M potassium dihydrogenphosphate, adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 750 ml methanol. Ultraviolet detection was used at an operation wavelength of 220 nm. Hydrolytic degradation was prevented during analysis by acidification of samples with the precipitation reagent. Thalidomide and phenacetin were found to have retention times of 7.9 and 15.0 min, respectively. Recoveries ranging from 79 to 84% were found for both components, with reproducibility relative standard deviations of 0.8–3% and repeatability coefficients of 1.2–3%. A mean correlation coefficient of 0.9995 was found for the linear calibration curve (n=2) of thalidomide with limits of quantitation of 0.222–21 mg/l. The method appeared to be feasible for pharmacokinetic studies with thalidomide.     

β-Cyclodextrin-modified covalent organic framework as chiral stationary phase for the separation of amino acids and β-blockers by capillary electrochromatography

Covalent organic frameworks (COFs) as a novel stationary phase have attracted much attention in the field of chromatography owing to their permanent nanoscale porosity, higher surface area, and exceptional stabilities. Here, a novel isocyanate-β-cyclodextrin-modified COF (MDI-β-CD-modified COF) was synthesized using isocyanate-β-cyclodextrin as the chiral selector and imine-based TpPa-1 COF as the matrix by a bottom-up strategy. The reaction condition and the structure of MDI-β-CD-modified COF were optimized and characterized by X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectra, nitrogen adsorption/desorption (Brunauer–Emmett–Teller [BET]), and thermogravimetric analysis (TGA). And then the coated open-tubular column (OT column) was prepared using MDI-β-CD-modified COF as chiral stationary phase (CSP) by in situ growth approach, which exhibited excellent stability and repeatability. For seven consecutive runs, the intraday and interday relative standard deviations (RSDs) were in range from 0.35% to 2.21% for the migration time of histidine. The column-to-column reproducibility ranged from 2.39% to 3.08%. Meanwhile, the separation of eight compounds including four amino acids and four β-blockers by capillary electrochromatography sufficiently verified the favorable chiral resolution properties of the MDI-β-CD-modified COF-coated OT column. This strategy of fabricating MDI-β-CD-modified COF-coated OT column expanded the application of imine-based COFs in chromatographic analytical fields.     

Determination of tetracyclines residues in honey using high-performance liquid chromatography with potassium permanganate-sodium sulfite-beta-cyclodextrin chemiluminescence detection

A novel method was developed for the simultaneous determination of tetracycline antibiotic (TCA) residues such as oxytetracycline (OTC), tetracycline (TC), and metacycline (MTC) by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection. The procedure was based on the chemiluminescent enhancement by TCAs of the potassium permanganate-sodium sulfite-beta-cyclodextrin system in a phosphoric acid medium. The separation was carried out with an isocratic elution using a mixture of acetonitrile and 0.001 M phosphoric acid. For the three TCAs, the detection limits at a signal-to-noise of 3 ranged from 0.9 to 5.0 ng/ml. The relative standard deviations for the determination of TCAs ranged from 3.1 to 7.4% within a day (n=11) and ranged from 2.2 to 8.6% in 3 days (n=9), respectively. The method was successfully applied to the determination of TCA residues in honey samples. The possible mechanism of the CL reaction was also discussed.     

Improved RP-HPLC method to determine neferine in dog plasma and its application to pharmacokinetics

Existing methods to determine neferine, a bisbenzylisoquinline alkaloid, either have no internal standard or lack selectivity, or take longer time. Here an improved reverse-phase high-performance liquid chromatographic (RP-HPLC) method was established in biological samples. The extraction recovery was 90.9% for neferine at concentration level of 0.2 microg/ml and 77.7% for dauricine (the internal standard) at 5 microg/ml in dog plasma, respectively. The linear quantification range of the method was 25-2000 ng/ml in dog plasma, with linear correlation coefficients greater than 0.999. The intra-day and inter-day relative standard deviations (R.S.D.s) for neferine at 50, 200 and 1000 ng/ml levels in dog plasma fell in the range of 3.0-5.4% and 4.3-9.5%, respectively. The RP-HPLC method was successfully applied to a pharmacokinetics study, in which experimental dogs received a single dose of neferine (5 mg/kg i.v. or 10 mg/kg p.o.). The pharmacokinetic result was presented.     

Determination of selected monohydroxy metabolites of 2-, 3- and 4-ring polycyclic aromatic hydrocarbons in urine by solid-phase microextraction and isotope dilution gas chromatography-mass spectrometry

Eighteen monohydroxy polycyclic aromatic hydrocarbon metabolites (OH-PAHs) representing polycyclic aromatic hydrocarbons (PAHs) containing up to four rings in human urine have been measured. The method includes the addition of carbon-13 labeled internal standards, enzymatic hydrolysis, and solid-phase microextraction followed by gas chromatography with high-resolution mass spectrometry. By using response factors calculated with the carbon-13 labeled standards, results are presented for calibration, relative standard deviations and analyte levels from an unspiked human urine pool. The method detection limits ranged from 0.78 ng/l for hydroxyphenanthrenes to 15.8 ng/l for 1-hydroxynaphthalene, and the recoveries ranged between 6% for hydroxychrysene and 47% for 1-hydroxypyrene. The relative standard deviation was lowest for 3-hydroxyphenanthrene at 2.4% and went up to 18.7% for 6-hydroxychrysene. The method was calibrated from 10 to 1200 ng/l. Eleven of the 18 metabolites were found in background pooled urine samples. This validated method is a convenient and reliable tool for determining urinary OH-PAHs as biomarkers of exposure to eight PAHs.     

HPLC-UV法定量测定发酵液中的S-腺苷-L-甲硫氨酸

本文采用高效液相色谱-紫外检测器（HPLC-UV）定量测定发酵液中S-腺苷-L-甲硫氨酸（SAM）的含量。结果表明,S-腺苷-L-甲硫氨酸浓度在0.1~1.0g/L时,其峰面积（Y）与相应的浓度（X）呈线性关系,线性方程为Y=13.937X－0.1949,相关系数为0.9969。S-腺苷-L-甲硫氨酸的平均回收率为99.89~101.7%,相对标准偏差为0.48~1.36%。该方法精密度、准确度好,稳定性高,能简便、快速、准确地测定发酵液中S-腺苷-L-甲硫氨酸的含量。