Run Precision Research Articles

Analytical performance of a new chemiluminescent phenytoin (ACS:180) assay.

The authors, as a beta testing site, evaluated the ACS:180-phenytoin chemiluminescent assay (Ciba-Corning Diagnostics Corp., Medfield, MA, U.S.A.) by comparing its performance with a widely used fluorescence assay for phenytoin (Abbott Laboratories, Abbott Park, IL, U.S.A.). The ACS:180-phenytoin assays were run on a ACS-180 analyzer and fluorescence polarization assays on a TDx analyzer. The within-run precision for ACS-phenytoin assay was determined using controls obtained from Ciba-Corning. The CVs were 2.9% for low control (mean = 5.5, SD = 0.16 microgram/ml, n = 10), 2.8% for the medium control (mean = 13.4, SD = 0.37 microgram/ml, n = 10), and 2.7% for the high control (mean = 24.6, SD = 0.66 microgram/ml, n = 10). The corresponding between run precisions were 4.1% for the low control (mean = 5.4, SD = 0.22 mg/ml, n = 10), 3.1% for the medium control (mean = 13.8, SD = 0.43 mg/ml, n = 10), and 2.9% (mean = 24.5, SD = 0.70 mg/ml, n = 10) for the high control. The assay was linear from 0.5 to 40 micrograms/ml of serum phenytoin concentrations with a detection limit of 0.24 microgram/ml. The recoveries were 93-97% for concentrations of phenytoin of 5-30 micrograms/ml. They also compared 111 serum specimens collected from patients receiving phenytoin. The concentrations of phenytoin ranged from none detected to 32.4 micrograms/ml. Using fluorescence polarization assay as x-axis (reference method) and ACS:180-phenytoin assay as y-axis, they obtained the following regression line: y = 1.0x - 0.26, r = 0.993. They conclude that the ACS-phenytoin assay has a good precision and that the results correlate well with the fluorescence polarization assay.

Microparticle-enhanced nephelometric immunoassay for caseinomacropeptide in milk

SummaryA microparticle-enhanced nephelometric immunoassay has been developed for the determination of caseinomacropeptide (CMP) in bovine milk. It is based on the nephelometric quantification of the competitive immunoagglutination of a microparticle–CMP conjugate with an anti-κ-casein (κ-CN) antiserum. This one step immunoassay was sensitive (detection limit in reaction mixture, 16μg/l), accurate (linear recovery of CMP in dilution overloading) and reproducible (CV 7–14% for within and between run precision). Because of the specificity of the polyclonal antiserum used, it was necessary to separate CMP from κ-CN by ultrafiltration before the quantification of bovine milk CMP. Under the conditions of milk ultrafiltration used, κ-CN was entirely retained (> 99·5%) but the concentration of CMP measured in milk ultrafiltrates was underestimated (by ∼25%) compared with its concentration in whole milk. Microparticle-enhanced nephelometric immunoassay of CMP, with a calibration range from 0·32 to 20 mg/1 for 20- fold diluted milk ultrafiltrate, allowed contamination of bovine milk by rennet whey as low as 5 ml/1 to be detected. Applied to ultrafiltrates from milk stored at 4 °C, this immunoassay also detected proteolysis of κ-CN not revealed by measurement of κ-CN concentration in milk. A statistical lower limit of 3·21 mg/1 was determined as the increase in CMP concentration in milk ultrafiltrates that indicated probable κ-CN proteolysis in the milk sample. Previously demonstrated to be an easy to perform method for assaying the main proteins of bovine milk, microparticle-enhanced nephelometric immunoassay thus also appeared to be appropriate to quantify CMP so as to detect slight contamination of milk by whey and to indicate the proteolysis of κ-CN during milk storage at low temperature.

A Novel Derivatization of Ethylene Glycol From Human Serum Using 4-Carbethoxyhexafluorobutyryl Chloride for Unambiguous Gas Chromatography-Chemical Ionization Mass Spectrometric Identification and Quantification

Ethylene glycol poisoning is a common problem and identification of ethylene glycol in serum is important for both medical and legal purposes. Other glycols like propylene glycol, 2,3-butanediol, and diethylene glycols are also abused. Common derivatization techniques to form cyclic ester of ethylene glycol work only for 1,2-diols and cannot derivatize other glycols like diethylene glycol. The authors describe novel derivatization technique of ethylene glycol (Molecular weight 62) using 4-carbethoxyhexafluorobutyryl chloride that produces a distinct protonated molecular ion peak at m/z 563 in chemical ionization mode, thus aiding in unambiguous confirmation of ethylene glycol. The authors' novel derivatization technique is not limited to 1,2-diols and can derivatize any glycol. It yields less volatile derivatives than any conventional derivatives and is free from interference from the serum matrix. Moreover, those glycols produce distinctively different mass spectra compared to ethylene glycol. Quantitation of ethylene glycol can be easily achieved by using 1,2-butanediol as an internal standard. The assay showed a within run and between run precision of 6.7% and 8.2% and a linearity from 1.1-36.1 mmol/L (70-2,240 micrograms/mliters) of serum ethylene glycol concentration.

CEDIA for Screening Drugs of Abuse in Urine and the Effect of Adulterants

The performance of the Microgenics CEDIA DAU assays for screening amphetamines, barbiturates, benzodiazepines, cocaine, opiates, phencylidine (PCP), and tetrahydrocannabinol (THC) was evaluated on the Boehringer Mannheim/Hitachi 717 in urine. Limits of detection ranged from 0.6 ng/mL for PCP, to 34.1 ng/mL for benzodiazepines. The average within run and total precision for these assays ranged from 1.3 to 7.3% for controls at cutoff concentrations, and control values at -25% and +25% of cutoffs. The rate separations by CEDIA between the negative and cutoff calibrators for all drugs were greater than corresponding EMIT II (Syva Co.) assays. The relative sensitivity and specificity of CEDIA as compared to EMIT II were 95.6 and 98.8%, respectively, on 13,535 urine samples. All positive samples, and those samples producing discordant results between the assays were confirmed by quantitative gas chromatography/mass spectrometry (GC/MS). Using SAMHSA cutoff limits (and including barbiturates and benzodiazepines at 300 ng/mL), the relative sensitivity and specificity of CEDIA vs. EMIT II were 96.7 and 98.8%, respectively. The overall sensitivity of CEDIA vs. GC/MS was 98.9% with 179 false positives, as compared to 96.2% with 189 false positives for EMIT II vs. GC/MS. The effect of adulterants added to urine to potentially invalidate screening results was also tested. CEDIA produced strong interferences for most drug assays in the presence of glutaraldehyde, detergent, and high concentrations of bleach and Drano. Minimal or selective interferences were seen with golden seal tea lemon juice, Visine, and low concentrations of bleach and Drano. Essentially no interference was observed with bicarbonate, sodium chloride, and vinegar.

Automated flow-injection serial dynamic dialysis technique in the study of drug binding with cyclodextrins

A flow-injection dynamic dialysis technique is presented for the determination of binding parameters of drugs to cyclodextrins (CDs). The automated system consists of a flow-injection unit, the sample loop which is the receiving compartment of a dialyser unit, and a home made timing module for operation control through two flow switching solenoid valves. The procedure of binding studies is rapid and yields reproducible results. Binding parameters of CD-micromolecule complexes were calculated using the Scatchard model. Typical examples of the binding of p-nitrophenol with α-CD ( K as = 1.58 × 10 3 M −1 at pH 7.4 and 2.06 × 10 3 M −1 at pH 9.0), salicylic acid with β-CD ( K as = 3.8 × 10 2 M −1 at pH 1.5 and 51 M −1 at pH 7.4) and ibuprofen with β-CD ( K as 2.2 × 10 2 M −1 at pH 2.5) are presented and the binding constants obtained are compared to literature values 1:1 stoichiometry was found in all cases and within the run precision ranged from 2 to 14% R.S.D. The between run precision for the binding of p-nitrophenol to α-CD was 2% ( n =3).

Synthetic IgG Receptor, Avid AL: Applications in Quantitative IgG Determination and in Immunoprecipitation

Abstract Flow injection analysis and low pressure liquid affinity chromatography are combined to develop a novel method suitable for the quantitation of immunoglobulins (IgG) in biological samples. The proposed system utilizes a novel synthetic IgG receptor affinity gel (Avid AL). This system, at an optimized sample loop size of 150 μL and binding and eluting buffer flow rates of 0.5 and 2.9 mL min−1 respectively, generate calibration curves (r > 0.990) linear up to at least 2.5 mg mL−1 (375 μg applied to column) of human IgG. Excellent recovery yields ranging between 99 - 101 % were obtained. The within run precision study gave a CV of 4.1 % (n=12) and 1.9 % (n=12) at human IgG concentrations of 0.36 and 1.32 mg mL−1 respectively. Comparison of the present method, using human IgG standards, gave excellent correlations (r > 0.993) both with a similar system but, using immobilized Protein A as the affinity support instead of Avid AL as well as with an independent radial immunodiffusion method. A single, ac...

Mechanized Toxicological Serum Tests in Screening Hospitalized Patients

A spectrum of quantitative and qualitative methods was adapted to the RA-1000/RA-XT selective analyser for the purpose of excluding or detecting common types of intoxication in the emergency laboratory of our primary care community hospital. Ethanol and salicylates (measured photometrically) and acetaminophen (measured immunologically by EMIT tox) were quantitatively analysed in serum. immunological group tests (EMIT tox) for barbiturates, benzodiazepines, tricyclic antidepressants and related compounds were used for qualitative analysis. Well established clinical chemical methods (aspartarte aminotransferase, alanine aminotransferase, creatine kinase, pseudocholinesterase, glucose and lactate) were applied to the serum samples using the same selective analyser. Within and between run precision, accuracy, recovery and detection ranges (linearity) fulfilled the recommendations of forefield toxicological analysis for all methods. Ethanol (g/l), measured photometrically with the RA-1000 analyser, agreed with the reference method (headspace gas-chromatography) with a correlation coefficient greater than 0.99 (y = 0.06 + 0.98x). Acetaminophen and salicylates showed correlation coefficients greater than 0.94 and greater than 0.99, when compared with manual colorimetric procedures (acetaminophen (mg/l): y = -3.22 + 0.896x; salicylates (mg/l): y = -2.1 + 1x). Qualitative group tests for barbiturates, benzodiazepines and tricyclic antidepressants measured with the RA-1000 analyser were in good agreement with the EMIT single test procedure. The ranges of the quantitative methods allowed quantification of analytes from therapeutic (non-toxic) to very high levels in undiluted samples (ethanol 0.05 up to 4 g/l; salicylates 32 up to 1200 mg/l and acetaminophen 1.9 up to 200 mg/l). The low detection limits of the qualitative tests allowed the recognition of compounds in plasma that were present in low concentrations and/or displayed only minor reactivity with the antibodies provided by the EMIT tox test kits. As a consequence, decision limits for all three group tests in serum were lowered to near the detection limit: (table: see text) For quantitative tests the lower limits of quantification were: (table: see text) The working reagents were stable for at least 14 days at 4-8 degrees C. Calibration curves were stable over the expiration period of reconstituted original reagents (6-12 weeks), also when working reagents were prepared in aliquots from stored reconstituted reagents. Application of the newly adapted programme to serum samples of nearly two hundred patients showed it to be suitable for screening patients in which intoxication is suspected or needs to be excluded.

Determination Of Serum Acetaminophen In Emergency Toxicology: Evaluation Of Newer Methods: Abbott Tdx and Second Derivative Ultraviolet Spectrophotometry

Newer methods for the determination of serum acetaminophen in emergency toxicology, the Abbott TDx immunoassay and second derivative ultraviolet spectrophotometry, were evaluated and compared to a high pressure liquid chromatographic procedure. The Abbott TDx immunoassay within-run and day to day precision yielded coefficients of variance below 2.7% and 7.2% respectively: Abbott TDx immunoassay results correlated well with those of high pressure liquid chromatographic in patient serums 15.0 to 333 mg/L acetaminophen; r2 = 0.954, n = 40. The Abbott TDx immunoassay assay was rapid, easy to perform, free of interferences from other drugs and exhibited no carryover from previous samples. The within run precision of the second derivative ultraviolet spectrophotometry method yielded coefficients of variance of less than 7.0%. Second derivative ultraviolet spectrophotometry results demonstrated good correlation with high pressure liquid chromatographic results in patient sera; r2 = 0.923, n = 40; however, performance deteriorated below 50.0 mg/L acetaminophen. Spectral interferences were noted at high concentrations of some drugs.

A multicenter evaluation of the Boehringer Mannheim/Hitachi 717 system

Analytical performance of the Boehringer Mannheim/Hitachi 717 system was evaluated in a multicenter study involving seven different laboratories. Fifty-five methods including end point chemistries, enzymes, ISE, TDM, DAU, and specific protein assays were assessed over a 7 month period. Methods on the analyzer exhibited excellent precision with CVs less than 2% for within run precision, and CVs less than 3% for between day precision for most analytes; linearity, which met or exceeded manufacturer's claims; minimal sample and reagent carryover, and no significant interference from hemolysis; icterus; and lipemia. Recovery of the assigned value for 10 analytes in SRM 909 was acceptable. Comparison of methods with other BM/Hitachi analyzers resulted in slopes close to unity (0.93-1.06); comparison to other clinical chemistry analyzers yielded slopes of 0.88-1.07. Excellent performance and diverse method applications make the BM/Hitachi 717 analyzer a suitable instrument for work station consolidation.

Rapid Spectrophotometric Determination of Plasma Carnitine Concentrations

A spectrophotometric enzymatic assay for plasma carnitine concentrations has been automated on the Monarch 2000. Prior to the assay, each plasma sample was divided into three fractions, ie, free carnitine, acid-soluble carnitine and total carnitine, in order to determine the concentration of both free and esterified carnitine. Using the method developed by Tachikawa et al (Seikagaku 56:998, 1984), each of the samples was then chromatographed on an anion exchange resin to eliminate those compounds which could adversely impact the accuracy of the enzymatic assay for carnitine. After the completion of these preparatory steps, 32 specimens were assayed in less than 16 min on the Monarch 2000 with a high degree of both accuracy and precision. The assay was linear over a wide concentration range (5.0-80 mumol/liter), with the lower limit of sensitivity being 5.0 mumol/liter. The coefficient of variation (CV%) of the within run precision was 2.1%, 2.8%, and 6.7% for the determinations of free carnitine, acid-soluble carnitine, and total carnitine, and 17.4% and 27.8% for the calculated values of short-chain and long-chain acylcarnitines, respectively. The CV% of the between run precision for the same fractions was 6.5%, 2.7%, 3.8%, 14.2%, and 14.3%, respectively. When authentic L-carnitine was added to the plasma, the mean recovery rate was 94.7 +/- 11.0%. Reference values were determined using plasma obtained from 40 healthy adult volunteers.

Fluorometric Analysis of Alkaline Phosphatase in Fluid Dairy Products

A new quantitative assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products including whole milk, low fat and skim milks, chocolate milk, and creams. ALP in the test sample hydrolyzes a nonfluorescent substrate, FluorophosR, to a highly fluorescent product. Product formation is monitored continuously during a short incubation period and enzyme activity is calculated from the rate of fluorescence increase. Total test time is 3 min. Reaction rates are linear up to 0.5% raw milk (equivalent to 5 μg phenol/ml/15 min) with a detection limit of 0.006% raw milk. Within and between run precision of the fluorometric method was assessed by repeated analysis of a pasteurized milk sample containing added mixed herd raw milk. The within run (N=10) mean was 190.4 mU/L, standard deviation (SD) 3.2, and a coefficient of variance (CV) of 1.7%. The procedure provides a rapid, sensitive, precise, and easy-to-use ALP assay, applicable to a wide variety of dairy products.

Intensity and precision of circadian wheel running in three outbred rat strains

The laboratory rat is widely used in many areas of neurobiologic and behavioral research. However, studies of circadian activity rhythms have usually employed other rodent species whose activity patterns are more amenable to measurement in the circadian paradigm. Nevertheless, the rat may be the optimal choice in certain investigations of circadian function because of the wealth of data on the neuroanatomy and neuropharmacology of this species. This study was undertaken to determine whether choice of rat strain could affect the quality of measured circadian activity data. Intensity of wheel running (wheel revolutions per day), precision of onset, and longevity of consistently interpretable running records were assessed in three common outbred rat stains, Long-Evans, Wistar, and Sprague-Dawley. Long-Evans rats showed greater intensity of wheel running while entrained and at four and eight weeks of free running in constant darkness. There was no difference in precision of running onset among the three strains. Thus, use of the Long-Evans strain, with its greater intensity of wheel running and similar precision of activity onset, may confer advantages in studies involving measurement of circadian activity rhythms in the rat.

Evaluation of the abbott adx total serum tricyclic immunoassay

The ADx total serum tricyclic antidepressant (TCA) fluorescence polarization immunoassay (Abbott Diagnostics) for the semi-quantitation of imipramine or amitriptyline and their respective N-demethylated metabolites in cases of TCA overdose was evaluated. The assay is linear from 75-1000 ng/mL total TCA in serum, and flaggs as "HI" all results exceeding 300 ng/mL. The within and between run precision of the assay for patient serum containing imipramine or amitriptyline and their metabolites gave CV's of less than 5.5% and 8.9%, respectively. A good correlation between the results of patient serum containing imipramine and desipramine simultaneously analyzed by ADx and gas liquid chromatography (GC) was observed, r2 = 0.964, n = 32. Results of patient serum containing amitriptyline and nortriptyline or doxepin and desmethyldoxepin analyzed by ADx and GC or GC-mass spectrometry were not well correlated; r2 = 0.738, n = 44 and r2 = 0.695, n = 21, respectively. The assay consistently flagged as "HI" serum with total imipramine and desipramine concentrations above 300 ng/mL by GC, and serum with greater than 360 ng/mL of amitriptyline and nortriptyline by GC/MS. No significant cross-reactivity was observed for drugs other than the TCA.

Assessment of vitamin B1, B2 and B6 status by coenzyme activation of red cell enzymes using a centrifugal analyser.

We describe a fully automated method for the assessment of vitamin B1, B2 and B6 status using a centrifugal analyser. The activation of the red cell enzymes transketolase, glutathione reductase and aspartate aminotransferase) by their respective coenzymes were measured in freshly prepared haemolysate. The enzyme catalytic activities in the sample were measured with (maximal activity) and without (basal activity) the coenzyme, and the percentage activation was calculated. The between run precision for red cell transketolase, glutathione reductase and aspartate aminotransferase were 8.5%, 10.3% and 9.5% respectively. When whole blood was stored at room temperature for 6 hours, red cell aspartate aminotransferase activity significantly decreased (n = 10, p less than 0.05). There were no significant changes in the activities of the other two enzymes. For a group of 30 healthy young subjects, the mean (standard deviation) values for the percentage activation of transketolase, glutathione reductase and aspartate aminotransferase were 11.9% (7.3), 35.1% (19.1) and 85.3% (18.0), respectively. The vitamin status of a group of 86 pregnant women was assessed by this method; 2.3%, 8.1% and 8.1%, respectively, of the pregnant women showed a higher percentage activation for transketolase, glutathione reductase and aspartate aminotransferase than that found in the young subjects. Both groups correlated well with respect to the basal activity and the percentage activation of each enzyme. Basal activity was inversely proportional to the percentage activation. It is therefore suggested that the basal activity can be used as a second criterion in the assessment of vitamin status.

A Direct Liquid Chromatography Method for Serum Caffeine Analysis

Abstract A rapid sensitive method for serum caffeine analysis is reported. One hundred microliters of serum is mixed with 50 microliters of the acetonitrile/internal standard mixture, centrifuged, and 10 microliters of the supernatant is injected into the liquid chromatograph. Caffeine and internal standard are eluted at 6.09 and 7.17 minutes respectively. The procedure is linear to at least 20 micrograms/milliliter. The within run precision was 2.3% and 3.8% (N=10) respectively for mean concentrations of 7.77 and 15.73 micrograms/milliter. Between day precision was 11.0% and 8.6% respectively for mean concentrations of 7.63 and 15.55 micrograms/milliliter. Recovery of caffeine over the analytical range averaged 104%.

Automated method for the determination of angiotensin-converting enzyme in serum.

A method for the determination of angiotensin-converting enzyme in serum (S-ACE; EC 3.4.15.1.) with use of 3-(2-furylacryloyl)-L-phenylalanyl-glycyl-glycine (FAPGG) as substrate has been adapted for the Cobas Bio microcentrifugal analyser. The method allows 24 determinations per hour in a sample volume of 28 microliters with a within run precision of less than 3% and a between run precision of less than 5%. The reaction is linear up to at least 470 U/l. The reference interval in 92 blood donors has been determined to 14-110 U/l. The method correlates well with the manual method of Hurst & Lovell-Smith (r = 0.982). We have found the method excellently suited for routine assay.

Enzymatic assay for methotrexate in erythrocytes.

Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection limit was 3 nmol/l. Within run and between-run precision was 7.4% and 13.5% for control 10 nmol/l and 1.2% and 3.2% for control 50 nmol/l. Recovery was 85-115% of MTX added to haemolysed erythrocytes. We found the method useful for pharmacokinetic studies of MTX in erythrocytes in MTX-treated patients.

A comparative study of linear array synthesis technique using a personal computer

Five computer programs for synthesizing low-sidelobe sum patterns from linear arrays are evaluated in terms of run time and precision. Three of the programs are based on the Dolph-Chebyshev synthesis procedure, in which all sidelobes are set at the same level. The other two programs are based on a discretized version of the Taylor synthesis procedure, in which far-out sidelobes are allowed to decay. The programs were written for use on small 8- and 16-bit personal computers. It was found that the fastest running programs are also the most precise. The only Chebyshev program that gave satisfactory precision for arrays as large as 100 elements is based on Bresler's nested product algorithm, and the only similarly acceptable Taylor program is based on Shelton's discretized synthesis.

Performance of a new rate-nephelometric assay for rheumatoid factor, and its correlation with tube-titer results for human sera and synovial fluid.

We evaluated a rate-nephelometric assay for rheumatoid factor (RF), as developed for use with the Beckman Immunochemistry System (ICS). Within- run precision (CV) for low-, mid-, and high-dilution samples (60-400 kilo-int. units/L) was 3.5, 1.5, and 1.6%; between-run precision was 3.4%. Analytical linearity was excellent. Biological interference resulted in some degree of nonlinearity in more than 70% of the patients' samples tested. Sensitivities were 96.5 and 94.1% and specificities were 98.5 and 95.3% for the ICS RF and Wampole slide methods, respectively, for a clinically defined population of 170 patients. Results for 100 ICS RF-positive samples correlated well with concomitantly measured Calbiochem-Behring tube-titers. Weekly measurement of Calbiochem-Behring. Hyland Diagnostics, and ICL Scientific tube-titer values for RF along with the ICS RF values on samples from the same patients indicated stable ICS RF values but showed at least +/- 1 tube dilution variances both within and between the tube-titer methods. Therefore, it may not be appropriate to compare a precise new method with a relatively imprecise comparison method. Treatment of RF-positive samples with dithiothreitol, which disrupts the pentameric character of immunoglobulin M, rendered the samples negative by repeat ICS RF assay and confirmed the method's specificity for pentameric immunoglobulin M-RF. Serum RF concentration as determined by ICS paralleled changes in clinical symptoms in a patient treated with both effective and relatively ineffective regimens, which suggests a useful role for the assay in monitoring efficacy of clinical treatment.

A simple and reproducible method for the estimation of triiodothyronine uptake (thyroxine binding index) using a new adsorbent

A simple, rapid and reproducible method for the determination of triiodothyronine uptake using heat-treated wheat flour (TWF) as adsorbent is described. Three ml of TWF suspension in barbital buffer containing 125I triiodothyronine is added to 0.1 ml of serum sample in a plastic tube. The mixture is shaken for 30 seconds and is left to settle for 12 minutes or alternatively is centrifuged for one minute at 600 X g. The 1.0 ml of supernatant is transferred to another tube for counting. This method is virtually time and temperature independent. The effects of altering the concentration of 125I triiodothyronine and TWF are reported. Within-run precision has been estimated for normal (C.V. 1.2), high (C.V. 1.2) and low (C.V. 1.8) ranges. Estimation of between -run precision gave similar results. Comparison of results obtained by this method and by Thyopac-3 reagent kit (Radiochemical Centre, Amersham, England) shows good correlation for Thyopac-3 values about 105, while for values below 105 the present method gives lower values than Thyopac-3.