Determination of theophylline in serum with the Seralyzer Aris reagent strip test evaluated

We evaluated a new Seralyzer Aris reagent strip test (Ames Div., Miles Labs.) for the determination of theophylline in human serum. The method is based on the monoclonal enzyme immunoassay with dry reagent chemistry. The analysis is rapid and simple to perform: results are available only 5-10 min after receipt of the sample. Intra-assay precision (CV) was 2.2-3.3% (n = 15) for theophylline concentrations of 5-25 mg/L; interassay CV was 5.9% (n = 19) at 15 mg/L. The results (y) agreed well with those by liquid chromatography (x): r = 0.949 (p less than 0.001), and y = 0.967x + 0.214. We conclude the method is useful for rapid evaluation of theophylline concentrations in asthmatic patients.     

Evaluation of a radioimmunoassay for estradiol in unextracted serum

We describe the performance of a commercial (Steranti/EIR) RIA reagent kit for measuring 17 beta-estradiol directly in serum. Day-to-day precision data for control sera were as follows: mean = 102.8 ng/L, CV = 6.8%, n = 20; mean = 231.1 ng/L, CV = 5.3%, n = 21; mean = 747.7 ng/L, CV = 9.4%, n = 21. Analytical recovery of added estradiol from seven different serum pools from men, to which three different concentrations of estradiol had been added, was (mean +/- SD): 98.6 +/- 7.0% at 107.5 ng/L added; 98.8 +/- 4.7% at 322.5 ng/L added; 108.2 +/- 4.8% at 645 ng/L added. Overall recovery of estradiol in these experiments (mean +/- SD for 21 determinations) averaged 101.9 +/- 7.0%. Assay of 32 serum specimens from women by both the direct (y) and an extraction method (x) gave the following linear regression statistics: y = 1.12x - 1.3, r = 0.998, Sy/x = 30.2 ng/L, mean y = 438.2 ng/L, mean x = 391.4 ng/L. Hemoglobin, bilirubin, and moderate lipemia do not interfere. Sensitivity of the direct assay was 2.6 ng/L. Compared with the extraction assay, the direct estradiol assay has advantages of speed and simplicity.     

Determination of vitamin E in microsamples of serum by liquid chromatography with electrochemical detection

In this procedure for determination of vitamin E by "high-performance" liquid chromatography with electrochemical detection, 25-microL serum specimens are deproteinized with ethanol. Vitamin E (alpha-tocopherol), its derivatives (beta- and gamma-tocopherols), and the internal standard (delta-tocopherol) are extracted into heptane and the extract is evaporated and the residue reconstituted with methanol before injection into the chromatograph. Within- and between-run CVs for an alpha-tocopherol concentration of 13.6 mg/L were 5.1% (n = 28) and 6.0% (n = 5), respectively. The standard curve is linear to 100 mg/L; the minimum concentration detectable is 0.1 mg/L. Analytical recovery ranged from 99.8% to 104.8%. In 36 specimens collected from apparently healthy subjects who were not taking vitamin supplements, alpha-tocopherol as determined by this method ranged from 4.3 to 9.7 mg/L, from 1.8 to 3.9 mg/L for beta- and gamma-tocopherols. Results by this method (y) and an HPLC-ultraviolet method (x) correlate reasonably (r = 0.81): y = 0.88x - 0.55 mg/L (n = 45). This procedure is adaptable to automated analysis, and the small sample requirement facilitates its applicability to neonates.     

Immunoturbidimetry of urinary albumin: prevention of adsorption of albumin; influence of other urinary constituents

I describe a simple, rapid immunoturbidimetric assay for low concentrations of albumin in urine (2 to 260 mg/L). However, in this assay, the human serum albumin (HSA) in the standards binds nonspecifically to the polystyrene or glass tubes. This nonspecific binding cannot be prevented by adding bovine serum albumin (BSA) to standards, because the anti-HSA antibody cross reacts with BSA. Adding Triton X-100 (1 mL/L) to standards effectively prevents this nonspecific binding of HSA from standards to both polystyrene and glass tubes. High concentrations of compounds found in urine from normal and diabetic subjects do not interfere with this assay if pH extremes can be avoided. The between-day CV is 4.8% at means = 18.8 mg/L and 2.0% at means = 183.1 mg/L. Measurements by this immunoturbidimetric method (y) correlate well with those obtained by a radioimmunoassay (x): y = 1.078x - 0.141 mg/L (n = 98; r = 0.984) and with those obtained by a radial immunodiffusion method (x'): y = 1.026x' - 0.117 mg/L (n = 98; r = 0.983). Urinary excretion of albumin by 25 healthy, nondiabetic subjects was less than 8 micrograms/min.     

Reflotron cholesterol measurement evaluated as a screening technique

We evaluated the analytical performance of Boehringer Mannheim Diagnostics' "Reflotron" analyzer for the measurement of cholesterol. Coefficients of variation (CVs) for whole-blood cholesterol were: within-day 2.0% and 2.2% at 1680 and 2670 mg/L, respectively; between-day 1.8% and 2.4% (n = 9 and 8). Results were similar for serum and heparinized or EDTA-treated single-donor plasma (CV 1.4% to 2.6%). CVs of results for two reconstituted commercial quality-control materials were 3.4% and 4.6%. Heparin and hematocrit were evaluated as interferents, and critical limits for interference were identified for bilirubin, hemoglobin, and triglyceride in blood and plasma or serum. When sample collection and analysis were controlled by trained personnel, results with the Reflotron (y) compared well with those by the Ektachem procedure (x) for both blood and serum samples: r = 0.950, y = 0.944x + 130 mg/L; and r = 0.955, y = 0.93x + 43.5 mg/L, respectively. The same comparability was observed when the analysis was performed by briefly trained high-school students: r = 0.980, y = 0.949x + 23 mg/L. Performance decreased when both collection and analysis were performed by laymen: r = 0.880, y = 0.870x + 186 mg/L.     

Simple and sensitive determination of urea in serum and urine.

In this method for serum and urinary urea determination, the same reagent is used without predilution of urine samples. The method is based on the pH increase resulting from the ammonia released by urease hydrolysis of urea. o-Cresolphthalein complexone is used to monitor the pH change colorimetrically. Urea concentration and absorbance at 570 nm are linearly related for concentrations as great as 600 mmol/L for urine samples and 100 mmol/L for serum. There are no clinically significant interferences from physiological substances or drugs, and precision and accuracy are excellent (CV approximately 2%, except at very low concentrations in serum; analytical recovery was 99% in urine, 100% in serum). Results by this method (y) and by the Astra method (x) for urine correlated well (y = 0.991x - 2.87, Sy/x = 9.21, r = 0.994), as did the results by this method and by the total enzymatic method (x') for serum (y = 1.002x' + 0.192, Sy/x' = 0.598, r = 0.997). This method is applicable to automated as well as manual instruments, and one-reagent or two-reagent formats can be used.     

Fluorometric determination of magnesium in serum with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone

This simple, rapid, sensitive fluorometric method for determining magnesium is based on formation of a fluorescent complex of magnesium with 2-hydroxy-1-naphthaldehyde salicyloyl-hydrazone in alkaline ethanolic medium (lambda ex = 420 nm, lambda em = 460 nm). The detection limit of the method is 0.05 microgram/L (2 nmol/L) and the relative standard deviations (CVs) are 0.8%, 1.1%, and 2.5% at magnesium concentrations of 50, 5.0, and 0.50 microgram/L, respectively. The standard curve is linear from 0 to 250 micrograms/L (0-10 mumol/L). We investigated the chemistry of the reaction, and have applied the method to determine magnesium in 100-fold-diluted, otherwise untreated serum samples. Within-run CVs for the method were 1.7%, 1.1%, and 1.3% at mean magnesium concentrations of 15.7, 26.4, and 36.2 mg/L, respectively. Day-to-day CVs were 2.7%, and 3.6% at mean magnesium concentrations of 10.1 and 25.5 mg/L, respectively. Samples from 96 hospitalized patients in intensive-care units were analyzed by the proposed method (y) and by an automated colorimetric method involving Magon sulfonate reagent (x). Linear regression analysis of the results yielded: y = 1.01x - 0.04 (r = 0.982, Sxy = 0.90).     

Modified magnesium method in the aca III: elimination of interference by bilirubin

Bilirubin interferes with the Du Pont magnesium method in the aca at 510 nm. We determined that bilirubin concentrations in serum samples up to 380 mg/L did not affect the absorbance measured in the aca III at 540 nm, and therefore we modified the Du Pont setting of spectrophotometer for the magnesium method to 540 and 600 nm. Accuracy and precision of the modified method were comparable with the unmodified method and with atomic absorption spectrophotometry. For comparison, we analyzed serum samples with normal (n = 37) and increased (n = 22) bilirubin concentration with the modified method in the aca III (y) and in the atomic absorption spectrophotometer (x). The results (range 0.56-1.25 mmol/L) were in good agreement (x = y = 0.84 mmol/L, D(x - y) = -0.001, SD = 0.004, t = 0.238, P greater than 0.50) and bilirubin did not interfere with the modified method.     

Colorimetric determination of potassium in whole blood, serum, and plasma

This spectrophotometric method for the direct determination of potassium in serum or plasma is based on the selective complexing of potassium by a specific macrocyclic polyether, with the subsequent formation of an ion-pair with a colored anion. The colored anion is extracted into an organic solvent, clarified by centrifugation, and then measured at 415 nm. The absorbance of the chromogen varies linearly with [K+] to at least 15 mmol/L. Results of this colorimetric method (y) correlate well with the results obtained by a flame-photometric method (y = 1.04x - 0.22, r = 0.97, n = 81), with CVs ranging from 2 to 4%. We observed no interferences from lipemia, added bilirubin, or various electrolytes. We also evaluated the use of this reagent in a new automated blood analyzer developed by Abbott, a two-dimensional centrifugal system (Clin Chem 31:1457-1463, 1985). Potassium determined with this system (y) correlated well with results by flame photometry: y = 1.02x + 0.02 (r = 0.94, n = 168). With this system one can use whole-blood specimens in measuring potassium.     

Centrifugal analysis with automated sequential reagent addition: measurement of serum calcium

We describe an automated method for calcium assay, for use with the Cobas-Bio centrifugal analyzer. Calcium is reacted with cresolphthalein complexone and the absorbance of the calcium--dye complex at 575 nm is measured. EDTA is then added to break up the calcium--dye complex and the absorbance at 575 nm is re-measured, to correct for endogenous color and turbidity. Day-to-day precision data, determined over four months, were as follows: mean = 92.9 mg/L, CV = 1.47%; n = 216; mean = 128.7 mg/L, CV = 1.72%; n = 216. Comparison of the Cobas-Bio method (y) with an atomic absorption spectrometric method (x) gave the following results: y = 1.012x--2.05, r = 0.991, Sy/x = 1.2, mean x = 92.63 mg/L, mean y = 91.69 mg/L, n = 74. Hemoglobin, bilirubin, or turbidity does not interfere. At the medical decision value (110 mg/L), the overall analytical error is 4.6 mg/L, which is less than the 5 mg/L allowable (95% confidence limit) error.     

Total urinary hydroxyproline determined with rapid and simple high-performance liquid chromatography.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).     

Homogenous enzyme immunoassay for cyclosporine in whole blood using the EMIT 2000 cyclosporine specific assay with the COBAS MIRA-plus analyzer.

We describe the evaluation of the EMIT 2000 cyclosporine specific assay kit, an enzyme-multiplied immunoassay for cyclosporine in whole blood, with a COBAS MIRA-plus analyzer. The enzyme used for the assay was glucose-6-phosphate dehydrogenase (EC 1.1.1.49 G6PDH) from Leuconostoc mesenteroides; the monoclonal antibody is fairly specific for cyclosporine, and is not reactive with most metabolites. The assay principle is based on competitive immunoassay with G6PDH-labeled cyclosporine and cyclosporine in sample to the anticyclosporine mouse monoclonal antibody binding site. The within-assay coefficient of variation (CV) of this method was 2.7-4.2% (n = 10) at the levels of 56.2-339.7 microg/L. Day-to-day CVs ranged from 4.2-8.1% at the levels of 47.2-350.2 microg/L. The within-day CVs ranged from 2.0-6.4% at the levels of 43.3-330.5 microg/L. The functional detection limit was 24.9 microg/L. Samples treated with pretreatment reagent were stable at least 5 hr. Calibration was stable at least 10 days. The analytical recovery was 81-109%. The correlation between values obtained with the EMIT 2000 cyclosporine specific assay kit (y) and fluorescence polarization immunoassay (FPIA) (TDxFLx) (x) was: y = 0.880x - 13.053 microg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 +/- 19.89 microg/L ((TDxFLx - EMIT 2000) +/- SD); for the FPIA (AxSYM) (x): y = 0.989 - 4.144 microg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 +/- 17.38 microg/L ((AxSYM - EMIT 2000) +/- SD); and for the radioimmunoassay (RIA, CYCLO-Trac SP) (x): y = 0.893 - 6.764 microg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 +/- 14.98 microg/L ((RIA - EMIT 2000) +/- SD) using the Bland-Altman technique.     

Definitive liquid-chromatographic demonstration that N-ethylglycine is the metabolite of lidocaine that interferes in the Kodak sarcosine oxidase-coupled method for creatinine

Patients receiving lidocaine may show false increases of serum creatinine as assayed by the single-slide method on the Kodak Ektachem 700. Bissell et al. (Clin Chem 1987;33:951) suggested that this interference was due to oxidation of N-ethylglycine (NEG), a previously uncharacterized metabolite of lidocaine, by the sarcosine oxidase preparation used in the Ektachem creatinine slide. To investigate this possibility, we synthesized NEG, added it to drug-free human serum, and analyzed the NEG-supplemented sera for creatinine with the Ektachem 700. We found the following linear relationships between creatinine bias (y, mg/L) and NEG concentration (x, mg/L) for first (I), third (III), and fourth (IV) generation slides: I: y = 1.70x - 0.8 mg/L (n = 13, r = 1.0) III: y = 0.39x - 0.3 mg/L (n = 3, r = 1.0) IV: y = 0.79x - 1.8 mg/L (n = 13, r = 1.0) Using HPLC, we directly demonstrated the presence of NEG in sera of patients receiving lidocaine and quantified NEG concentrations in sera from four of these patients. The increasing artifactual bias in creatinine with increasing NEG concentration unequivocally confirmed that NEG is responsible for the lidocaine-associated interference in the Kodak Ektachem single-slide creatinine method.     

Rapid quantification of C-reactive protein by centrifugal analysis

I describe a rapid, simple immunoturbidimetric method for determining C-reactive protein in serum. With the Instrumentation Laboratory Multistat III microcentrifugal analyzer, quantitative results are obtained automatically after a few minutes of reaction time. Within-run and between-run coefficients of variation ranged from 2.5 to 12.7% at C-reactive protein concentrations of 55 to 69 and 14 to 25 mg/L, respectively, normal values being less than 10 mg/L. Comparison with the commercially available radial immunodiffusion method (y) yields the regression equation y = 1.011x - 2.112 (r = 0.979, n = 100).     

Rapid quantitative method using spin columns to measure porphobilinogen in urine

BACKGROUND: Large increases of urinary porphobilinogen (PBG) indicate acute porphyria, which may be due to acute intermittent porphyria, variegate porphyria, or hereditary coproporphyria. These conditions are relatively rare but share symptoms with more common conditions, such as acute surgical abdomen, and often must be ruled out rapidly. Reported quantitative methods for PBG measurement are time-consuming and inconvenient. We developed a rapid quantitative method that uses resin-packed spin columns to measure PBG in urine. Method: We applied urine to anion exchange resin in a spin column, then performed centrifugal separation and washing. PBG was eluted in 1 mol/L acetic acid and reacted with Ehrlich's reagent. After 5 min, we measured absorbance at 525, 555, and 585 nm. PBG concentration (mg/L) was calculated as 88 (A(555) - (1/2)(A(525) + A(585))). RESULTS: The reportable PBG concentration range was 0.2-15 mg/L. Between-day (total) imprecision (CV) was 8.4% at 1.2 mg/L and 3.5% at 4.4 mg/L. Comparison with our established method (x) yielded a Deming regression equation: y = 1.04x - 0.01 mg/L (R(2) = 0.98; S(y,x) = 0.87 mg/L). No interference was noted from urobilinogen or highly colored urine specimens. CONCLUSIONS: This method for PBG measurement is more rapid and precise than other methods. This test can serve as a quick screening test and facilitates batch analysis for routine quantitative testing.     

Measurement of lipase activity by a differential pH technique

This is a new electrochemical method for determination of lipase activity in biological fluids, including serum, plasma, and duodenal juice. Advantages of turbidimetric methods--short reaction time, and small sample and reagent volumes--are combined with those of titrimetric methods: measurement of absolute activity (i.e., no standardization required), saturated substrate conditions, and direct measurement of reaction products. The proposed method is easy, inexpensive, and takes only 3 min. Precision is good: CV = 3.74% within day and 7.3% between days at the clinical-decision concentration, CV = 1.86% within day and 4.65% between days for above-normal lipase activities. The standard curve is linear up to 4500 U/L. Results (y) correlate well with those by turbidimetry (x): y = 0.9287x - 65.3 (r = 0.9719). Reference values are between 0 and 130 U/L.     

A dry-reagent strip for quantifying carbamazepine evaluated

We examined a new colorimetric homogeneous immunoassay for carbamazepine based on the apoenzyme reactivation immunoassay system (ARIS) principle. The test, in dry-reagent strip format, is to be used with the Ames Seralyzer reflectance photometer. Within-run CVs (n = 20) were 3.0%, 2.7%, and 2.8% at 3.0, 6.1, and 12.1 mg/L; between-run CVs (n = 15, in 15 days) were 4.1%, 2.7%, and 1.9% at 6.0, 9.1, and 12.1 mg/L. Mean analytical recovery was 99.9 (SD 2.3)%. Results by this test (y) for clinical plasma specimens (n = 96) compared very well with those obtained by fluorescence polarization immunoassay (y = 1.01 x - 0.02, r = 0.995) and by liquid chromatography (y = 0.99 x + 0.14, r = 0.990). Bilirubin (45 mg/L), uric acid (145 mg/L), and various anticoagulants at about fourfold the usual concentrations did not interfere. High concentrations of cholesterol (4.9 g/L), triglycerides (3.8 g/L), and hemoglobin (4 g/L) caused slight positive interference. Carbamazepine-10,11-epoxide cross reacted only at greater than or equal to 5 mg/L. The two-point calibration line was validly stored for at least three weeks. Free carbamazepine also can be measured. The test is convenient and rapid (test time 80 s), and thus is particularly useful in all clinical settings where prompt testing is needed.     

A more simple, rapid and sensitive fluorimetric method for the determination of isoniazid and acetylisoniazid in serum. Application for acetylator phenotyping

A simple, rapid and sensitive fluorimetric method for isoniazid determination in serum is described. The method is based on the reaction of isoniazid with 2-hydroxy-1-naphthaldehyde in acidic medium in the presence of excess of scandium. Within-run precision (CV) was 1.5%, 1.0% and 1.2% at mean isoniazid concentration in serum of 0.244, 1.94, and 25.9 mg/l respectively (n = 10); between-run precision (CV) was 3.0%, 2.6%, and 1.9% at mean isoniazid concentration of 0.265, 1.93, and 26.2 mg/l respectively (n = 10). The linearity of the method extended over the range of 0-300 mg isoniazid/l serum. The detection limit (defined as three times the SD of the mean blank) for the method is 0.008 mg/l of serum. Samples from 80 tuberculous patients treated with isoniazid were analysed by the proposed method (y) and by the modified spectrofluorimetric method of Miceli et al (x). Linear regression analysis of the results yielded the equation y = 0.98x + 0.05 (r = 0.986, Sxy = 0.22).     

An enzymatic method for 5-fluorocytosine

We developed an enzymatic method for determination of 5-fluorocytosine in serum, using creatine iminohydrolase (EC 3.5.4.21), the Cobas-Bio analyzer, and an extant ammonia method. Analytical recovery (y) of drug added to serum (x) was good, with y = 0.97x-0.7, Sy.x = 3.6, r = 0.997 (n = 65) over the range 6.25 to 150 mg/L. Comparison with an HPLC method (x) showed good agreement: y = 0.98x + 1.34, Sy.x = 3.7 (n = 37), as analyzed with the Deming debiased regression. Precision was good, CVs being less than 3% for within-run and less than 6% for between-run controls. Ammonia, amphotericin B, glucose, urea, and hemolysis do not interfere, but bilirubin shows analyte-dependent interference and lipemia interferes when triglycerides exceed 5 g/L. This assay is accurate, inexpensive, and easy to perform. It can be easily adapted for routine or emergency use.     

Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer

In this new method for determining urinary protein, the reaction is complete within 10 min at 37 degrees C. This method is applicable to automated as well as manual measurements. Protein concentration and absorbance at 600 nm are linearly related throughout a wide range of concentrations, 10 to 16 000 mg/L. However, the chromogenicity of the gamma-globulins in this method is 70% of that of albumin, as estimated from results by a biuret method. Within-run CVs were less than 3.3%; the day-to-day CV was 2.9%. Errors due to interfering components in urine are less than 2%. The normal range for urinary protein as measured by this method was from 28 to 141 mg/day. Results by this method (y) and by a trichloroacetic acid-biuret method (x) correlated well (n = 80, r = 0.995; y = 0.99x - 2.9).