Automated stopped-flow analyser in clinical chemistry: determination of albumin with bromcresol green and purple.

We describe an adaptation of a microcomputer-controlled stopped-flow analyzer for automated analysis of albumin in serum. The immediate reaction between bromcresol green or purple and albumin is used to develop a routine procedure with 12 s reaction time. The approach shows excellent linearity to 68 g/l, good sensitivity, a relative SD of less than 0.5%, mean recovery 99.1%, high sample throughput (180 prediluted samples/h) and no significant interferences. The 'reaction time' related overestimation of albumin that appears in prolonged procedures is eliminated, and results with this method correlate well with those obtained using the more specific immunonephelometric method.

Automated flow-injection pseudotitration of strong and weak acids, ascorbic acid and calcium, and catalytic pseudotitrations of aminopolycarboxylic acids by use of a microcomputer-controlled analyser.

A simple, inexpensive, fully automated spectrophotometric system for flow-injection pseudotitrations is used to perform acid-base, redox, complexometric and catalytic "titrations". Peak widths (in time units) in the range 10-100 sec can be measured with a precision of better than 0.3% in most cases. Strong and weak acids in the range 5.0 x 10(-4)-1.0 x 10(-2)M are measured by using sodium hydroxide-Bromothymol Blue "titrant". Ascorbic acid (1 x 10(-4)-1 x 10(-2)M) is "titrated" with 2,6-dichlorophenolindophenol, and calcium (5.0 x 10(-4)-5.0 x 10(-2)M) with EDTA, with calmagite as indicator in the presence of magnesium. Aminopolycarboxylic acids (5 x 10(-6)-1 x 10(-2)M) are measured by use of catalytic indication based on the manganese-catalysed periodate-diethylaniline reaction. The ascorbic acid method has been applied to analysis of pharmaceutical preparations, and the calcium method to water analysis.

Automated determination of crude protein, phosphorus, calcium, iron, and magnesium in feeds by using stopped-flow analyzer.

Methods are described for determination of crude protein, phosphorus, calcium, iron, and magnesium in feeds, using an automated microprocessor-based stopped-flow analyzer. Crude protein is determined by a reaction-rate procedure based on the ammonia-sodium phenate-hypochloride Berthelot reaction. Phosphorus determination is based on a phosphomolybdenum blue reaction-rate method. o-Cresolphthalein complexone, ferrozine, and calmagite are used as colorimetric reagents for calcium, iron, and magnesium, respectively. The methods are precise with relative standard deviations less than 1%, rapid with analysis rates of 110-266 samples per hour, sensitive, and require less than 1 mL sample and reagent volumes. Results for feed samples are comparable with those obtained by official AOAC methods.

Total calcium and magnesium determined in serum with an automated stopped-flow analyzer.

We describe the measurement of total calcium and magnesium in serum with an automated microcomputer-controlled stopped-flow analyzer. The calcium method is based on the cresolphthalein complexone procedure, with 2-amino-3-methyl-1-propanol as the alkalinizing agent. The assay, performed on 60-fold prediluted samples, requires 50 microL of serum. Absorbance is measured at 580 nm for 1 s, after a 5-s delay. Response is linearly related to concentration up to 5 mmol/L; analytical recovery averaged 97.8%. Within-day CVs were 0.7 to 1.5%, day-to-day CVs 1.8 to 2.5%. Results compared well with those by continuous-flow Technicon SMA II method. A sample throughput of as many as 260 samples per hour is possible. The magnesium determination, a complexometric procedure, involves magnesium/calmagite complex in an alkaline reagent mixture and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate calcium interference. Prediluted serum samples are used (100 microL of serum diluted 25-fold), and absorbance at 520 nm is linear with concentration to 50 mg/L. Within-run CVs were 0.5 to 1.1%, and day-to-day 1.3 to 3.8%; analytical recovery was 99.3%. Results compared well with those by atomic absorption spectrometry (r = 0.994). A delay time of 10 and a measurement time of 2.5 s allows for a throughput of as many as 180 samples per hour.

Corticosteroid determination in skin preparations by a reaction rate method.

A reaction rate method for the determination of beta-methasone, betamethasone valerate, triamcinolone acetonide, and fluocinolone acetonide is described. The method is based on a modification of the widely accepted blue tetrazolium reaction. Analysis times of 30--70 sec are required. Relative standard deviations of 0.3--1.9% are obtained, and the analytical working curves are linear. Analysis of pharmaceutical skin preparations by the new method gave results that correlated well with the time-consuming standard equilibrium method. Analysis of betamethasone and betamethasone valerate mixtures by measuring absorbance values at two different times was performed also.

Microcomputer controlled titration for determination of protein nitrogen in feeds and wheat.

The determination of protein nitrogen in feeds and wheat by microcomputer controlled titration is described. The method involves direct titration of ammonia with standard hypochlorite titrant in the presence of bromide. The titrant is delivered by an automatic buret, and the microcomputer controlled, automatically computed potentiometric end points are precise to 0.1% over a 5-fold concentration range of nitrogen. Digestions performed with both mercury and copper catalysts show comparable results. Samples are weighed before digestion by an electronic balance interfaced to the computer which records sample number and weight. An automatic pipet aliquots, dilutes, and buffers samples directly from the digestion tubes; the samples can be immediately titrated with the automatic titrator. The results for protein in NBS standards and check feed samples from an offical testing program compare closely with average values reported for these standards. Results show that feed and wheat samples contained 10-100% protein. Precision for successive aliquots of the same digests is 0.1-0.4%relative standard deviation; precision for multiple digestions of the same sample is 0.1-0.8%.

Spectrometric rapid sampling/mixing system for analytical/clinical methods.

A rapid sampling/mixing system has been designed which conforms to certain criteria necessary for its use as a clinical analyser. These include low solution volume (<100mul) for each determination on a sample, rapid cycle time (<2 sec to take an aliquot, mix and transfer reactants to an observation cell), good precision (reproducibility better than 0.2%), and ready automation (requiring only two electronic signals to perform a complete cycle). This device has been incorporated into an automated spectrophotometer to be used for a variety of clinical methods. Equilibrium methods for the determination of calcium and albumin are presented that require measurement times of only 6 and 7 sec, respectively. A reaction-rate procedure for total protein is presented that requires only 3 sec per sample. Precisions obtained with these procedures are typically better than 1% in the normal serum range. Results on samples from hospital patients are compared with values obtained on an SMA 12/60 instrument.

Reaction-rate method for the determination of phosphorus in agricultural products.

A new automated reaction-rate method for the quantitative determination of phosphorus in grains and feeds is presented. The chemical methodology has been investigated, resulting in a high analytical throughput with precise, accurate results. The phosphomolybdenum blue reaction is used for the reaction-rate determination. After digestion of the samples by the official AOAC block digestion procedure, automated instrumentation is used for precise and rapid combination of the reactants and transfer of the mixed solution to an automated spectrophotometer. The rate of formation of the product during 5 sec is automatically determined and compared with rates obtained for phosphorus standards to determine the phosphorus content of the sample. Relative standard deviations of about 0.3% are obtained and results for determinations of phosphorus in grains and feeds are accurate as indicated by comparison with the average obtained by all laboratories participating in the AAFCO check sample programme.

Adaptation of the EMIT serum digoxin assay to a mini-disc centrifugal analyzer.

We have modified the EMIT (Syva) serum digoxin assay for use with a mini-disc centrifugal analyzer. A minicomputer is used to control data acquisition, calculations, and readout for 14 cuvettes simultaneously. Thus, it is not necessary for time each individual assay manually or to plot calibration curves on graph paper. We use incubation times and dilutions similar to those used for the manual procedure, but we have changed the buffer solution, decreased the measurment time, and decreased the required serum volume per assay fivefold. We evaluated day-to-day and within-run precision, using Syva calibrators and control sera. EMIT assay values for radioimmunoassay control sera correlate well with the supplier's stated values.

Automated enzymatic determination of L-lactate in serum, with use of a miniature centrifugal analyzer.

We describe an automated enzymatic reaction-rate method for spectrophotometric determination of lactate in serum with a miniature centrifugal analyzer. The L(+) -lactate is selectively oxidized in the presence of lactate dehydrogenase (EC 1.1.1.27) and NAD+ to form NADH, which is measured from its absorption. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer-generated calibration curve with aqueous lithium lactate standards. Lactate concentrations in the range 0.32-1.6 mug/4 mul (80-400 mg/liter) of sample were determined with relative errors and coefficient of variation of 4.8%. Analytical recovery of lactate added to pooled serum was 89-112% (average, 101%). Comparison with a kit ("Rapid Lactate") method gave a correlation coefficient squared of 0.979 over a concentration range of 39-779 mg/liter.

Adaptation of "EMIT" technique for serum phenobarbital and diphenylhydantoin assays to the miniature centrifugal analyzer.

We used a miniature centrifugal analyzer in a spectrophotometric rate-measurement mode to determine the anticonvulsant drugs phenobarbital and diphenylhydantoin in serum, by use of a modified enzyme immunoassay ("EMIT", Syva Corp.) We decreased reagent cost per determination by at least sixfold by means of microscale techniques. Also, the analysis rate is increased by measuring multiple samples simultaneously. Our method requires only 3 mul of serum for duplicate determinations. Replicate analyses of sera containing phenobarbital and diphenylhydantoin gave reaction rates with a CV of 1.5%. Run-to-run CV was 15%. Analytical recovery for drug-supplemented serum samples was 98%, and results for a series of samples compared well with results obtained by gas chromatography (for phenobarbital, r = 0.95; for diphenylhydantoin, r = 0.91).

Automated enzymic determination of ethanol in blood, serum, and urine with a miniature centrifugal analyzer.

We describe an automated spectrophotometric reaction-rate method for determination of ethanol in serum and urine with a miniature centrifugal analyzer. The theanol is selectively oxidized in the presence of alcochol dehydrogenase and NAD+ to form NADH, which is measured by the rate of change of its absorbance. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer-generated working curve based on aqueous ethanol standards. Blood, serum, or urine specimens need not be deproteinized. The method permits duplicate analysis of at least 30 samples per hour. Coefficients of variation and relative errors are about 2-3% for ethanol concentrations of 0.3-3.0 mug per 2 mul of sample. Analytical recovery of ethanol added to serum is 92-103% (average, 98.5%). Comparisons with distillation-oxidation, gas-chromatographic, and conventional enzymic procedures give satisfactory agreement.