Analysis Of Quality Control Samples Research Articles

Analysis of hydroxylated and N-dealkylated metabolites of terfenadine in microsomal incubates by liquid chromatography–mass spectrometry

This report describes an assay for the H 1-receptor antagonist, terfenadine, and its two primary metabolites, terfenadine alcohol (TOH) and azacyclonol (AZ), using positive-ion, electrospray ionization–liquid chromatography–mass spectrometry. The assay was developed in support of kinetic studies of terfenadine oxidative metabolism in human liver and intestinal microsomes, which required quantification of incubate metabolites at low nanomolar concentrations. Terfenadine metabolites were extracted from basified microsomal incubates into methylene chloride. Reconstituted extracts were subject to liquid chromatographic separation on a cyano-reverse phase column. The [M+H] + ions of terfenadine, terfenadine metabolites, and internal standard were monitored in the effluent by quadrupole mass spectrometry. The assay demonstrated linearity over an incubate concentration range of 5–250 and 12.5–1250 ng/ml for the metabolites and the parent drug, respectively. The respective limits of detection and quantitation for all three analytes were 1.5 and 5 ng/ml of microsomal incubate. Replicate analysis of quality control samples exhibited intra-day coefficients of variation ranging from 3.3% to 7.8% for the three analytes. The corresponding inter-day coefficients of variation ranged from 4.2% to 8.6%. The reproducibility and sensitivity of the assay, combined with the selectivity of mass spectrometric detection, should allow an accurate kinetic characterization of terfenadine oxidation mediated by the high affinity CYP3A enzymes in human liver and intestinal microsomes.

Validation of a method for the determination of zolpidem in human plasma using LC with fluorescence detection

A sensitive and selective high-performance liquid chromatography (HPLC) method was developed for the determination of zolpidem in human plasma. Zolpidem and the internal standard (trazodone) were extracted from human plasma that had been made basic. The basic sample was loaded onto a conditioned Bond Elut C18 cartridge, rinsed with water and eluted with methanol. Forty microliters were then injected onto the LC system. Separation was achieved on a C18 column (150×4.6 mm, 5 μm) with a mobile phase composed of acetonitrile:50 mM potassium phosphate monobasic at pH 6.0 (4:6, v/v). Detection was by fluorescence, with excitation at 254 nm and emission at 400 nm. The retention times of zolpidem and internal standard were approximately 4.7 and 5.3 min, respectively. The LC run time was 8 min. The assay was linear in concentration range 1–400 ng/ml for zolpidem in human plasma. The analysis of quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated excellent precision with relative standard deviations (RSD) of 3.7, 4.6, and 3.0%, respectively ( n=18). The method was accurate with all intraday ( n=6) and overall ( n=18) mean concentrations within 5.8% from nominal at all quality control sample concentrations. This method was also performed using a Gilson Aspec XL automated sample processor and autoinjector. The samples were manually fortified with internal standard and made basic. The Aspec then performed the solid phase extraction and made injections of the samples onto the LC system. Using the automated procedure for analysis, quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated acceptable precision with RSD values of 9.0, 4.9, and 5.1%, respectively ( n=12). The method was accurate with all intracurve ( n=4) and overall ( n=12) mean values being less than 10.8% from nominal at all quality control sample concentrations.

Validation of a method for the determination of (R)-warfarin and (S)-warfarin in human plasma using LC with UV detection

A sensitive and selective chiral high-performance liquid chromatography (HPLC) method was developed for the determination of (R)-warfarin and (S)-warfarin in human plasma. (R)- and (S)-warfarin and the internal standard (oxybenzone) were extracted from human plasma that had been made acidic with 1 N sulfuric acid into ethyl ether. The ethyl ether layer was removed and evaporated, and the residue was reconstituted in 200 μl of acetonitrile. A 50-μl aliquot was injected onto the HPLC system. Separation was achieved on a β-cyclodextrin column (250×4.6 mm, 5 μm) with a mobile phase composed of acetonitrile:glacial acetic acid:triethylamine (1000:3:2.5, v/v/v). Detection was by ultraviolet absorbance at 320 nm. Late-eluting peaks were diverted from the analytical column by using a β-cyclodextrin precolumn (50×4.6 mm, 5 μm) and a column switching device. The retention times of (R)- and (S)-warfarin and the internal standard were approximately 7.7, 6.9 and 4.0 min, respectively. The run time was 15 min. The assay was linear in concentration ranges of 12.5–2500 ng/ml for (R)- and (S)-warfarin in human plasma. The analysis of quality control samples for (R)- and (S)-warfarin (25.0, 400 and 2000 ng/ml) demonstrated excellent precision with relative standard deviations (R.S.D.) for (R)-warfarin of 10.9, 2.8, and 2.8%, respectively ( n=18), and for (S)-warfarin of 7.0, 2.4 and 2.6%, respectively ( n=18). The method was accurate with all overall ( n=18) mean concentrations being less than 6.0% from nominal at all quality control sample concentrations.

Quantitative and qualitative determination of estrogen sulfates in human urine by liquid chromatography/tandem mass spectrometry using 96-well technology.

A sensitive and robust method to determine five estrogen sulfates in human urine has been developed employing high-throughput solid-phase extraction with 96-well technology, and HPLC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. The five estrogen sulfates determined include three major endogenous estrogen sulfates in the human, estrone 3-sulfate (E1-3S), estriol 3-sulfate (E3-3S), and 17 beta-estradiol 3-sulfate (E2-3S), and two biochemical synthetic estrogen sulfates, 17 beta-estradiol 17-sulfate (E2-17S) and 17 beta-estradiol 3,17-disulfate (E2-3,17S). For E2-3,17S, E3-3S, and E2-17S, external standard calibration was used for quantitation, and for the remaining two compounds, internal standard calibration using a stable isotopic labeled internal standard was employed. A total of 96 samples may be prepared with 96-well C18 extraction disk plate techniques performed by a robot within 25 min including the time for evaporation of solvent. The lower level of quantitation (LOQ) for these estrogen sulfates in human urine was determined at 0.2 ng/mL based on 100-microL aliquots of human urine using the optimum tuning parameters for each individual selected precursor ion/product ion transition. The assay was validated with a linear concentration range of 0.2-200 ng/mL, and the interassay accuracy, intraassay precision, and interassay precision do not exceed 8.6%, 12%, and 12%, respectively, by analysis of quality control samples at five concentration levels including the LOQ of 0.2 ng/mL, from four 96-well plates. The target endogenous test articles were qualitatively determined by comparing the full-scan LC/MS/MS mass spectra and retention time in test samples and reference standards. The LOQ is significantly improved compared to previous reports for the targeted compounds using LC/MS/MS. The described simple and automated sample preparation procedure recovered 91% of the target compounds. A total of 192 samples can be analyzed within 1 day (22 h). The method can measure the endogenous estrogen sulfates in urine from both gravid and nongravid subjects.

Identification and quantitation of despropionyl-bezitramide in postmortem samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry.

A sensitive and specific method for the determination and quantitation of (despropionyl) bezitramide in postmortem samples using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method is the result from a simple methodological transfer of a liquid chromatographic method with fluorescence detection (LC-FL) previously developed in our laboratory. A liquid-liquid back-extraction procedure using n-hexane isoamyl alcohol (93:7, v/v) as the extraction solvent is performed for a basic sample cleanup. N-Methyldespropionyl bezitramide is used as the internal standard. Chromatographic separation of the analytes of interest is achieved on a Hypersil ODS 5-micron column, using a 80:20 (v/v) mixture of 1.0 mM ammonium acetate and methanol/acetonitrile (50:50, v/v) and 1.0 mM ammonium acetate as the mobile phase. To obtain as high a sensitivity and selectivity as possible, a selected reaction-monitoring mass spectrometric technique is applied. In addition, low-energy collisional-activated dissociation (CAD) product ion spectra are recorded for a few samples. Calibration graphs are prepared for blood and urine, and good linearity is achieved over a concentration range of 1-150 ng/mL. The intra- and interassay coefficients of variation (CV%) for the analysis of quality control samples at 10 and 50 ng/mL concentration levels do not exceed 10.2% and percent of targets are within 12.1%. Postmortem samples (blood, urine, stomach contents, bile, liver, and kidney) from three fatalities, all suspected victims of drug overdoses, are analyzed, and the results are reported. The results obtained with LC-ESI-MS/MS are in close agreement with those obtained using the LC-FL method. Moreover, the isolates' identity and structure are confirmed by the CAD product ion spectra, thus allowing to make unequivocal conclusions about the prior intake of bezitramide by the three subjects.

Determination of linezolid in plasma by reversed-phase high-performance liquid chromatography

An HPLC–UV method was developed for assay of linezolid in dog, rat, mouse, and rabbit plasma. Linezolid and the internal standard were extracted on a solid phase cartridge (SPE) and separated on a reversed-phase column (C8, 4.6×150 mm, 5 μm) with 20% acetonitrile in water as mobile phase. The SPE quantitatively recovered linezolid and the internal standard from plasma samples. The chromatographic peak height ratio or peak area ratio based on UV absorbency at 251 nm was used for quantitative analysis. The assay procedures were simple and the assay was specific and had adequate precision and accuracy. Calibration standards with concentrations over the range of 0.01–20 μg/ml were validated for routine sample analysis to support the pharmacokinetic and toxicology studies with linezolid in dog, rat, mouse, and rabbit. Analysis of quality control samples showed the coefficients of variation were usually <10% and the measured and theoretical concentrations differed by <10% in most assays. Linezolid in the plasma samples was stable when stored at below −20°C for at least 63 days, at room temperature (22–23°C) for up to 24 h, and after three freeze–thaw cycles. This HPLC method has been successfully used in multiple laboratories to assay plasma samples from pharmacokinetic and toxicology studies with linezolid in the animal species.

High-performance liquid chromatographic assay for the quantitation of irbesartan (SR 47436/BMS-186295) in human plasma and urine

A selective, accurate, precise and reproducible high-performance liquid chromatographic assay was developed for the quantitation of irbesartan, an angiotensin II antagonist, in human plasma and urine samples. The method involved solid-phase extraction of irbesartan and internal standard (I.S.) using a 100-mg Isolute CN cartridge. A portion of the eluate was injected onto an ODS analytical column connected to a fluorescence detector that was set at an excitation wavelength of 250 nm and an emission wavelength of 371 nm. The mobile phase consisted of 50% acetonitrile and a 50% weak phosphate–triethylamine solution, pH 3.5, at a flow-rate of 0.8 ml/min. The assay was linear from 1 to 1000 ng/ml with both plasma and urine. In either matrix, the lower limit of quantitation was 1 ng/ml. The analyses of quality control samples indicated that the nominal values could be predicted with an accuracy >95%. The inter- and intra-day coefficients of variation for the analyses in both matrices were <8%. Irbesartan was stable in both human plasma and urine for at least seven months at −20°C. The application of the assay to a pharmacokinetic study is described.

Radioimmunoassay for quantitation of 2',3'-didehydro-3'-deoxythymidine (D4T) in human plasma.

2',3'-Didehydro-3'-deoxythymidine (D4T, or stavudine) has been recently approved for the treatment of AIDS. In the present study, a specific and sensitive radioimmunoassay (RIA) was developed for the quantitation of D4T in human plasma. The RIA is a double-antibody competitive binding assay which uses anti-D4T antiserum raised in rabbits as the primary antibody, a tritiated derivative of D4T as the radioactive tracer, and goat anti-rabbit immunoglobulin G as the secondary antibody. No cross-reaction between D4T and various nucleoside analogs was detected. The limit of quantitation of the method approximated 20 ng/ml. Replicate analyses of quality control samples (40 to 3,500 ng/ml) had satisfactory intra- and interassay precision (coefficient of variation, 1.7 to 16.7%) and accuracy (deviation, -9.5 to +21.0). This newly developed RIA was successfully used in the monitoring of plasma drug levels in healthy volunteers receiving an oral dose of D4T.

Stereoselective analysis of nadolol in human plasma.

A stereoselective method, involving a single liquid-liquid extraction step, was developed and validated for the analysis of nadolol in human plasma. The assay involved extraction of nadolol and desmethyl-nadolol (as internal standard (IS)) from alkalinized plasma into dichloromethane. The organic solvent was separated and evaporated under nitrogen at 40 degrees C. A chiral derivatization scheme with (R)-(-)-napthylethylisocyanate (50 microL of 0.1% solution in dichloromethane for 60 min) was employed to convert the enantiomers of nadolol into the corresponding diastereomeric derivatives. The residue was reconstituted in the mobile phase and injected onto a C-18 column. The mobile phase was a mixture of methanol:tetrahydrofuran:water (52:7:41 by vol) containing about 0.001% v/v of both phosphoric acid and tetramethylethylenediamine. Fluorimetric detection was performed at excitation 230 and emission 330 nm. The assay was specific for the enantiomers of nadolol and the lower limit of quantitation was 2 ng/mL for each of the enantiomers. Analysis of quality control samples resulted in precision estimates of 7% RSD for inter-assay and 10.1% RSD for intra-assay and the predicted concentrations deviated less than 9.4% of the nominal values for the four enantiomers. The extraction recoveries of the individual enantiomer was about 70%. Stability of nadolol enantiomers were established for four freeze/thaw cycle periods and in the autosampler at 5 degrees C for at least 116 h.

Lead and cadmium levels in human milk and blood

Lead and cadmium levels were determined (with AAS) in blood and milk obtained at 6 weeks after delivery from women living in the vicinity of a copper and lead metal smelter and in a control area. Analyses of lead and cadmium were also performed in blood samples obtained at delivery. Accuracy of the analysis was confirmed by the analysis of quality control samples. In general, blood and milk levels of lead and cadmium were low in both areas. At 6 weeks after delivery the lead levels in blood and milk were 32 ± 8 and 0.7 ± 0.4 μg Pb/l, respectively (total mean ± S.D., n = 75). Cadmium levels in blood and milk were 0.9 ± 0.3 and 0.06 ± 0.04 μg Cd/l, respectively ( n = 75). At delivery the lead levels in blood of women in the smelter area were higher, 38.7 μg Pb/l, than the blood lead levels in women from the control area, 32.3 μg Pb/l, ( P < 0.001). At 6 weeks after delivery there was no difference in blood lead levels between the two groups. In contrast, the lead levels in milk were higher in women from the smelter area, 0.9 μg Pb/l, than in women from the control area, 0.5 μg Pb/l, ( P < 0.001). No differences in blood cadmium levels were found between the two groups. Milk cadmium levels in women from the control area, 0.07 μg Cd/l, were somewhat higher ( P < 0.01) than in women from the smelter area, 0.05 μg Cd/l. Blood cadmium levels were significantly influenced by smoking habits. The mean blood cadmium level for smokers was 1.12 μg Cd/l compared with 0.87 μg Cd/l for non-smokers.

Quantification of 3′-amino-3′-deoxythymidine, a toxic catabolite of 3′-azido-3′-deoxythymidine (zidovudine) in human plasma by high-performance liquid chromatography using precolumn derivatization with fluorescamine and fluorescence detection

A specific and sensitive high-performance liquid chromatographic (HPLC) assay with precolumn derivatization and fluorescence detection was developed for the determination of 3′-amino-3′-deoxythymidine (AMT), a recently characterized toxic catabolite of 3′-azido-3′-deoxythymidine (zidovudine, AZT). After extraction with tert.-butyl methyl ether—1-butanol (6:4, v/v) and back-extraction into basified aqueous phase, the solution was neutralized with phosphate buffer and the compound derivatized with fluorescamine. Fluorescamine-labeled AMT was chromatographed on a reversed-phase C 18 column using a mixture of phosphate buffer—methanol—acetonitrile (47:48:5, v/v/v) as mobile phase and fluorescence detection (excitation wavelength 265 nm, emission wavelength 475 nm). The limit of quantification was 3 ng/ml using a 200-μl plasma sample. The recovery of the extraction procedure averaged at 94.4% in the range of 3–200 ng/ml. The HPLC analysis time required per sample was 16 min. Replicate analyses of quality control samples (5–80 ng/ml) gave satisfactory intra- and inter-assay precision (coefficient of variation varied from 1.9 to 6.7%) and accuracy.

Importance of quality control in a gas chromatographic method to characterize crude oils

AbstractA quality control scheme has been developed to achieve reproducible capillary GC characterization of crude oils and petroleum condensates. The method uses an internal standard to quantify the amount of non‐eluted material in the crude oil sample. Correlation between the gas chromatographic results and actual distillation was 2 %. After repeated use, however, weekly analysis of the same standard crude oil did not give the correct composition. This was a result of discrimination, which worsened with time, as a result of leaks in the septum or the graphite ferrules. More reproducible results were obtained by performing frequent analyses of quality control samples to ensure that the gas chromatographic system was operating properly. The use of quality control charts was a convenient way of ensuring correct operation and identifying the need for corrective action on the gas chromatographic system.

Determination of Salmeterol in Rat and Dog Plasma by High-Performance Liquid Chromatography with Fluorescence Detection

Salmeterol (as the hydroxynaphthoate) Is the first of a new class of long-acting β-adrenergic receptor agonists with both broncho- dilator and anti-inflammatory actions. A sensitive, accurate, and precise high-performance liquid chromatographic method for the determination of salmeterol in rat and dog plasma is described. Samples are prepared by solid-phase extraction and, after chromatography of the extracts on a reversed-phase styrene/divinylbenzene analytical column, salmeterol is detected by fluorescence monitoring (excitation wavelength, 230 nm; emission wavelength, 305 nm). The method Is sensitive to 1 ng/mL, at which concentration the coefficient of variation was 16.3% in a single assay run. Repeated analyses of quality control samples, nominally at 2 ng/mL, were carried out over a number of assay runs with a coefficient of variation of 10.4%. The method is specific for salmeterol with respect to endogenous plasma components and identified metabolites. The assay was applied to the analysis of salmeterol in plasma of rats and dogs from pharmacokinetic studies.

Liquid chromatography of the potential memory-enhancing agent CL 275,838 and its main metabolites, using a post-column photochemical reactor and fluorimetric detection

On irradiation with short-wavelength ultraviolet light, the potential memory-enhancing compound CL 275,838 (I) and its desbenzyl derivative CL 286,527 (metabolite II) are cleaved into the highly fluorescent derivative CL 228,346 (metabolite IV). This reaction was exploited for the sensitive and selective detection of these compounds in human and animal plasma, after reversed-phase high-performance liquid chromatography on a Supelco LC18 DB column (15 cm × 4.6 mm I.D.) at room temperature. The parent compound and its metabolites were isolated from plasma constituents using the Sep-Pak C 18 Plus cartridge, with satisfactory recovery (76–90%) and selectivity. The detection limits were ca. 1.25, 5 and 0.3 ng/ml for I, II and IV, respectively, using 1 ml of plasma. The validation procedure, which includes analysis of multiple ascending calibration curves based on between-day values and replicate analysis of quality control samples analysed with each standard curve, indicated acceptable precision and accuracy of the method within the concentration ranges investigated, the overall coefficient of variation and relative error being less than 10%. The method was successfully applied to plasma samples from healthy volunteers and animals after single of multiple doses of compound I. Metabolites II and IV were detectable in plasma of all species, the former at higher concentrations than the parent compound and metabolite IV. Together with the fact that metabolite II retains much of the parent compound's biological activity in vivo and in vitro, this suggests that it may contribute to the pharmacological effects of compound I.

High-performance liquid chromatographic determination of 2′,3′-didehydro-3′-deoxythymidine, a new anti-human immunodeficiency virus agent, in human plasma and urine

Sensitive and selective high-performance liquid chromatographic techniques have been developed for the determination of 2′-3′-didehydro-3′-deoxythymidine, d4T (BMY-27857), in human plasma and urine. The methods had linear standard curves over the concentration ranges 0.025–25.0 and 0.5–100 μg/ml for the plasma and urine matrices, respectively. Both methods used solid-phase extraction for isolating d4T and the internal standard, thymidine oxetane, from the biological matrix. In addition, the analytical column, mobile phase, instrumentation and chromatographic conditions used for both methods were identical. The ultraviolet absorbance of the column effluent was monitored at 266 nm. Results of analysis of quality control samples indicated that the intra-assay precision values, as measured by percent relative standard deviation, were within 12 and 3%, and accuracy samples deviated less than 10 and 5% from nominal values for the plasma and urine assays, respectively.

Determination of Ondansetron in Plasma and Its Pharmacokinetics in the Young and Elderly

Ondansetron is a 5-hydroxytryptamine3 receptor antagonist for the treatment of chemotherapy- and radiotherapy-induced nausea and emesis. A sensitive, accurate, and precise HPLC method for the determination of ondansetron in plasma is described. Samples are prepared by solid-phase extraction and, after chromatography of the extracts on a silica analytical column, ondansetron is detected by UV absorbance at 305 nm. The method is sensitive down to 1 ng/mL, at which concentration the coefficient of variation was 6.2% in a single assay run. Repeated analyses of quality control samples, nominally at 2 ng/mL, were carried out over a number of assay runs with a coefficient of variation of 5.5%. The method is specific for ondansetron with respect to endogenous plasma components, identified phase I metabolites, and some co-administered chemotherapeutic drugs. In sustained use over several months, and in support of the clinical development of ondansetron, the method has been shown to be robust. An application of the assay in the investigation of the pharmacokinetics of ondansetron in the young and elderly is described.

High-performance liquid chromatographic procedures for the determination of temafloxacin in biological matrices

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination of temafloxacin and its trace level metabolites in biological matrices. Plasma samples are ultrafiltered after addition of an internal standard in a displacing reagent containing sodium dodecyl sulfate and acetonitrile. Plasma ultrafiltrates or diluted urines are chromatographed on a reversed-phase analytical column, using an ion-pair chromatographic mobile phase and fluorescence detection. The chromatographic system allows resolution and quantitation of temafloxacin's oxidative metabolites, which collectively account for less than 2% of the administered dose. The mean intra-assay coefficient of variation for determination of temafloxacin concentrations in plasma ranging from 0.05 to 10.0 μg/ml was 0.7%. The procedure was implemented at four laboratories for the analysis of over 12 000 samples from clinical studies. Inter-assay coefficients of variation estimated from routine analyses of quality control samples in these studies averaged 4% or lower for concentrations in the 0.15–10 μg/ml range. The limit of quantitation of the procedure is approximately 10 ng/ml; inter-assay coefficients of variation at 15 ng/ml averaged under 9%. Calibration curves were reproducible and highly linear, with correlation coefficients typically averaging over 0.9995. An alternative, more complex procedure, involving methylene chloride extraction, which extends the detection limits to below 1 ng/ml, is also described.

Lead and cadmium levels in blood samples from the general population of Sweden

Lead and cadmium was determined in whole blood samples obtained from 473 nonoccupationally exposed adult persons in Sweden in 1980. Analyses were performed using atomic absorption spectrophotometry equipped with an electrothermal atomization unit. Accuracy of the analysis was confirmed by the analysis of quality control samples. Blood lead concentrations were shown to be significantly influenced by sex, smoking habits, and alcohol consumption. Current male smokers had a median blood lead level of 92 μg Pb/liter, as compared to 77 μg Pb/liter for nonsmokers. For females the corresponding values were 69 μg Pb/liter and 57 μg Pb/liter for current smokers and nonsmokers, respectively. Highly significant correlations were found between stated alcohol consumption and blood lead in most of the different sex and smoking categories. People living in apartments close to streets with heavy traffic in Stockholm had slightly, but not significantly, higher blood lead levels when compared to people living in areas of this city with low traffic density. Blood cadmium levels were very strongly affected by smoking habits. A significant correlation existed between the number of cigarettes consumed daily and blood cadmium concentration. The median blood cadmium level for nonsmoking males was 0.2 μg Cd/liter (⩽0.2, detection limit) and for females 0.3 μg Cd/liter. About 90% of all nonsmokers had cadmium concentrations in blood below 0.6 μg Cd/liter, whereas about 90% of the current male and female smokers had cadmium concentrations in blood of 0.6 μg Cd/liter or more.

Simple Manual Procedure for Determination of Serum Triglycerides

We describe a modified method for determining serum triglycerides (triacylglycerols), which is based on the heptane extraction procedure of Gottfried and Rosenberg [Clin. Chem. 19, 1077 (1973)] with the stable saponification, oxidation, and color development reagents of Neri and Frings [Clin. Chem. 19, 1201 (1973)]. This modified method eliminates one heating step, reduces saponification time to 5 min, absorbances are read at room temperature, and the calibration curve is linear to 3.0 g/liter. A sample comparison between the proposed method and the automated Block and Jarrett [Am. J. Med. Technol. 35, 1 (1969)] procedure showed no significant difference (r equals 0.98). The coefficient of variation (47 duplicate samples) for the modified method was 6.3%. Further validation was obtained from analysis of quality-control samples; the proposed method gave equivalent values.