Inter-batch Coefficient Of Variation Research Articles

Development of a time-resolved immunochromatographic test strip for rapid and quantitative determination of GFAP in serum.

Glial fibrillary acidic protein (GFAP) in serum has been shown as a biomarker of traumatic brain injury (TBI) which is a significant global public health concern. Accurate and rapid detection of serum GFAP is critical for TBI diagnosis. In this study, a time-resolved fluorescence immunochromatographic test strip (TRFIS) was proposed for the quantitative detection of serum GFAP. This TRFIS possessed excellent linearity ranging from 0.05 to 2.5 ng/mL for the detection of serum GFAP and displayed good linearity (Y = 598723X + 797198, R2 = 0.99), with the lowest detection limit of 16 pg/mL. This TRFIS allowed for quantitative detection of serum GFAP within 15 min and showed high specificity. The intra-batch coefficient of variation (CV) and the inter-batch CV were both < 4.0%. Additionally, this TRFIS was applied to detect GFAP in the serum samples from healthy donors and patients with cerebral hemorrhage, and the results of TRFIS could efficiently discern the patients with cerebral hemorrhage from the healthy donors. Our developed TRFIS has the characteristics of high sensitivity, high accuracy, and a wide linear range and is suitable for rapid and quantitative determination of serum GFAP on-site.

Development of a time-resolved immunochromatographic test strip for rapid and quantitative determination of retinol-binding protein 4 in urine.

Urine retinol-binding protein 4 (RBP4) has recently been reported as a novel earlier biomarker of chronic kidney disease (CKD) which is a global public health problem with high morbidity and mortality. Accurate and rapid detection of urine RBP4 is essential for early monitor of impaired kidney function and prevention of CKD progression. In the present study, we developed a time-resolved fluorescence immunochromatographic test strip (TRFIS) for the quantitative and rapid detection of urine RBP4. This TRFIS possessed excellent linearity ranging from 0.024 to 12.50 ng/mL for the detection of urine RBP4, and displayed a good linearity (Y = 239,581 × X + 617,238, R2 = 0.9902), with the lowest visual detection limit of 0.049 ng/mL. This TRFIS allows for quantitative detection of urine RBP4 within 15min and shows high specificity. The intra-batch coefficient of variation (CV) and the inter-batch CV were both < 8%, respectively. Additionally, this TRFIS was applied to detect RBP4 in the urine samples from healthy donors and patients with CKD, and the results of TRFIS could efficiently discern the patients with CKD from the healthy donors. Thedeveloped TRFIS has the characteristics of high sensitivity, high accuracy, and a wide linear range, and is suitable for rapid and quantitative determination of urine RBP4.

Abstract 4560: Assay-ready tissue-engineered cancer microspheres for high-throughput screening

Abstract For drug discovery, new in vitro cancer models are needed to obtain more translatable study outcomes in a low-cost and high-throughput manner. For this purpose, 3D cancer spheroids have been established as more effective than 2D models. Current commercial techniques, however, rely heavily on self-aggregation of dissociated cells and cannot replicate key features of the native tumor microenvironment, particularly due to a lack of control over extracellular matrix components and heterogeneity in size and aggregate-forming tendencies. Also, current spheroidal techniques are typically limited to one spheroid per well, therefore providing a narrow range of cell numbers per well, disadvantageous for assay development in drug screening. Here, we overcome these challenges by coupling tissue engineering toolsets with microfluidic technologies to create engineered cancer microspheres and sorting desired numbers of microspheres into assay-ready well-plate format. To form the engineered cancer microspheres, MCF7 (non-metastatic) and MDA-MB-231 (metastatic) breast cancer cells were encapsulated within poly(ethylene glycol)-fibrinogen hydrogels using our previously developed microfluidic platform. Highly uniform cancer microspheres (intra and inter-batch coefficient of variation ≤ 5%) with high cell densities (over 20 × 106 cells/ml) were produced rapidly, which is critical for use in drug testing. The microspheres supported the 3D culture of both breast cancer cell lines over at least 14 days in culture. Encapsulated cells displayed cell type-specific differences in morphology, proliferation, metabolic activity, ultrastructure, and overall microsphere size distribution and bulk stiffness. To prepare assay-ready pre-plated microspheres, a COPAS FP flow cytometer was used for its ability to analyze and sort large sample particles such as tumor spheroids and hydrogel cancer microspheres generated in this study. When using a 96-well plate, the sorting rate varied from 2.5 - 6 microspheres per second, depending on the sample concentration. When sorting a desired number of microspheres per well, the accuracy was greater than 95% as verified visually by microscopy. Viability of sorted microspheres was verified 24 hours post-sort. Shipping conditions were established that maintained cell viability for remote use in drug testing. Methods for compound addition by pinning and imaging were tested and optimized. Using these approaches, the microsphere system was shown to be compatible with an automated liquid handling system for administration of drug compounds; MDA-MB-231 microspheres were distributed in 384 well plates and treated with chemotherapeutic drugs. Expected responses were quantitated using CellTiter-Glo® 3D and detected using automated imaging. Overall, our results demonstrate initial applicability for the tissue-engineered cancer microspheres for drug screening. Citation Format: Yuan Tian, Yongwoon Kim, Wen J. Seeto, Shantanu Pradhan, Dmitriy Minond, Rock Pulak, Elizabeth A. Lipke. Assay-ready tissue-engineered cancer microspheres for high-throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4560.

Development of a time-resolved immunochromatographic strip for rapid and quantitative determination of deoxynivalenol.

Deoxynivalenol (DON) contamination of food crops and feeds is almost impossible to avoid completely; however, through best management practices, this risk can be effectively managed and maximumly mitigated. Accurate and rapid detection of DON contamination as early in the entire value chain as possible is critical. To achieve this goal, we developed a DON test strip based on time-resolved fluorescence immunoassay (TRFIA) and a specific DON monoclonal antibody for the rapid quantification of DON in food crops and feeds. The strip displayed a good linearity (R 2 = 0.9926), with a limit of quantification of 28.16 μg/kg, a wide linear range of 50 ~ 10,000 μg/kg. The intra-batch coefficient of variation (CV) and the inter-batch CV was <5.00 and 6.60%, respectively. This TRFIA-DON test strip was applied to detect DON in real samples, and the accuracy and reliability were confirmed by liquid chromatography-mass spectrometry (LC-MS/MS). Results showed that the relative standard deviation between the DON strips and LC-MS/MS was <9%. The recovery rates in corn samples ranged from 92 to 104%. The established TRFIA-DON test strip had the characteristics of high sensitivity, high accuracy, and a wide linear range which was suitable for rapid and quantitative determination of DON in food crops and feeds at both on-site and laboratory.

Abstract 175: Production of cancer tissue-engineered microspheres for high-throughput screening

Abstract There is a need for new in vitro systems that enable pharmaceutical companies to collect more physiologically-relevant information on drug response in a low-cost and high-throughput manner. For this purpose, three-dimensional (3D) spheroidal models have been established as more effective than two-dimensional models. Current commercial techniques, however, rely heavily on self-aggregation of dissociated cells and are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of control over extracellular matrix components and heterogeneity in shape, size, and aggregate forming tendencies. In this study, we overcome these challenges by coupling tissue engineering toolsets with microfluidics technologies to create engineered cancer microspheres. Specifically, we employ biosynthetic hydrogels composed of conjugated poly(ethylene glycol) (PEG) and fibrinogen protein (PEG-Fb) to create engineered breast and colorectal cancer tissue microspheres for 3D culture, tumorigenic characterization, and examination of potential for high-throughput screening (HTS). MCF7 and MDA-MB-231 cell lines were used to create breast cancer microspheres and the HT29 cell line and cells from a stage II patient-derived xenograft (PDX) were encapsulated to produce colorectal cancer (CRC) microspheres. Using our previously developed microfluidic system, highly uniform cancer microspheres (intra-batch coefficient of variation (CV) ≤ 5%, inter-batch CV &amp;lt; 2%) with high cell densities (&amp;gt;20×106 cells/ml) were produced rapidly, which is critical for use in drug testing. Encapsulated cells maintained high viability and displayed cell type-specific differences in morphology, proliferation, metabolic activity, ultrastructure, and overall microsphere size distribution and bulk stiffness. For PDX CRC microspheres, the percentage of human (70%) and CRC (30%) cells was maintained over time and similar to the original PDX tumor, and the mechanical stiffness also exhibited a similar order of magnitude (103 Pa) to the original tumor. The cancer microsphere system was shown to be compatible with an automated liquid handling system for administration of drug compounds; MDA-MB-231 microspheres were distributed in 384 well plates and treated with staurosporine (1 μM) and doxorubicin (10 μM). Expected responses were quantified using CellTiter-Glo® 3D, demonstrating initial applicability to HTS drug discovery. PDX CRC microspheres were treated with Fluorouracil (5FU) (10 to 500 μM) and displayed a decreasing trend in metabolic activity with increasing drug concentration. Providing a more physiologically relevant tumor microenvironment in a high-throughput and low-cost manner, the PF hydrogel-based cancer microspheres could potentially improve the translational success of drug candidates by providing more accurate in vitro prediction of in vivo drug efficacy. Citation Format: Elizabeth A. Lipke, Wen J. Seeto, Yuan Tian, Mohammadjafar Hashemi, Iman Hassani, Benjamin Anbiah, Nicole L. Habbit, Michael W. Greene, Dmitriy Minond, Shantanu Pradhan. Production of cancer tissue-engineered microspheres for high-throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 175.

Reproducibility of identical solid phantoms.

.SignificanceTissue-like solid phantoms with identical optical properties, known within tolerant uncertainty, are of crucial importance in diffuse optics for instrumentation assessment, interlaboratory comparison studies, industrial standards, and multicentric clinical trials.AimThe reproducibility in fabrication of homogeneous solid phantoms is focused based on spectra measurements by instrument comparisons grounded on the time-resolved diffuse optics.ApproachEpoxy-resin and silicone phantoms are considered as matrices and both employ three different instruments for time-resolved diffuse spectroscopy within the spectral range of 540 to 1100 nm. In particular, we fabricated two batches of five phantoms each in epoxy resin and silicone. Then, we evaluated the intra- and interbatch variability with respect to the instrument precision, by considering the coefficient of variation (CV) of absorption and reduced scattering coefficients.ResultsWe observed a similar precision for the three instruments, within 2% for repeated measurements on the same phantom. For epoxy-resin phantoms, the intra- and the interbatch variability reached the instrument precision limit, demonstrating a very good phantom reproducibility. For the silicone phantoms, we observed larger values for intra- and interbatch variability. In particular, at worst, for reduced scattering coefficient interbatch CV was about 5%.ConclusionsResults suggest that the fabrication of solid phantoms, especially considering epoxy-resin matrix, is highly reproducible, even if they come from different batch fabrications and are measured using different instruments.

Reliability of urinary charged metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry

Currently, large-scale cohort studies for metabolome analysis have been launched globally. However, only a few studies have evaluated the reliability of urinary metabolome analysis. This study aimed to establish the reliability of urinary metabolomic profiling in cohort studies. In the Tsuruoka Metabolomics Cohort Study, 123 charged metabolites were identified and routinely quantified using capillary electrophoresis-mass spectrometry (CE-MS). We evaluated approximately 750 quality control (QC) samples and 6,720 participants’ spot urine samples. We calculated inter- and intra-batch coefficients of variation in the QC and participant samples and technical intraclass correlation coefficients (ICC). A correlation of metabolite concentrations between spot and 24-h urine samples obtained from 32 sub-cohort participants was also evaluated. The coefficient of variation (CV) was less than 20% for 87 metabolites (70.7%) and 20–30% for 19 metabolites (15.4%) in the QC samples. There was less than 20% inter-batch CV for 106 metabolites (86.2%). Most urinary metabolites would have reliability for measurement. The 96 metabolites (78.0%) was above 0.75 for the estimated ICC, and those might be useful for epidemiological analysis. Among individuals, the Pearson correlation coefficient of 24-h and spot urine was more than 70% for 59 of the 99 metabolites. These results show that the profiling of charged metabolites using CE-MS in morning spot human urine is suitable for epidemiological metabolomics studies.

Establishment and Application of a Dual-Labeling Time-Resolved Fluorescence Immunoassay Method for Simultaneous Detection of the Troponin I-C Complex and Full-Size-Troponin I.

Background: The measurement of cardiac troponin I (cTnI) is widely used in the diagnosis of acute myocardial infarction (AMI). Although existing cTnI detection methods measure total cTnI, the significance of undegraded full-size-cTnI levels is still not well-understood. In this study, we have established a novel dual-labeling time-resolved fluorescence immunoassay (TRFIA) technique that simultaneously detects the cTnI-C complex and full-size-cTnI, allowing us to explore the clinical value of full-size-cTnI determination.Methods: An antibody against the 23–43 amino acid region of cTnI protected by endogenous cTnC is coupled to magnetic beads to provide a solid-phase antibody for capturing all cTnI. An antibody against cTnC in the cTnI-C complex labeled with Eu3+ was used to detect the cTnI-C complex, and an antibody labeled with Sm3+ near the C-terminal 190–203 amino acids of cTnI was used to detect full-size-cTnI. Through dual-labeling TRFIA, cTnI-C complex, full-size-cTnI, and the full-size-cTnI/cTnI-C ratio can be detected simultaneously. The dual-labeling TRFIA technique was used to analyze serum samples collected at different times during treatment and compare their full-size-cTnI/cTnI-C ratios.Results: The sensitivity for the cTnI-C-TRFIA complex was 0.02 ng/mL, the measurement range was 0.02–40 ng/mL, the average intra-batch coefficient of variation (CV) was 4.35%, and the inter-average CV was 6.23%. The correlation coefficient between cTnI-C-TRFIA and commercial cTnI-CLIA methods was R2 = 0.8887. The sensitivity for full-size-cTnI-TRFIA was 0.04 ng/mL, the measurement range was 0.04–40 ng/mL, the average intra-batch CV was 4.95%, and the average inter-batch CV was 7.79%. The correlation coefficient between full-size-cTnI-TRFIA and commercial cTnI-CLIA methods was R2 = 0.7247.Conclusions: Dual-labeling full-size-cTnI/cTnI-C-TRFIA analysis is helpful for determining the length of time of chest pain before admission and the degree of continuous release of cTnI in the myocardium. Thus, it is more for early prognosis than just detecting cTnI.

Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry.

BackgroundCohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography–mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population.MethodsMetabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid.Results and conclusionsIn QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies.

Prediagnostic plasma adiponectin and survival among patients with colorectal cancer.

526 Background: Adiponectin is a hormone secreted by adipose tissue and has been demonstrated to possess anticarcinogenic, anti-inflammatory and insulin-sensitizing effects. Circulating adiponectin has been shown to be inversely associated with the risk of colorectal cancer (CRC) in prospective studies. However, the association of prediagnostic adiponectin with survival among patients with established colorectal cancer is unclear. Methods: We conducted a prospective study of 621 incident colorectal cancer cases from the Nurses’ Health Study and Health Professionals Follow-up Study to evaluate the association between prediagnostic plasma adiponectin and mortality. Plasma adiponectin levels were determined by enzyme-linked immunosorbent assay from ALPCO Diagnostics. The interbatch coefficient of variation from quality control samples randomly interspersed among the case samples was 8.6%. We examined the associations between quartiles of plasma adiponectin and mortality using a Cox proportional hazards model adjusted for established and putative risk factors. All statistical tests were two sided. Results: After a median follow-up of 9 years, there were 267 (43%) total deaths and 130 (21%) CRC deaths in the total study cohort. Compared with patients in the lowest quartile of adiponectin, patients in the highest quartile had a multivariate hazard ratio (HR) for CRC-specific mortality of 2.70 [95% confidence interval (CI), 1.54-4.75; Ptrend = 0.001]. The corresponding multivariate HR for overall mortality was 1.64 (95% CI, 1.14-2.36; Ptrend = 0.007). These associations were reasonably consistent in analyses according to subgroups defined by age, gender, body mass index, stage, grade and site of primary cancer. Similar results were yielded after excluding patients diagnosed within 4 years of blood collection (Ptrend = 0.027). Conclusions: Prediagnostic adiponectin is associated with an increased risk of colorectal cancer- specific and overall mortality. Further studies are needed to elucidate the mechanistic basis for these findings and determine the potential role of adiponectin as a prognostic marker in colorectal cancer.

A versatile method for protein-based antigen bioanalysis in non-clinical pharmacokinetics studies of a human monoclonal antibody drug by an immunoaffinity liquid chromatography–tandem mass spectrometry

A versatile immunoaffinity liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to quantify the total concentration of a protein-based antigen in non-clinical pharmacokinetics (PK) studies of a human monoclonal antibody drug. The method combines using magnetic beads that have been coated with a commercial anti-human Fc region antibody to capture an immune complex of the antigen and antibody drug, with subsequent digestion and quantification of the antigen-derived tryptic peptide via LC–MS/MS. Although a typical immunoassay or an immunoaffinity LC–MS/MS assay requires an antigen-specific antibody that uses a different epitope from the antibody drug, this method requires only a commercial anti-human Fc region antibody. The method was applied to quantify total receptor activator of nuclear factor-κB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL. The assay was validated as fit-for-purpose and found to be accurate (<115% interbatch accuracies) and precise (<15%, interbatch coefficient of variation) across a range of 3.13–200ng/mL RANKL. Commercial enzyme-linked immunosorbent assay (ELISA) kit was not able to determine the total RANKL because interference by denosumab decreased recovery. In contrast, the antibody drug had less effect on the LC–MS/MS method. The method now provides a bioanalytical platform for developing other protein-based antigen assays in the early drug stage.

Quantitative Determination of Deoxynivalenol (DON) Using the Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA)

An indirect competitive Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) was established by using anti-deoxynivalenol (DON) polyclonal antibody and coating antigen DON-bovine serum albumin (BSA) for detection of DON in cereals. DON-BSA was coated on Acceptor bead, the kit also contains donor bead sensitizer particles coated with streptomycin. The optional test conditions and analytical performance of the method were studied. Working concentration ranged from 0.007 to 100 ng/ml and the sensitivity for detection was 0.007 ng/ml. The intra- and inter-batch coefficient of variation (CV) of the assay were both below 5%. The recovery rate of artificially contaminated cereals ranged from 81.1% to 110.9% while the mean recovery of DON from cereals was 94.4%. This study suggests that DON-AlphaLISA method is a good method with high sensitivity and rapidity for quantitative analyzing DON in cereals.

An immunoaffinity liquid chromatography–tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum

An immunoaffinity liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap–release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3 nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay.

Dual-label time-resolved fluoroimmunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A

A dual-label time-resolved fluoroimmunoassay (TRFIA) for simultaneous quantification of aflatoxin B1 (AFB1) and ochratoxin A (OTA) is described. For this, microtitration wells were coated with AFB1-horse radish peroxidase (HRP) and OTA-bovine serum albumin. The standards and samples were loaded on the coated plates, and diluted antibodies and Eu3+- and Sm3+-labeled IgG were then added. Our results showed that the sensitivity of TRFIA for AFB1 was 0.02 microg/L (range 0.02-100 microg/L). The intra- and inter-batch coefficient of variation (CV) was 3.2 and 7.3%, respectively, and the average recovery rate was 88.1%. On the other hand, the sensitivity of OTA was 0.05 microg/L (range 0.05-50 microg/L), the intra- and inter-batch CV was 2.9 and 7.9%, respectively, and the average recovery rate was 89.9%. In the AFB1/OTA-TRFIA, AFB1 and OTA did not mutually interfere. The correlation coefficients between the dual-label AFB1/OTA-TRFIA and the single-label AFB1-TRFIA or OTA-TRFIA were 0.972 and 0.981, respectively, indicating that the results were consistent. Our study suggests that AFB1/OTA-TRFIA allows the simultaneous detection of AFB1 and OTA; is a simple, fast, and economic method for screening large quantities of samples, and has good prospects of application.

The determination of plasma oxalate concentrations using an enzyme/bioluminescent assay

An enzyme/bioluminescent assay for the determination of oxalate in plasma is described in which NADH, a reaction product of the enzymic degradation of oxalate by oxalate decarboxylase and formate dehydrogenase, is determined using a commercially available bioluminescent system. In contrast to most previously documented methods, this sensitive and specific assay requires minimal sample preparation allowing oxalate concentrations to be determined within 2 h of sample collection. The limit of detection for plasma samples is 0.8 μmol/l. The recovery of oxalate added to plasma averaged 99%. The inter-batch coefficient of variation, calculated by analysis of a plasma sample from a uraemic patient (oxalate concentration = 45.8 μmol/l) on 8 occasions, over a period of 5 wk, was 3.2%. Plasma oxalate concentrations in 35 normal subjects ranged from < 0.8–1.5 μmol/l, which is in excellent agreement with values obtained by in vivo isotope dilution studies. Plasma oxalate was found to be strikingly elevated in a group of uraemic patients maintained on regular haemodialysis.

An improved benzophenone procedure for the microdetermination of 5,5-diphenylhydantoin in blood

A modification of the benzophenone procedure of Wallace for the determination of 5,5-diphenylhydantoin in blood has been developed. It is extracted from blood with dichloroethane, returned to alkali, washed with n-heptane, and oxidized with permanganate in screw capped tubes containing a layer of n-heptane. The absorption of the oxidation product is measured spectrophotometrically on an expanded scale using semi-microcuvettes. This procedure eliminates specialized glassware, reduces sample volume, extractant volumes, extraction and oxidation periods, and by washing the final extract with n-heptane non-specific interference is removed. The method is simple, reliable, highly specific and suitable for routine use. Sample requirement is 100–200 μl for a single determination with a detection threshold of less than 0.1 mg/100 ml. The intrabatch coefficient of variation is 1.2% at a blood level of 4.0 mg/100 ml and 7.0% at 0.5 mg/100 ml. The interbatch coefficient of variation for a control serum over one month was 3.8% at 1.31 mg/100 ml.