Radioimmunoassay for arylsulfatase A in urine

Purified arylsulfatase A (EC 3.1.6.1) from human urine was radioiodinated under conditions that caused no significant loss of antigenic activity. We used this labeled arylsulfatase A (specific radioactivity 4-7.5 Ci/g) together with nonlabeled enzyme and rabbit antiserum produced against homogeneous enzyme to develop a radioimmunoassay for arylsulfatase A in urine. A solid-phase, second-antibody technique (Immunobead Second Antibody; Bio-Rad Laboratories) was used to separate free enzyme from antigen-antibody complexes. The working range of the assay was 0.1-4.0 ng of enzyme; within- and between-assay CVs were around 10%, and the analytical recovery was 105.5% (SD 7.7%). The lower limit of detection was 0.08 ng of arysulfatase A per assay, substantially less than that of typical activity-based assays. Over a wide range of urinary arylsulfatase A activities, results by this method agreed well (r = 0.99) with those obtained by activity assays. We measured the enzyme in urines of 59 healthy volunteers and 92 patients with different diseases, including a group of colorectal cancer cases, to determine whether this could serve as a reliable marker for cancer of the colon; however, urinary excretion of arylsulfatase A by most patients with colon cancer was within normal limits.     

Rapid measurement of estrogens and their metabolites in human serum by liquid chromatography-tandem mass spectrometry without derivatization

Objectives

The steroids estradiol (E2), estrone (E1), and estriol (E3) are the major estrogens. E1/E2 and their metabolite 16-hydroxyestrone (16-OHE1, known to be carcinogenic) could be involved in the development of many cancers including human breast cancer. The aim of the current study was to develop a rapid and simple high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to simultaneously measure E1, E2, E3 and 16-OHE1 in human serum without the need for solid phase extraction or derivatization.

Methods

An API-5000 triple-quadrupole mass spectrometer coupled with electrospray ionization (ESI) source and Shimadzu HPLC system was used employing isotope dilution with deuterium-labeled internal standard (IS) for each analyte. Quantitation by multiple reaction monitoring (MRM) analysis was performed in negative ion mode.

Results

The limits of detection were 1.0 pg/mL for E1 and 16-OHE1 and 2.0 pg/mL for E2 and E3. Within-day CVs were < 6.5% for all analytes tested and between-day CVs ranged from 4.5% to 9.5%. Recovery ranged from 88% to 108%.

Conclusion

This method allows for the simultaneous measurement of four estrogens in human serum within 8 min. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing, micro sample requirement, and high throughput.     

Direct chemiluminescence immunoassay for estradiol in serum

In this simple, reliable, fast solid-phase chemiluminescence immunoassay for directly measuring (i.e., without prior extraction) estradiol-17 beta in serum, a monoclonal antibody is used that binds estradiol with high affinity (Ka = 10(10) L/mol), and does not bind other steroids tested, the highest cross reactivity observed being 0.1% for estradiol-17 alpha. In this system the monoclonal antibody is bound to the wells of microtiter plates via a second antibody directed against the monoclonal antibody. Fifty microliters of serum and estradiol-displacing agents are added, followed by 100 pg of estradiol-isoluminol conjugate, and the label is measured by luminometry after the binding reaction. The sensitivity of the assay is 180 pmol per liter of serum, and the effective working range at less than or equal to 10% CV is 270 to 6700 pmol/L. Analytical recovery of added estradiol averaged 99.7% (SD 6.5%). Within- and between-assay CVs ranged between 5 and 12.7%. Thirty-five unknown serum samples can be assayed within 4 h. Results correlated well with those obtained with a direct RIA: r = 0.94 (n = 149). This assay opens new perspectives for chemiluminescence immunoassays.     

External quality assessment of assays for cortisol in plasma: use of target data obtained by gas chromatography/mass spectrometry

We describe procedures for measuring cortisol in plasma and serum by isotope dilution and mass spectrometry. A method that incorporated solvent extraction, derivatization, and gas chromatography/high-resolution mass spectrometry provided data of good precision; interassay CVs were generally 3 to 4% for the concentration range of 100-650 nmol/L. Replacing solvent extraction with extraction on a column of Lipidex 1000 or extraction by immunoadsorption yielded data in excellent agreement with the first method. Plasma and serum pools were analyzed to provide target data for use in the U.K. National External Quality Assessment Scheme for cortisol assays. Routine laboratory assays, as judged by comparison with mass-spectrometric data, were generally positively biased except for analysis of a charcoal-stripped plasma supplemented with cortisol. The results emphasize the importance of using unadulterated plasma or serum pools in assessments of steroid assay procedures.     

Therapeutic drug monitoring on COBAS INTEGRA 400--evaluation results

The COBAS INTEGRA 400 (Roche Diagnostics GmbH) is a random access analyzer with a consolidated test menue for routine clinical chemistry, specific proteins, drugs of abuse screening and therapeutic drug monitoring (TDM) and different measuring technologies. It was the aim of the present study to evaluate the suitability of this instrument as dedicated analyzer for TDM. Eight assays based on three different technologies were included: Acetaminophen (enzymatic method), Amikacin/ Phenytoin/ Free Phenytoin/ Lidocaine (fluorescence polarization immunoassays; FPIA), Digitoxin/Digoxin (kinetic interaction of microparticles in solution; KIMS). The study comprised the determination of imprecision according to NCCLS EP-T protocol, method comparison and linearity studies. The assays were compared with the corresponding methods on AxSYM or TDx analyzers (Abbott Laboratories). For Acetaminophen and Amikacin COBAS INTEGRA 700 was used as additional comparison instrument. The results are summarized in a table (table 6). Precision results are well acceptable with within-run CVs < 5% and total CVs < 6% except for Digitoxin and Digoxin which show a somewhat higher imprecision at low concentrations. Results obtained for Acetaminophen and Amikacin on COBAS INTEGRA 400 and 700 show excellent agreement. A good comparability is also found between COBAS INTEGRA 400 and AxSYM or TDx methods with slight systematic deviations for Acetaminophen, Amikacin and Free Phenytoin. The lower correlation coefficient for the digoxin method comparison can be attributed to two discrepant samples. Linearity throughout the range studied which covered > 80% of the measuring range was confirmed for the five assays tested (Digitoxin, Digoxin, Lidocaine, Free Phenytoin, Phenytoin) based on the acceptance criteria of +/- 10% deviation of the measured values from the theoretical values. Based on the analytical performance of the TDM tests studied it can be concluded that the COBAS INTEGRA 400 is very well suited for routine TDM analysis.     

Indirect vs direct measurement of magnesium and zinc in erythrocytes

We evaluated three methods (two indirect and one direct) for determining the magnesium (Mg) and zinc (Zn) content of erythrocytes, to compare methodologic differences and to establish a method suitable for use in field studies. For the indirect methods, erythrocytes in whole blood were lysed by adding either de-ionized water (I) or nitric acid, 2 mol/L (II). For the direct method (III), erythrocytes were isolated by density centrifugation, washed, then digested in concentrated HNO3. Mg and Zn concentrations were measured by atomic absorption spectrophotometry in plasma and whole blood for the indirect methods, and in the pellet for the direct method. Hematocrit and hemoglobin were measured, and erythrocytes were sized and counted on all samples. Within-run CVs for the three methods ranged from 2.2% with method III for Mg to 5.4% with method I for Zn. CVs for reproducibility of the analytical procedures ranged from 2.6% with method II for Zn to 4.2% with method I for the two cations. Analytical recoveries of added Mg and Zn ranged from 93.8 to 104.7%. When values for the three methods were compared, those by method I were significantly (p less than 0.05) lower than those by methods II and III. Values obtained by method II were 100.1% for Mg and 102.4% for Zn of those by the direct method. Thus, the indirect method with 2 mol/L HNO3 lysing solution provides a reproducible, reliable, accurate, and simple technique for measuring Mg and Zn in erythrocytes.     

Simplified liquid-chromatographic assay of amiodarone and desethylamiodarone after solid-phase extraction

We describe a rapid, simplified isocratic "high-performance" liquid-chromatographic method for simultaneous measurement of the antiarrhythmic drug amiodarone and its major metabolite, desethylamiodarone, in small volumes of sera (100 microL). Compared with liquid-liquid extraction, the solid-phase method of extraction saves time and glassware and improves reproducibility for small sample volumes. Amiodarone and desethylamiodarone could be measured at concentrations as low as 250 micrograms/L. Standard curves for the drug and metabolite are linear over the range of concentrations found in our patients. Within-run CVs (n = 6) ranged from 2.7% to 4.5% for amiodarone and from 4.0% to 5.7% for desethylamiodarone over the range of 250 to 4000 micrograms/L. Between-run CVs (n = 12) were 8.3% and 5.7% for amiodarone and desethylamiodarone, respectively. Commonly used cardiovascular medications do not interfere with the assay.     

Enlightenment about the new Architect-i2000 estradiol (Abbott Laboratories) immunoassay during in vitro fertilization

OBJECTIVE: We assessed a new estradiol (E2) immunoassay on the Architect-i2000 (Abbott Laboratories) for monitoring ovulation stimulation for IVF-ET and re-establishing clinical cut-off points. The method has been modified to improve E2 measurements especially at normal and low concentrations. DESIGN AND METHOD: E2 was determined for 552 samples, from 83 women, presenting normal follicular status and undergoing 100 cycles of IVF treatment. We assessed the value of this assay for down-regulation of E2 concentration limit using gonadoliberin-releasing hormone agonist (GnRHa), and monitoring of the ovarian hyperstimulation, expected range of E2 per mature follicle prior to the administration of exogenous hCG and day 3 concentration limit. We compared results with our routine method (E2-6II Advia-Centaur; Siemens-Diagnostics) for which decision-making values were known. RESULTS: Considering E2 concentrations obtained with the new Architect-i2000 assay for patients treated with GnRHa for 2 weeks, the cutoff-point for ovarian down-regulation should be set down at 110 pmol/L to maintain 100% of sensitivity. Considering day 3 concentration limit determination, results were not significantly different from those obtained with our routine method. The mean E2 values per mature follicle fell into the range generally expected. CONCLUSION: E2 determination with the new E2 Architect-i2000 assay could be used to monitor ovulation, in patients undergoing IVF-ET, in combination with transvaginal ultrasound.     

Amylase in urine as measured by a single-step chromolytic method

We studied a new single-step direct chromolytic method (Behring D.A.T.) for measuring urinary amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) activity, comparing results with those by a similar, but two-step, procedure that requires an auxiliary (coupling) enzyme. The two methods gave virtually identical relative responses to purified human pancreatic and salivary amylases. Assay of four quality-control materials to evaluate the total (day-to-day) precision of the new method yielded CVs of 4 to 7%, similar to those of the comparison method for each of the four quality-control samples. Amylase activity was measured by both methods in 110 random (i.e., untimed) urine specimens. Linear regression analysis provided a slope and y-intercept of 0.947 and 4 U/L (x = comparison method, y = direct method), respectively, and a standard error of the estimate of 25 U/L for specimens in which the amylase activities ranged from 11 to 1465 U/L (mean = 358 U/L) by the comparison method. The mean rate of amylase excretion in 2-h timed urine specimens from 95 healthy volunteers, as measured by the new method, was 7.18 (SD = 3.18) U/h, and the nonparametric (95% confidence interval) reference interval was 1.6 to 15.2 U/h. We consider the new method a promising alternative to other kinetic assays that require the use of auxiliary enzymes.     

雌二醇化学发光免疫测定法的建立

目的　建立灵敏度高 ,测定范围宽的检测人血清雌二醇 (E2 )的化学发光免疫分析法 (CLIA)。方法　采用竞争抑制法 ,用碱性磷酸酶标记抗原 ,金刚烷 (CSPD)增敏化学发光体系作为酶底物。结果　敏感度为 2 .0pg/ml;在 10～ 10 0 0pg/ml之间可以准确定量 ;用不同浓度的E2 质控血清测定精密性 ,批内变异 <8% (n =2 0 ) ,批间变异 <10 % (n =2 0 )。与雌醇、雌酮、雌三醇(E3 )的交叉反应 <1% ,与睾酮、可的松等无交叉反应。试剂具有良好的稳定性 ,在 4℃保存 14个月其整体变化幅度 <10 %。与BeckmanAccessTm及其配套试剂比较有较好的相关性。结论　方法灵敏度高 ,特异性强 ,稳定性好 ,检测范围宽 ,准确性和重复性好。     

Clinical vitamin B6 analysis: an interlaboratory comparison of pyridoxal 5'-phosphate measurements in serum

BACKGROUND: Recent investigations into the role vitamin B(6) plays in reducing risk of stroke and cardiovascular disease have heightened interest in vitamin B(6) intake and its relationship to clinical status indicators. Because a true reference method and certified reference materials are lacking, little is known about the relative analytical performance of clinical vitamin B(6) assays. METHODS: Ten laboratories experienced in clinical vitamin B(6) analysis participated in a 3-day analysis of 69 serum and 3 aqueous specimens for pyridoxal 5'-phosphate (PLP). Laboratories used either HPLC-based or enzymatic assays. Results were analyzed for imprecision, recovery, and bias relative to consensus means. RESULTS: Among laboratories, mean within-day CVs (3 specimens x 3 measurements/day) were 0.6%-37% and between-day CVs (20 specimens x 1 measurement/day x 3 days) were 1.4%-26%. Mean recoveries of added PLP were 53%-144%, and mean sample pool mixing recoveries were 75%-119%. Consensus means calculated for 20 serum specimens gave mean relative biases between measurement of -10.0% to 24.3% among participating laboratories over a range of 15.8-319 nmol/L PLP. Measurement imprecision and biases were evaluated against empirically derived performance criteria based on biological variation. Three of 10 laboratories met optimum imprecision requirements and had 90% or more of measurements satisfy optimum criteria for biases among methods. All 10 laboratories met minimum imprecision requirements, but 25%-53% of the results reported by 4 of the 7 suboptimal laboratories failed to satisfy the minimum criteria for bias. CONCLUSION: Agreement among vitamin B(6) methods is good, but large differences in laboratory proficiency exist, pointing to the need for vitamin B(6) reference materials and external quality assurance programs.     

Direct chemiluminescence immunoassay of estradiol in saliva

A sensitive and simple direct solid-phase chemiluminescence immunoassay is described for estradiol in saliva. In this assay, a second antibody is bound to the wells of microtiter plates. Either buffer with standards or saliva (100 microL) is incubated in these wells with monoclonal anti-estradiol antibody and with estradiol-isoluminol conjugate. Incubation time is 2 h. Chemiluminescence of the bound fraction is measured in a manually operated luminometer (Biocounter). The assay has a detection limit of 3.8 pmol/L; analytical recovery of added estradiol is 96.8% (SD 7.0%); within- and between-assay CVs range between 2.5% and 12.7%. Forty unknown saliva samples can be assayed and results calculated within 4.5 h. Results of a slightly modified procedure-with black microtiter plates and a prototype of an automated plate reader (Luminoskan)--compare well with those of the described method (r = 0.97). Because steroid-binding globulins have been found in saliva, the effect of displacing agents on the results of the direct chemiluminescence assay is described.     

Multicentre performance evaluation of the E170 module for modular analytics.

The E170 module was evaluated at 13 sites in an international multicentre study. The objective of the study was to assess the analytical performance of 49 analytes, and to collect feedback on the system's reliability and practicability. The typical, within-run coefficients of variation (CVs) for most of the quantitative assays ranged between 1 and 2% while a range of 2-4% was achieved with the infectious disease methods. Total precision CVs were found to be within the manufacturer's expected performance ranges, demonstrating good concordance of the system's measuring channels and a high reproducibility during the 2-4-week trial period. The functional sensitivity of 11 selected assays met the clinical requirements (e.g., thyreotroponin (TSH) 0.008 mU/l, troponin T 0.02 microg/l, total prostate-specific antigen (PSA) 0.03 microg/l). The E170 showed no drift during an 8-hour period and no relevant reagent carryover. Accuracy was confirmed by ring trial experiments and method comparisons vs. Elecsys 2010. The reliability and practicability of the system's hardware and software met with, or even exceeded, the evaluator's requirements. Workflow studies showed that E170 can cover the combined workload of various routine analysers in a variety of laboratory environment. Throughput and sample processing time requirements were achieved while personnel 'hands-on-time' could be reduced.     

Evaluation of a radioimmunoassay of urinary cortisol without extraction

We evaluated the Kallestad "Quanticoat" cortisol RIA for direct (no extraction) measurement of urinary free cortisol, which requires no solvent extraction. An analytical-recovery study showed a linear regression of y (measured) = 0.65x (added) + 37.5 micrograms/L (Sy.x = 21.4 micrograms/L, r = 0.978, n = 48). Recovery appeared to vary with the urine used and with the concentrations of cortisol added. Within- and between-run CVs were less than or equal to 4.1% and less than or equal to 3.8%, respectively. Cross reactivities were low, except for prednisolone (20.5%). This no-extraction method gave higher values for urinary free cortisol than did either an RIA method involving extraction or an HPLC method. A comparison study with the HPLC (x) and with the method involving extraction (x') gave the following Deming-debiased regression equations: y = 1.60x + 68.8 (Sy.x = 34.4, n = 29) and y = 1.33x' + 0.69 (Sy.x = 40.3, n = 66), respectively. We conclude that the no-extraction method may give misleading results for patients' diagnosis or management if this cross reactivity is not taken into account.     

Evaluation of analytical methods and workflow performance of the Architect ci8200 integrated serum/plasma analyzer system

BACKGROUND: The Architect ci8200 is an integrated serum analyzer for photometric, electrochemical and immunological assays. Several assays of each category and the workflow performance of the system were compared with established laboratory procedures in two laboratories. METHODS: Measurements were compared with the ELECSYS 2010 (Roche Diagnostics) for CEA, PSA, FPSA, AFP, folate, vitamin B12, with the CENTAUR (Bayer) for TSH, T4, FT4, FSH and Estradiol, with the LIAISON (DiaSorin) for TSH, FT4 and FT3, with the Behring Nephelometer BN II (Dade-Behring) for ferritin, and with the INTEGRA 800 (Roche Diagnostics), and the AU640 (Olympus) for clinical chemistry assays. Workflow studies were performed to compare times of analysis required for defined analytical workloads. RESULTS: The coefficients of variation (CVs) for within-run imprecision were between 3% and 6% for CEA, PSA, FPSA, AFP and ferritin, and between 3% and 11% for TSH, FT4, FT3, folate and vitamin B12. The CVs for day-to-day imprecision for immunoassays were between 3% and 10%, except for vitamin B12 (CVs 11-13%) and FT4 (CV 10% -13%). For clinical chemistry tests corresponding CVs for within-run imprecision were < 1%, except for HDL, triglyceride, creatinine, ALT, LD and lipase (CVs<2%) and bicarbonate (CV 3%-6%) and magnesium (CV < 3%). The CVs for day-to-day imprecision for clinical chemistry tests were < 1%, except for sodium, CO(2), magnesium, phosphorus, glucose, uric acid, HDL, triglyceride, ALT, AST CK, lipase with CVs < 6% and for CO(2)<11%. Dilutional linearity testing of seven immunoassays and five clinical chemistry analytes resulted in recovery rates of 90-110%. Correlation studies with 15 immunoassays and 25 clinical chemistry tests showed acceptable agreements with established methods. Work flow analyses demonstrated a net gain in time of analysis up to 109 min depending on the size of the sample batch analyzed with the Architect ci8200 as the main analyzer as compared to the currently installed routine laboratory equipment. Median turn-around times were 7 and 30 min for chemistry assays and immunoassays, respectively, when ordered as STAT analyses, and 18 min when chemistry assays were ordered as routine determinations. CONCLUSIONS: Assays on the Architect ci8200 performed well, fulfilling quality control requirements as defined for instance by German quality control guidelines (RiliBAK). Method comparisons showed acceptable agreements with established assays. Workflow studies using the Architect ci8200 documented shorter times of analyses as compared with the conventionally established laboratory routine demonstrating the potential of integrated chemistry/immunoassay analyzers to provide faster and more efficient performance.     

Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Given the role of folate in many disorders, intracellular distribution of folate vitamers is of potential clinical importance. In particular, accumulation of non-methyltetrahydrofolates due to altered partitioning of folate metabolism at the level of methylenetetrahydrofolate is of interest. METHODS: We describe a positive-electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows determination of erythrocyte folate vitamer distribution by accurately measuring both 5-methyltetrahydrofolate (5-methylTHF) and non-methyl folate vitamers. Whole blood lysates are deconjugated in ascorbic acid solutions, deproteinized, purified using folate-binding protein affinity columns, concentrated by solid-phase extraction (SPE) and evaporation, and separated on a C18 column within 6 min. RESULTS: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4 nmol/L (signal-to-noise >10). Intra- and inter-assay CVs for 5-methylTHF were 1.2% and 2.8%, respectively. Intra- and inter-assay CVs for non-methylTHF as a group were 1.6% and 1.5%, respectively. Recovery results were 97-107%. We measured 8-72% non-methyl folate vitamers in volunteers (n=5) with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype. Concentrations ranged from 117 to 327 nmol/L and 23 to 363 nmol/L for 5-methylTHF and non-methylTHF vitamers, respectively. We measured 0-2% non-methylTHF vitamers in MTHFR 677 CC genotype volunteers. In addition, we found that storage of whole-blood samples in ascorbic acid at low pH resulted in 53-90% loss of the non-methylTHF fraction. CONCLUSION: This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.     

Automated chemiluminescence-immunoassay for aldosterone during dynamic testing: comparison to radioimmunoassays with and without extraction steps

BACKGROUND: Measurements of aldosterone have become more common since the recognition that primary aldosteronism is a more frequent cause of hypertension than previously believed. Our aim was to compare concentrations reported by 4 assays for samples obtained after saline infusion during dynamic testing. METHODS: We tested 104 participants (27 with primary aldosteronism, 30 with essential hypertension, and 47 healthy controls) with the intravenous saline infusion test (2.0 L isotonic saline over 4 h), with repetitive sampling. In all blood samples, aldosterone concentration was measured by an in-house RIA after extraction and chromatography, by 2 commercially available RIAs without extraction (Aldosterone Maia, Adaltis; Active Aldosterone, Diagnostics Systems Laboratories) and by an automated CLIA (Advantage, Nichols Institute Diagnostics). RESULTS: Correlation coefficients for results of pairs of assays ranged from 0.74 to 0.98. Agreement between commercial assays and in-house RIA was best at the low to intermediate concentrations after saline infusion. Mean (SD) Adaltis and DSL RIA results were 2- to 3-times higher [healthy participants: 78 (25) ng/L and 56 (18) ng/L, respectively] than those obtained by Nichols CLIA [17 (8) ng/L] and in-house RIA [23 (18) ng/L]. Aldosterone concentrations measured by the Nichols CLIA were below the limit of detection (limit of the blank) in 27 of 47 healthy participants. CONCLUSIONS: Aldosterone concentrations reported by the Adaltis and DSL nonextraction RIAs were consistently higher than those produced by the Nichols CLIA and the in-house RIA. The convenient Nichols CLIA showed better agreement with the in-house RIA, but the concentrations in healthy participants were frequently undetectable by this method. Uncritical application of cutoff values from the literature must be avoided.     

External evaluation of LIAISON tumour marker assays on the fully automated chemiluminescent LIAISON immunoassay analyser

The LIAISON immunoassay analyser was tested in a multicentre evaluation performed by 8 laboratories. The analytes evaluated were CA 15-3, CA 19-9, CA 125II, AFP, CEA, NSE and PSA. Excellent results were obtained for within-run and between-run precision with most assays showing within-run CVs < 5% and between-run CVs between 4 and 8%. The linearity of all assays was acceptable, however, for PSA, NSE and CA 19-9 a recovery > 110% was obtained for some of the samples tested. None of the assays revealed a high-dose hook effect. Method comparisons were performed by using the routine method of the respective study centre. Results generally showed an acceptable agreement between the LIAISON system and the different methods of comparison. The reference ranges for all assays were found to be in accordance with data known from the literature. All assays showed similar results for serum, heparinised plasma and EDTA plasma. Additionally, two experiments were performed with only one of the analytes tested: the sample-to-sample carry-over, using the CA 19-9 assay (3.3 x 10(-6)-2.3 x 10(-5)) and the functional sensitivity for the PSA assay (0.2 ng/ml).     

Analyses for progesterone in serum by gas chromatography/mass spectrometry: target data for external quality assessment of routine assays

We describe a procedure for measuring progesterone in plasma and serum by isotope dilution and mass spectrometry. Extraction with use of a microcellulose-coupled antiserum is followed by conversion to the 3-enol heptafluorobutyrate and analysis by gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring, at a resolution of 5000. Interassay CVs were 1.5 to 5.4% for the concentration range 13 to 43 nmol/L. Analyses of various serum volumes showed excellent linearity. Accurate determination of progesterone added to serum was demonstrated. Plasma and serum pools were analyzed to provide target data for use in the U.K. national external quality-assessment scheme for progesterone assays. Direct, non-extraction radioimmunoassays and those incorporating solvent extraction both showed a positive bias with respect to data obtained by the present procedure, but the bias was more marked with the direct assays.     

Quantitative determination of estradiol fatty acid esters in human pregnancy serum and ovarian follicular fluid.

BACKGROUND: Lipophilic estradiol derivatives carried by lipoprotein particles in blood may mediate antioxidant or endocrine effects. We developed a new quantitative method to determine the concentration of circulating lipophilic estradiol fatty acid esters in human early- and late-pregnancy serum and in ovarian follicular fluid. METHODS: After extraction from serum or follicular fluid, estradiol fatty acid esters were separated from nonesterified estradiol by Sephadex LH-20 column chromatography. The estradiol ester fraction was hydrolyzed by saponification and further purified by several chromatographic steps. The hydrolyzed estradiol esters were measured by time-resolved fluoroimmunoassay. RESULTS: The average estradiol fatty acid ester concentration in serum increased 10-fold during pregnancy, from 40.4 pmol/L (expressed as pmol/L estradiol; range, 25.0-64.2 pmol/L) in early pregnancy (n = 8) to 404 pmol/L (196-731 pmol/L) in late pregnancy (n = 10). The ratio of estradiol ester to nonesterified estradiol remained relatively constant during pregnancy, at 0.4-0.6%. In 10 follicular fluid samples, the mean estradiol ester concentration was 106 nmol/L (56.9-262 nmol/L). Compared with serum, a greater proportion of estradiol in follicular fluid (3.0-10%) was in the esterified form. CONCLUSION: The new method provides a means to measure circulating estradiol fatty acid ester concentrations in human pregnancy serum.