Between-assay Variability Research Articles

Optimized Enzyme-Linked Immunosorbent Assay for Anti-PEG Antibody Detection in Healthy Donors and Patients Treated with PEGylated Liposomal Doxorubicin.

There is considerable interest in quantifying anti-PEG antibodies, given their potential involvement in accelerated clearance, complement activation, neutralization, and acute reactions associated with drug delivery systems. Published and commercially available anti-PEG enzyme-linked immunosorbent assays (ELISAs) differ significantly in terms of reagents and conditions, which could be confusing to users who want to perform in-house measurements. Here, we optimize the ELISA protocol for specific detection of anti-PEG IgG and IgM in sera from healthy donors and in plasma from cancer patients administered with PEGylated liposomal doxorubicin. The criterion of specificity is the ability of free PEG or PEGylated liposomes to inhibit the ELISA signals. We found that coating high-binding plates with monoamine methoxy-PEG5000, as opposed to bovine serum albumin-PEG20000, and blocking with 1% milk, as opposed to albumin or lysozyme, significantly improve the specificity, with over 95% of the signal being blocked by competition. Despite inherent between-assay variability, setting the cutoff value of the optical density at the 80th percentile consistently identified the same subjects. Using the optimized assay, we longitudinally measured levels of anti-PEG IgG/IgM in cancer patients before and after the PEGylated liposomal doxorubicin chemotherapy cycle (1 month apart, three cycles total). Antibody titers did not show any increase but rather a decrease between treatment cycles, and up to 90% of antibodies was bound to the infused drug. This report is a step toward harmonizing anti-PEG assays in human subjects, emphasizing the cost-effectiveness and optimized specificity.

#4179 DIFFERENCES IN PROTOCOLS FOR MEASURING GLOMERULAR FILTRATION RATE USING IOHEXOL

Abstract Background and Aims When assessing kidney function, measurement of glomerular filtration rate (mGFR) using an exogenous marker such as iohexol is the gold standard. In this study, we aim to identify similarities and differences between iohexol-based mGFR protocols. Method Detailed data on iohexol measurement protocols were obtained using a standardized survey sent to centres in Europe and the US. It was completed by 15 participants. Data are reported as number and percentage (n, %). Results In the participating centres, measurements are performed after referral by a nephrologist (n=6, 40%), other specialties (n=5, 33%) or for research purposes (n=4, 27%). Most common indications for mGFR are evaluation of kidney donors (n=5, 33%), drug dosing (n=4, 27%), abnormal body composition (n=3, 20%) and transplant evaluation (n=4, 27%). Most participants perform measurements in the morning (n=10, 67%), with patients withholding caffeine (n=5, 33%), fasting (n=4, 27%) or avoiding heavy meals (n=3, 20%). Most centres use an IV iohexol dose of 5 mL 300 mg I/mL (n=10, 67 %) or a dose based on weight (n=2, 13%). The timing of sample collection is shown in Figure 1, most often a single sample per time point (n=12, 80%). Iohexol is measured by LC-MS (n=8, 53%) or LC-UV (n=7, 47%). Within-assay variability ranges between <2% (n=3, 20%) and 6-8% (n=1, 7%) and the between-assay variability ranges between <2% (n=2, 13%) and 6-8% (n=2, 13%). When asked about assumptions and corrections, most centres make a one-compartment assumption in their PK model (n=8, 53%), others making two-compartment (n=2, 13%) or measurement-dependent (n=1, 7%) assumptions. mGFR is standardized for body surface area according to the DuBois-formula (n=6 (40%), Haycock and Schwarz formula (n=2, 13%) or not-specified (n=7, 47%). Some participants correct their measurements for eGFR (n=9, 60 %). Most centres participate in external quality control (Equalis, n=12, 80%). Conclusion There is a large variation in protocols for iohexol-based mGFR, which highlights the need for a standardized mGFR protocol before widespread use in clinical routine.

Parathyroid hormone measurement in chronic kidney disease: Impact of inter-method variability on mineral bone disease assessment

BackgroundParathyroid hormone (PTH) is measured routinely as part of Chronic Kidney Disease Bone and Mineral Disorders (CKD-MBD) assessment. Multiple PTH assays exist with known differences resulting in CKD-MBD guidelines recommending treatment based on assay-specific thresholds. The study objectives are to assess between manufacturer and within manufacturer variability of PTH assays and the impact of assay variability on the assessment of CKD-BMD using both vendor defined and empirically derived thresholds. MethodsData were collected from Ontario, Canada’s Proficiency Testing Program (24 challenge vials, 115–133 laboratories all using secondary generation PTH assays. Mean PTH and precision by the coefficient of analytical variation (CVa) were calculated. For each vial, whether the manufacturer’s mean value exceeded the vendor-defined and empirically-derived upper limit of normal (ULN) was recorded and the concordance between assays was determined. ResultsAcross all laboratories, the mean PTH range was 12.0 ± 3.9 pmol/L and the mean CVa was 30%. The percent of vials with a mean PTH exceeding manufacturer’s specific ULN varied substantially between manufacturers. Only 58% of vials had complete concordance as to whether mean PTH was above assay-specific ULNs. This increased to 83% using the empirically derived ULN. ConclusionsCKD-BMD assessment and management will depend on the PTH assay. The between-assay variability is reduced but not eliminated when empirically derived reference intervals are used. Improvements in PTH measurement are required in order to ensure consistent patient care.

Demonstration of reciprocal diurnal variation in human serum T3 and rT3 concentration demonstrated by mass spectrometric analysis and establishment of thyroid hormone reference intervals.

Background:There has been a wide range of reference intervals proposed in previous literature for thyroid hormones due to large between-assay variability of immunoassays, as well as lack of correction for collection time. We provided the diurnal reference intervals for five thyroid hormones, namely total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), free triiodothyronine (FT3), and reverse T3 (rT3), measured in serum samples of healthy participants using a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method.Methods:Couplet serum samples (a.m. and p.m.) were collected from 110 healthy females and 49 healthy males. Healthy volunteers were recruited from four participating centers between 2016 and 2018. Measurements of thyroid hormones were obtained by LC-MS/MS analysis.Results:Our study revealed significant uptrend in AM to PM FT4 (p < 0.0001) samples, downtrend in AM to PM TT3 (p = 0.0004) and FT3 samples (p < 0.0001), and AM to PM uptrend in rT3 samples (p < 0.0001). No difference was observed for TT4 between AM and PM. No significant sex differences were seen for any of the five thyroid hormones.Conclusion:When diagnosing thyroid disorders, it is important to have accurate measurement of thyroid hormones, and to acknowledge the diurnal fluctuation found, especially for FT3. Our study highlights the importance of standardization of collection times and implementation of LC-MS/MS in thyroid hormone measurement.

Effect of faecal calprotectin assay variability on the management of inflammatory bowel disease and potential role of faecal S100A12

AimsTo prospectively evaluate whether between-assay variability of different faecal calprotectin (f-Cp) assays influences diagnostic accuracy for inflammatory bowel disease (IBD) in a cohort of patients with confirmed IBD and irritable...

The feasibility of harmonizing gluten ELISA measurements

Many publications have highlighted that routine ELISA methods do not give rise to equivalent gluten content measurement results. In this study, we assess this variation between results and its likely impact on the enforcement of the EU gluten-free legislation. This study systematically examines the feasibility of harmonizing gluten ELISA assays by the introduction of: a common extraction procedure; a common calibrator, such as a pure gluten extract and an incurred matrix material. The comparability of measurements is limited by a weak correlation between kit results caused by differences in the selectivity of the methods. This lack of correlation produces bias that cannot be corrected by using reference materials alone. The use of a common calibrator reduced the between-assay variability to some extent, but variation due to differences in selectivity of the assays was unaffected. Consensus on robust markers and their conversion to “gluten content” are required.

Searching for a BNP standard: Glycosylated proBNP as a common calibrator enables improved comparability of commercial BNP immunoassays

BackgroundCirculating B-type natriuretic peptide (BNP) is widely accepted as a diagnostic and risk assessment biomarker of cardiac function. Studies suggest that there are significant differences in measured concentrations among different commercial BNP immunoassays. The purpose of our study was to compare BNP-related proteins to determine a form that could be used as a common calibrator to improve the comparability of commercial BNP immunoassay results. MethodsBNP was measured in 40 EDTA-plasma samples from acute and chronic heart failure patients using five commercial BNP assays: Alere Triage, Siemens Centaur XP, Abbott I-STAT, Beckman Access2 and ET Healthcare Pylon. In parallel with internal calibrators from each manufacturer, six preparations containing BNP 1–32 motif a) synthetic BNP, b) recombinant BNP (E. coli), c) recombinant nonglycosylated proBNP (E. coli), d) recombinant His-tagged (N-terminal) nonglycosylated proBNP (E. coli), e) recombinant glycosylated proBNP (HEK cells), and f) recombinant glycosylated proBNP (CHO cells) were also used as external calibrators for each assay. ResultsUsing the internal standards provided by manufacturers and for five of six external calibrators, up to 3.6-fold differences (mean 1.9-fold) were observed between BNP immunoassays (mean between-assay CV 24.5–47.2%). A marked reduction of the between-assay variability was achieved, when glycosylated proBNP expressed in HEK cells was used as the common calibrator for all assays (mean between-assay CV 14.8%). ConclusionsOur data suggest that recombinant glycosylated proBNP could serve as a common calibrator for BNP immunoassays to reduce between-assay variability and achieve better comparability of BNP concentrations of commercial BNP immunoassays.

Validation of an electrophoretic method to detect albuminuria in cats.

Objectives The aims of this study were to validate a semi-automated high-resolution electrophoretic technique to quantify urinary albumin in healthy and diseased cats, and to evaluate its diagnostic performance in cases of proteinuria and renal diseases. Methods Urine samples were collected from 88 cats (healthy; chronic kidney disease [CKD]; lower urinary tract disease [LUTD]; non-urinary tract diseases [OTHER]). Urine samples were routinely analysed and high-resolution electrophoresis (HRE) was performed. Within-assay and between-assay variability, linearity, accuracy, recovery and the lowest detectable and quantifiable bands were calculated. Receiver operating curve (ROC) analysis was also performed. Results All coefficients of variation were <10%, percentage recovery was between 97% and 109% with a high linearity (r = 0.99). HRE allowed the visualisation of a faint band of albumin and a diffused band between alpha and beta zones in healthy cats, while profiles from diseased cats were variable. Albumin (mg/dl) and urine albumin:creatinine ratio (UAC) were significantly ( P <0.05) different between healthy and diseased cats. After ROC analysis, UAC values of 0.035 and 0.074 had a high sensitivity and high specificity, respectively, to classify proteinuria and identify borderline proteinuric cats. Moreover, a UAC of 0.017 had a high sensitivity in distinguishing between healthy and diseased cats. However, UAC was not able to distinguish between renal (CKD) and non-renal diseases (LUTD/OTHER), probably owing to the pathophysiology of CKD in cats, which is characterised by low-grade proteinuria and less glomerular involvement than in dogs. Conclusions and relevance HRE is an accurate and precise method that could be used to measure albuminuria in cats. UAC was useful to correctly classify proteinuria and to discriminate between healthy and diseased cats. HRE might also provide additional information on urine proteins with a profile of all proteins (albumin and globulins) to aid clinicians in the diagnosis of diseases characterised by proteinuria.

PTH-075 The effect of storage conditions on the stability of faecal calprotectin

Introduction Faecal calprotectin (f-Cp) is a sensitive non-invasive marker of intestinal inflammation used to help distinguish between inflammatory bowel disease (IBD) and functional bowel disorders such as IBS, thereby reducing the number of unnecessary endoscopic and radiological investigations in patients presenting with chronic diarrhoea. It has previously been reported that calprotectin is stable in faeces for up to seven days at room temperature (RT). A nationwide study was undertaken via the UK NEQAS Faecal Markers of Inflammation external quality assessment (EQA) programme in order to further investigate the stability of f-Cp. Method Complete 24 h stool collections were provided by three IBD patients. Each stool was homogenised and split into six portions, each of which was stored under different conditions: i) RT (∼20˚C) for seven days; ii) RT for 14 days; 4°C for seven days; 4°C for 14 days and; two portions immediately frozen at –40°C. Following storage, the portions not already in the freezer were frozen at –40°C. Sub-aliquots of each of the six portions from each patient were dispatched to the participants of the UK NEQAS Faecal Markers of Inflammation EQA scheme for analysis. Results Forty-four scheme participants returned results. Mean f-Cp concentrations in the baseline specimens were 9.3–95.4 µg/g [patient 1], 204–449 µg/g [patient 2] and 5.5–66.4 µg/g [patient 3]. F-Cp levels decreased over the period of 14 days when the stool samples were stored at RT but to varying degrees depending upon the initial concentration and the assay used for analysis. For the most severely-affected method (Thermo Fisher ELiA) f-Cp levels decreased by ∼50% following storage at RT for 14 days in the specimen from patient 2. No decrease was observed for the same assay at lower f-Cp concentrations [patients 1 and 3] under the same conditions. No effect of storage was observed at either RT or 4°C for the users of the Buhlmann, Immundiagnostik, Calpro (ELISA) and Buhlmann Quantum Blue assays at lower f-Cp concentrations [Patients 1 and 3]. Storage for 14 days at 4°C had no significant effect for the Buhlmann ELISA, Calpro ELISA and Quantum Blue users at higher f-Cp concentrations (Patient 2). A decrease of ∼10% was observed for the Thermo Fisher ELiA and Immundiagnostik ELISA assays stored under the same conditions. Conclusion The conditions under which stool samples are transported and stored prior to calprotectin analysis can affect the results obtained in the laboratory depending upon the baseline f-Cp level and the particular assay being used for analysis. Our data also further highlights the between-assay variability that exists amongst f-Cp kits. Disclosure of interest None Declared.

Standardizing vitamin D assays: the way forward.

For a number of years it has been widely assumed that measurement of serum 25-hydroxyvitamin D [25(OH)D] concentration is the best approach to assessing an individual's vitamin D status. However, it has also been recognized that there is substantial within-assay variation in 25(OH)D measurement and even greater between-assay variability. Such assay variation clearly confounds attempts to define what constitutes the diagnosis of hypovitaminosis D. Importantly, assay variability makes pooling of 25(OH)D results from different studies in systematic reviews for the specific purpose of determining dose-response and/or clinical cut points at best problematic. Therefore, to develop and implement evidence-based clinical guidelines, it is essential that 25(OH)D measurement be standardized in both clinical and research laboratories. In this Perspective we outline a way forward toward achieving this goal-the Vitamin D Standardization Program (VDSP).

Between-assay variability of faecal calprotectin enzyme-linked immunosorbent assay kits

Faecal calprotectin (f-Cp), a marker of intestinal inflammation, can be used to distinguish between functional and organic bowel disease. F-Cp, following extraction, is commonly quantified using enzyme-linked immunosorbent assays (ELISAs) but there are no data comparing the different f-Cp assays or sample extraction devices. We, therefore, evaluated and compared the performance of the Immunodiagnostik, Bühlmann and Eurospital f-Cp ELISA assays as well as the Roche, Immunodiagnostik and ScheBo Biotech commercial faecal extraction devices. We also briefly report results from a pilot f-Cp external quality assurance (EQA) scheme. Imprecision, linearity, recovery, drift and limit of quantitation of the f-Cp assays were evaluated and between-assay variability assessed. The three commercial sample extraction devices were compared with the manual weighing method. Four faecal samples were distributed as part of a pilot EQA scheme to 15 laboratories using quantitative ELISA f-Cp assays. The three f-Cp assays demonstrated adequate intra-/interbatch imprecision, linearity and recovery. The cross-comparison study and EQA data demonstrated that, for the same sample, the Bühlmann assay reports up to 3.8 times higher f-Cp concentrations than the Immunodiagnostik and Eurospital assays. On average, the commercial extraction devices led to a 7.8-28.1% under-recovery of f-Cp in comparison to the manual weighing method. Laboratories should be aware of the lack of the assay standardization, as demonstrated by the between-assay variability. A comparison between f-Cp concentrations reported by these assays and clinical markers of disease severity is required in order to determine their diagnostic accuracy. The EQA scheme represents the first available programme for f-Cp.

Quantification of anti-Mullerian hormone (AMH) in dried blood spots: validation of a minimally invasive method for assessing ovarian reserve

Biological markers of ovarian reserve have the potential to advance research on fecundability, infertility and reproductive aging. Anti-Müllerian hormone (AMH) has emerged as a clinically useful measure of ovarian reserve, but the requirement for venous blood is an obstacle to application in non-clinical settings. This paper validates a new method for quantifying AMH in dried blood spot (DBS) samples--drops of whole blood collected on filter paper following a simple finger stick. Matched serum and DBS samples were obtained from n=101 women of reproductive age, and AMH values were compared using regression analyses and scatter plots. The precision, reliability, linearity, recovery and lower detection limit of the DBS assay were evaluated, as well as the stability of AMH in DBS across a range of storage conditions. There was a strong agreement between AMH concentrations measured in DBS and serum samples across the entire assay range. Analysis of within-assay (percent coefficient of variation, 4.7-6.5%) and between-assay (3.5-7.2%) variability indicated a high level of assay precision and reliability, respectively. The minimum detectable dose of AMH was 0.065 ng/ml. Concentrations of AMH remained stable in DBS samples stored for 2 weeks at room temperature, and for 4 weeks when refrigerated. The DBS assay performs at a level that is comparable to serum-based methods, with the advantage of lower burdens and costs associated with blood collection that may be advantageous for research in clinical as well as non-clinical settings on the causes and consequences of variation in ovarian reserve.

A study of longer-time stability of plasma free metanephrines

Previous stability studies of plasma free metanephrines do not extend beyond one month. For retrospective evaluation and documentation purposes, knowledge of stability for more prolonged storage periods is required. A panel of seven plasma samples was aliquoted and stored at -20 and -80°C. Aliquots were thawed and assayed by high-performance liquid chromatography with electrochemical detection at regular intervals during three years. A final set was assayed after five years at -80°C. Results were evaluated by repeated-measures analysis of variance. After a stable period of over one year, an upward trend for plasma free metanephrine and a downward trend for normetanephrine was observed. For both analytes, measurement results were parallel between samples during the study. Concentrations in samples stored at -20°C did not differ from those stored at -80°C. Storage at either -20 or -80°C must be considered as safe for at least one year. The residual variation with respect to time of the concentrations of metanephrines was identical in all of the samples assayed and almost did not exceed previously determined within-run variation. This suggests that between-assay variability is the cause of the overall trends and not sample deterioration. Moreover, between-assay variability manifesting itself as drift or trend remained within the range of earlier observed between-assay variation. Despite this, the assay for plasma metanephrines remains capable of detecting catecholamine overproduction in plasma samples that have been stored for prolonged periods of time.

Harmonization of growth hormone measurements with different immunoassays by data adjustment

The aim of our study was to evaluate the between-assay variability of commercially available immunoassays for the measurement of human growth hormone (hGH). In addition, we asked whether the comparability of the diagnosis of childhood onset growth hormone deficiency could be improved by adjusting hGH results by statistical methods, such as linear regression, conversion factors, and quantile transformation. In archived sera from 312 children and adolescents (age: 17 days-17 years) hGH values between 0.01 and 16.5 ng/mL were determined by using the following immunoassays: AutoDELFIA (PerkinElmer), BC-IRMA (Beckman-Coulter), ELISA (Mediagnost), IMMULITE 2000 (Siemens), iSYS (IDS), Liaison (DiaSorin), UniCel DxI 800 Access (BeckmanCoulter) and "In house"-RIA (Tübingen). The assays differed in median hGH concentrations by as much as 5.44 ng/mL (Immulite), and as little as 2.67 ng/mL (BC-IRMA). The mean difference between assays ranged from 0.35 to 2.71 ng/mL, whereas several samples displayed differences up to 11.4 ng/mL. The best correlation (r=0.992) was found between AutoDELFIA and Liasion, the lowest (r=0.864) was between an in-house RIA and iSYS. The between-assay CV (mean ± SD) of values within the cut-off range was 24.3% ± 7.4%, resulting in an assay-dependent diagnosis of growth hormone deficiency (GHD) in more than 27% of patients. Yet, adjustment of this data by linear regression or a conversion factor reduced the CV below 14%, and the ratio of assay-dependent diagnoses below 8%. Using quantile transformation, the CV and ratio were reduced to 11.4% and <1%, respectively. hGH measurements using different assays vary significantly. Linear regression, conversion factors, or particularly quantile transformation are useful tools to improve comparability in the diagnostic procedure for the confirmation of GHD in childhood and adolescence.

Simultaneous determination of acyclovir, ganciclovir, and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine in human plasma using high‐performance liquid chromatography

Acyclovir, ganciclovir and (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine are active in vitro against the Epstein-Barr virus (EBV) but their in vivo anti-EBV activity is not well understood. We developed a novel, sensitive high-performance liquid chromatography assay with ultraviolet detection for measuring acyclovir, ganciclovir and (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine in human plasma to identify quantitative relationships between in vitro anti-EBV activity and therapeutic response. Characteristics of the assay include a low plasma volume (200 microL), perchloric acid protein precipitation, use of penciclovir as the internal standard, run times less than 8 min and a 50 ng/mL lower limit of quantification. The within- and between-assay variability is 0.7-4.8 and 1.0-7.9%, respectively. Accuracy for all three drugs ranges from 89.5 to 106.4% for four quality controls (50, 100, 1000 and 10,000 ng/mL). This assay supports pharmacokinetic and pharmacodynamic studies of candidate anti-EBV drugs in children and adults with EBV infections.

Interlaboratory comparison study of serum total testoserone measurements performed by mass spectrometry methods

Background Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays. Methods Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)–tandem mass spectrometry (HPLC–MS/MS, GC–MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis. Results The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid–liquid extraction for sample preparation or GC–MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range. Conclusions The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.

Monoclonal antibody based electrochemical immunosensor for the determination of ochratoxin A in wheat

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I 50 and detection limits were 0.05–2.5 and 0.1–7.5 μg L −1, 0.35 (±0.04) μg L −1 and 0.9 (±0.1) μg L −1, 60 and 100 μg L −1 in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I 50 in real samples was 0.2 μg L −1 corresponding to 1.6 μg/kg in the wheat sample with a detection limit of 0.4 μg/kg (calculated as blank signal −3σ). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation ( r = 0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley.

SimpliRED D-dimer Assay: Comparability of Capillary and Citrated Venous Whole Blood, Between-assay Variability, and Performance of the Test for Exclusion of Deep Vein Thrombosis in Symptomatic Outpatients

SimpliRED D-dimer Assay: Comparability of Capillary and Citrated Venous Whole Blood, Between-assay Variability, and Performance of the Test for Exclusion of Deep Vein Thrombosis in Symptomatic Outpatients -

Determination of a novel indolylpiperazine anti-migraine agent in rat, monkey, mouse and rabbit plasma by high-performance liquid chromatography with electrochemical detection

A specific, accurate, precise and reproducible assay for the quantitation of a novel indolylpiperazine anti-migraine agent (I) in plasma from various animal species is described. The method involves addition of internal standard (I.S.) and 1.0 M sodium carbonate to the plasma sample, vortex-mixing and extraction with ethylene dichloride. The organic layer is then back-extracted in a buffer consisting of 0.1 M tetramethylammonium hydroxide (TMAH), pH 3.0 and 0.1 M (NH 4) 2HPO 4, pH 3.0, in water. The aqueous layer is injected on to a Zorbax cyano analytical column with a mobile phase consisting of acetonitrile, methanol and water (15:5:80, v/v/v) with 0.01 M TMAH, pH 3.0 and 0.01 M (NH 4) 2HPO 4, pH 3.0. The eluate is monitored by electrochemical detection at 0.9 V (guard cell), 0.5 V (detector 1) and 0.8 V (detector 2). The retention times of I and I.S. were 7 and 10 min, respectively. In drug-free control plasma, there were no interfering peaks seen at the retention times of I or I.S. The standard curve was linear over the concentration range of 5–500 ng/ml in rat, monkey, mouse and rabbit plasma. The lower limit of quantitation in all four matrices was 5.0 ng/ml. Within- and between-assay variability of quality control samples was less than 9% relative standard deviation and the predicted concentration of the quality control samples deviated by less than 15% from the nominal concentration. The stability of I was established for up to 36 h in the autosampler tray, up to 10 months in plasma at −20°C and up to 2 h in plasma at room temperature. The assay is validated for determination of I in plasma.

Evaluation of an Enzyme-Immunometric Assay for Serum α-Glutathione S-Transferase

A commercially available enzyme-immunometric assay for serum alpha-glutathione S-transferase (GST) was evaluated. Endogenous serum alpha-GST diluted linearly within the calibration range. However, we recommend that the sample and second antibody reagent are always added sequentially in the assay to avoid hook effect. Between-assay variability was below 7% across the calibration range and the upper limit of the reference range in adults (n = 219) was 11.4 micrograms/L. Within-individual variability in serum alpha-GST concentrations measured over a 4-6 week period in 4 healthy adults was small. Serum alpha-GST concentrations did not change significantly 6 h after a therapeutic dose of paracetamol. Studies in two patients after liver transplantation showed that serum alpha-GST is a better discriminant of acute changes in liver function than conventional tests. Serum alpha-GST concentrations were unaffected by gross muscle damage, extrahepatic inflammation, or haemolysis and thus appear to be more liver specific than transaminase activities. The effect of renal impairment on serum alpha-GST concentrations requires further investigation.