Inter-assay Error Research Articles

Long-term stability of sufentanil quantified by UPLC-MS-MS in human plasma frozen for 11 years at -20°C.

The long-term stability of drug concentrations in human plasma samples when stored under normal laboratory conditions over several years is important for research purposes and clinical re-evaluation, but also for forensic toxicology. Fifty human plasma samples from a former clinical trial were re-analyzed after storage at -20°C for 11 years. Plasma samples were extracted using solid-phase extraction. Isotope labelled sufentanil-D5 was used as internal standard. Sufentanil plasma concentrations were determined by ultra-performance liquid chromatography (UPLC) with gradient elution, followed by tandem mass spectrometry with electrospray ionization. The linear dynamic range (LDR) was 25 - 2500 pg/mL, the limit of detection was 10 pg/mL, and the lower limit of quantification was 25 pg/mL. Intra- and inter-assay error did not exceed 6%. The deviation of the measured sufentanil plasma concentrations between the reanalysis and the first analysis was -63 ± 14% (mean ± SD). Therefore, sufentanil concentrations in human plasma were not stable in samples frozen at -20°C over 11 years.

Long-Term Stability of Hydromorphone in Human Plasma Frozen at -20°C for Three Years Quantified by LC-MS/MS.

The long-term stability of drugs under normal laboratory storage conditions (−20°C) for years is important for research purposes, clinical re-evaluation, and also for forensic toxicology. To evaluate the stability of the analgesic opioid hydromorphone, 44 human frozen plasma samples of a former clinical trial were reanalyzed after at least three years. Blood samples were disposed using solid-phase extraction with an additional substitution of stable isotope labelled hydromorphone as an internal standard. Hydromorphone concentrations were determined by ultra-performance liquid chromatography (UPLC) with gradient elution, followed by tandem mass spectrometry with electrospray ionization. Calibration curves demonstrated linearity of the assay in the concentration range of 0.3–20 ng/mL hydromorphone. The limit of detection of the hydromorphone plasma concentration was 0.001 ng/mL, and the lower limit of quantification was 0.3 ng/mL. Intra- and interassay errors did not exceed 16%. The percentage deviation of the measured hydromorphone plasma concentrations between the reanalysis and the first analysis was −1.07% ± 14.8% (mean ± SD). These results demonstrate that hydromorphone concentration in human plasma was stable when the samples were frozen at −20°C over three years. This finding is of value for re-evaluations or delayed analyses for research purposes and in pharmacokinetic studies, such as in forensic medicine.

Highly sensitive detection of rabbit IgG by electron spin resonance using CuS nanoparticles as probe

Abstract Water soluble copper sulfide (CuS) nanoparticles were synthesized using l -cysteine as the ligand. Multiple biotins were conjugated to the antibody of rabbit IgG, and the streptavidin was attached to the CuS nanoparticles. The Cu2+ ions enclosed in the nanoparticles were used as the electron spin resonance (ESR) probes and detected with ESR spectrometer. The immunoassay reaction was resulted in the formation of the coating antibody attached to the microplate well, the detecting antibody labeled with the biotins, and the streptavidin attached to the CuS nanoparticles. After the immunoassay reaction was performed, large amount of Cu2+/Cu+ ions inside the nanoparticles were released with the help of diethyldithiocarbamate (DDC) and the Cu2+-DDC complex formed. The Cu2+-DDC complex was extracted into n-butanol, which was used as the analytical sample. Both ESR and UV–vis signals were collected for the analytical sample. The double logarithm standard curve was well simulated with a linear regression equation. The limits of detection of the rabbit IgG were 1.76 pg/mL and 2.36 pg/mL by ESR and UV–vis method, respectively. The detection range using ESR as the detector was from 8.8 pg/mL to 500 ng/mL, covering almost 5 magnitude orders of the rabbit IgG concentrations. The rabbit serum was analyzed and the rabbit IgG concentration was found to be 7.76 mg/mL. The reproducibility of the present method was good enough with the intra-assay error within 3.4 % and the inter-assay error within 11.2 %. The spiked serum samples were analyzed and the experimental results indicated that the recoveries were from 108.2 to 113.7%.

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

Detection of survival motor neuron protein in buccal cells through electrochemiluminescence-based assay.

Spinal muscular atrophy (SMA) is a severe autosomal recessive disorder affecting one in every 10,000 live births. The disease is characterized by loss of alpha-motor neurons in the spinal cord that leads to progressive atrophy and weakness of limb and trunk muscles. This neuromuscular disorder results from deletions and/or mutations within the survival motor neuron 1 (SMN1) gene, leading to a pathologically decreased expression of functional full-length SMN protein. Here we report on the investigation to measure SMN protein levels through electrochemiluminescence immunoassay (ECLIA). This simple assay is a highly quantitative method able to measure SMN protein levels in human, mouse, and rat samples throughout a wide working range with low intra- and interassay error. The sensitivity for human SMN is 30 pg/mL and provides a new tool for the set up of high-throughput screening for basic research. Moreover, we describe a novel tool for a noninvasive assessment of SMN in buccal cells derived from healthy donors, SMA carriers, and SMA patients. The availability of a validated quantitative ECLIA should improve the investigation of novel compounds for the treatment of SMA.

A high throughput electrochemiluminescence assay for the quantification of frataxin protein levels

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease affecting 1 in 50,000 people and is caused by a GAA-trinucleotide expansion in the frataxin gene located on chromosome locus 9q13 which results in a markedly reduced expression of frataxin, a small mitochondrial protein. The exact function of frataxin is still unknown and currently there is no approved treatment available. In the near future there will be a high demand for measuring frataxin protein levels due to the development of therapeutic strategies for FRDA based on manipulating frataxin expression levels in vivo. In this paper we describe the development of an electrochemiluminescence assay (ECLIA) to measure frataxin protein levels in a 96-well plate format. The ECLIA for frataxin is able to measure human and mouse samples and is highly quantitative, accurate and reproducible, with low intra- and inter-assay error throughout a wide working range. The assay has an excellent precision and provides a new tool for the set up of high-throughput screening for basic research and for clinical studies with FRDA patients.

An alternative method to assess 24-h ruminal in vitro neutral detergent fiber digestibility

Interassay error caused by the inconsistent nature of rumen fluid inoculum confounds comparisons of forage in vitro neutral detergent fiber (NDF) digestibility (NDFD) analyzed in different repetitions or laboratories. Our objective was to determine if priming rumen fluid and allowing it to produce a standard amount of gas before inoculating samples improved assay repeatability. In 2 experiments, we compared interassay error of NDFD estimates between several in vitro assays. In both experiments, dried, ground (1mm) alfalfa samples (0.5g) sealed in bags were placed in 125-mL Erlenmeyer flasks and incubated with in vitro media and 10mL of rumen fluid. In experiment A, rumen fluid was collected from a cannulated cow fed a high forage diet and prepared one of 2 ways; rumen fluid was either used immediately after it was collected and strained through cheese cloth (GVA), or strained fluid was combined with buffer, reducing solution, and 12.5mg of cellulose/mL of rumen fluid and allowed to produce a consistent amount of gas before inoculation (RPA). The assay was repeated 5 times, with 13 samples per method. In experiment B, inoculum was prepared one of 3 ways; RPA, GVA except rumen fluid was collected and pooled from 2 cows (GVB), or RPA with fluid pooled from 2 cows. The in vitro assays were repeated 5 times with 8 samples per method. Neutral detergent fiber was analyzed using a forage fiber analyzer and 24-h NDFD was determined as: NDFD (% of NDF)=100×[(NDF0h−NDFresidue)/(NDF0h)]. Data for each experiment were analyzed using a mixed model procedure and repetition sum of squares for each technique was determined and compared with an F-test to assess technique interassay error. In both experiments, rumen fluid priming significantly reduced repetition sums of squares, 51.2 versus 503 and 23.3 versus 164, compared with the respective GVA or GVB. However, priming significantly decreased NDFD values, 22.5 versus 24.8 and 23.9 versus 26.6%, compared with GVA and GVB, respectively. Priming rumen fluid with cellulose improved in vitro NDFD assay precision, but depressed in vitro NDFD.

Modification of a rumen fluid priming technique for measuring in vitro neutral detergent fiber digestibility

Recently, we developed an alternate method to measure in vitro neutral detergent fiber (NDF) digestibility (ivNDFD) based on a primed rumen fluid inoculum. Pretreating rumen fluid inoculum with cellulose and holding the inoculum until it generated 0.3mL of gas/mL of rumen fluid before inoculating forage samples improved ivNDFD assay repeatability but depressed ivNDFD means. Our objective in this study was to determine if pretreating rumen fluid with a mixture of carbohydrates and urea would affect the ivNDFD mean and variance. We also used the modified procedure as a reference assay to calibrate near-infrared reflectance spectroscopy (NIRS) to predict 24-, 30-, and 48-h ivNDFD. Two experiments were completed. In experiment A, 3 ivNDFD assays modified from the method of Goering and Van Soest were evaluated over 24, 28, 48, 54, and 72h by using dried, ground alfalfa (1mm) or wheat straw (0.5g) sealed in Ankom F57 forage fiber bags. Bags were placed individually in 125-mL Erlenmeyer flasks and incubated with Goering and Van Soest media and 10mL of rumen fluid. Rumen fluid was collected before feeding from 2 cannulated cows fed a high-forage diet and was prepared in 1 of 3 ways: 1) pooled rumen fluid was strained and used immediately to inoculate flasks (modified Goering and Van Soest method); 2) strained, pooled fluid was combined with buffer, reducing solution, and 1.25mg of primer/mL of rumen fluid and allowed to produce 0.12mL of gas/mL of rumen fluid before sample inoculation [Combs-Goeser (CG) method]; or 3) the CG method was used without the primer mixture (unprimed method). The assay was repeated 5 times, with 5 time points (24, 28, 48, 54, and 72h) and 2 subsamples per time point for each method. Neutral detergent fiber was analyzed using an Ankom200 forage fiber analyzer and ivNDFD was determined as follows: ivNDFD (% of NDF) = 100×[(NDF0h–NDFresidue)/(NDF0h)]. Results were analyzed using a mixed model procedure, and data were blocked by method to obtain repetition sums of squares, which were compared by an F-test to assess interassay error. Repetition sums of squares were reduced with the CG method compared with the Goering and Van Soest method (19 vs. 228), and mean ivNDFD estimates were similar at 28, 48, and 54h. In experiment B, 24-, 30-, and 48-h ivNDFD data for 54 feeds were determined in triplicate using the CG method, and corresponding samples were then scanned with an NIRS instrument. Calibrations were computed using partial least squares regression techniques. The NIRS calibration equation R2 values were 0.93, 0.93, and 0.89 for 24-, 30-, and 48-h ivNDFD. Results suggest that the modified ivNDFD method using rumen fluid primed with a mixture of carbohydrate and urea (CG method) reduced interassay error.

Lateral-flow immunoassay for the frataxin protein in Friedreich’s ataxia patients and carriers

Friedreich’s Ataxia (FA) is an inherited neurodegenerative disease caused by reduction in levels of the mitochondrial protein frataxin. Currently there are no simple, reliable methods to accurately measure the concentrations of frataxin protein. We designed a lateral-flow immunoassay that quantifies frataxin protein levels in a variety of sample materials. Using recombinant frataxin we evaluated the accuracy and reproducibility of the assay. The assay measured recombinant human frataxin concentrations between 40 and 4000 pg/test or approximately 0.1–10 nM of sample. The intra and inter-assay error was <10% throughout the working range. To evaluate clinical utility of the assay we used genetically defined lymphoblastoid cells derived from FA patients, FA carriers and controls. Mean frataxin concentrations in FA patients and carriers were significantly different from controls and from one another ( p = 0.0001, p = 0.003, p = 0.005, respectively) with levels, on average, 29% (patients) and 64% (carriers) of the control group. As predicted, we observed an inverse relationship between GAA repeat number and frataxin protein concentrations within the FA patient cohort. The lateral flow immunoassay provides a simple, accurate and reproducible method to quantify frataxin protein in whole cell and tissue extracts, including primary samples obtained by non-invasive means, such as cheek swabs and whole blood. The assay is a novel tool for FA research that may facilitate improved diagnostic and prognostic evaluation of FA patients and could also be used to evaluate efficacy of therapies designed to cure FA by increasing frataxin protein levels.

Alternative immunoassay for tartrate-resistant acid phosphatase isoform 5b using the fluorogenic substrate naphthol ASBI-phosphate and heparin

Objective: Our purpose was to develop a specific immunoassay for tartrate-resistant acid phosphatase (TRACP) 5b using naphthol ASBI phosphate (N-ASBI-P) as a selective substrate for isoform 5b and heparin as a selective inhibitor of isoform 5a. Methods: Serum TRACP 5a and 5b and recombinant TRACP 5a were used to optimize and standardize the immunoassay for specificity, linearity, analytical recovery, sensitivity and reproducibility. Serum N-telopeptide cross-links (NTX) and bone alkaline phosphatase (bone ALP) were also measured. The clinical sensitivity and specificity were assessed in healthy control subjects and patients with osteoarthritis (OA), rheumatoid arthritis (RA) and endstage renal disease (ESRD). Results: TRACP 5b specificity was achieved at pH 6.3 with 2.5 mmol/l substrate and 25 U/ml heparin. Isoform 5b specificity was increased over our original immunoassay using 4-nitrophenyl phosphate (4-NPP) without heparin. The alternative immunoassay was linear with 110% analytical recovery and no serum matrix effects. The average intra-assay error was 10.65%; the average inter-assay error was 10.11% for values of 1–3 U/l and 6.5% for values of 7–11 U/l. Mean serum TRACP 5b in OA and RA were not significantly different from control using either immunoassay. Mean serum TRACP 5b was significantly increased in ESRD with both immunoassays. Serum TRACP 5b levels correlated significantly with NTX and bone ALP in the disease groups, but not always in the control group. Conclusion: This alternative immunoassay for TRACP 5b activity is highly specific. It should have applications in evaluating patients with bone disease and will improve our understanding of the biological significance of TRACP 5b expression in health and disease.

Development and validation of a gas chromatography–mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography–mass spectrometry (GC–MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam ( N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid–liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N, O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/μl plasma for BUP, 0.125 and 12.5 ng/μl for NBUP and N-DMFNZ, 0.125 and 5 ng/μl for FNZ, and between 0.025 and 50 ng/μl for 7-AFNZ. The limit of quantification was 0.025 ng/μl plasma for 7-AFNZ and 0.125 ng/μl for the four other compounds. A good reproducibility (intra-assay CV=0.32–11.69%; inter-assay CV=0.63–9.55%) and accuracy (intra-assay error=2.58–12.73%; inter-assay error=0.83–11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC–MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.

Development and validation of a gas chromatography?mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography–mass spectrometry (GC–MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid–liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/μl plasma for BUP, 0.125 and 12.5 ng/μl for NBUP and N-DMFNZ, 0.125 and 5 ng/μl for FNZ, and between 0.025 and 50 ng/μl for 7-AFNZ. The limit of quantification was 0.025 ng/μl plasma for 7-AFNZ and 0.125 ng/μl for the four other compounds. A good reproducibility (intra-assay CV=0.32–11.69%; inter-assay CV=0.63–9.55%) and accuracy (intra-assay error=2.58–12.73%; inter-assay error=0.83–11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC–MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.

A new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity in children.

The evaluation of estrogenic status is necessary for many physiological and pathological conditions in pediatric as well as adult endocrinology. Because current immunoassays exclusively measure E2--and with a sensitivity that is insufficient for prepubertal children--we developed a new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity. This assay is based on human uterine cervix carcinoma cells, HeLa cells, that do not naturally express E2 receptor. These cells were transfected with plasmids encoding the human ERalpha or beta, along with an estrogen-responsive promoter fused to the luciferase gene, and called HELNalpha and HELNbeta for HeLa estrogen-responsive element luciferase neomycin alpha and beta. HELNalpha and HELNbeta are able to respond to estrogens and various compounds having estrogenic activity but, because of the importance of ERalpha in the reproductive function, we chose to work with the HELNalpha cell line. The luciferase activity we obtained was compared with an E2 standard curve specific for each serum sample and established with stripped serum. The estrogenic bioactivity was expressed in picograms of E2 equivalents, and the detection limit was < 1 pg x ml(-1) E2 equivalents. The intra and interassay error was lower than 10% and 20%, respectively. We measured estrogenic bioactivity in 18 normal prepubertal boys (age = 9.7 +/- 2.4 yr), 18 normal prepubertal girls (age = 9.2 +/- 1.7 yr) and 18 normal pubertal girls (age = 13.6 +/- 1.8 yr). The estrogenic bioactivity in the prepubertal girls was significantly higher than in the boys, i.e. 3.53 +/- 2.23 pg x ml(-1) vs. 1.44 +/- 0.87 pg x ml(-1) (P < 0.01). A significant difference was found between the pre- and pubertal girls, i.e. 3.53 +/- 2.23 pg x ml(-1) vs. 26.77 +/- 18.32 pg x ml(-1) (P < 0.01). This ultrasensitive bioassay measures total estrogenic bioactivity of serum with very high sensitivity. It has numerous potential applications in pediatric and adult endocrinology. In addition, this assay may help to evaluate excess estrogenic activity related to aromatase overexpression or contamination by environmental chemicals.

Rapid Detection of D-dimer Using a Fiber Optic Biosensor

We describe a rapid and sensitive method for detection and quantification of D-dimer and other crosslinked fibrin degradation products (XL-FDPs), which are present in elevated concentrations in patients with sepsis and thrombotic disorders. The method utilizes a sandwich fluoroimmunoassay immobilized in the sensing region of an evanescent wave biosensor. Physiological concentrations of D-dimer and high molecular weight XL-FDP could be determined in buffer and plasma samples on calibrated fibers in 11 min. Samples from septic patients were assayed using ELISA and the fiber optic method; concentrations determined by fiber optic assay were strongly correlated with those determined by ELISA (r = 0.918); intra- and inter-assay errors were comparable to those from ELISAs. Given its accuracy and rapid response time, this fiber optic biosensor shows great potential for development as a diagnostic tool.

High Performance Liquid Chromatography of Dextromethorphan And Its Main Metabolite in Human Urine

Dextromethorphan (DM) is 0-demethylated to its main metabolite, dextrorphan (DO), following a polymorphic reaction that depends on the isoenzyme, cytochrome P-il502Di5 [C'i'P2D6). Traditionally, phenotyping of this isoenzyme utilises debrisoquine, individuals with rapid or normal metabolism of debrisoquine are described as 'extensive metabolisers" and those with slower drug metabolism are known as "poor metabolisers". Drug oxidation phenotyping with DM has been found to be in complete concordance with results of phenotyping with debrisoquine. A sensitive High Performance Liquid Chromatography method with both fluorescent and UV detection was developed for the analysis of [JM and [JD in human urine. Prior to analysis by HPLC, the urine samples were subjected to enzymatic hydrolysis of the dextrorphan glucuronides followed by extraction into chlarofarmtisopropanolmixture. Standard curves for both compounds were linear over concentrations ranging from 0 to 500 ng/ml with a mean linear regression value of 0.00 for DM and 0.97 for DO. The intra-assay error was 4.07% and 7.05% for DM and DD respectively and the interassay error was 10.16% and 7.13% for DM and DO respectively. Following ingestion of 15 mg dextromethorphan hydrobromide, a preliminary oxidation phenotyping was performed on 8 volunteers. The metabolite ratio was calculated as MR = 0-3 hr urinary output of unchanged DM/ 0-3 hr urinary output of DO. The total urinary output of DM ranged from 0 to 4.8'' mg whereas that of DE) ranged from 0.57 to 21.00 mg. The MR values were between 0 to 0.220 in 7 of the subjects tested while 1 subject showed a relatively high MR value of 1.376. This was consistent with the finding that in this same subject the urinary output of DM was the highest, and the output of D0 was the lowest among those tested, thus indicating that this subject is possibly a poor metabiliser of DM. It is concluded that the assay is sensitive for the measurements of levels of DM and D0 in human urine and hence will be utilised in a large scale study of the frequency distribution of drug oxidation phenotyping with DM in a Malaysian population.

Functional evaluation of erythrocytic antibodies by photometric detection of erythrophagocytosis in the monocyte monolayer assay

In haemolytic disease of newborn (erythroblastosis fetalis) the in vivo behaviour of erythrocytic IgG antibodies is of particular significance. The monocyte monolayer assay (MMA), which determines by microscopy the number of erythrocytes phagocytosed by monocytes, is an important functional test for the qualitative assessment of erythrocytic IgG antibodies. We set up a photometric MMA and compared it with the microscopic MMA. In both tests we employed commercially available rhesus antibody sera and examined 8 sera of pregnant women with mild and severe courses of haemolytic disease of newborn by the photometric MMA. Good agreement was found between the microscopic and photometric MMA (r = 0.93). Over and above this the photometric MMA correlated with the course of haemolytic disease of newborn in 7 out of 8 cases (with one false-positive finding). The photometric technique permits rapid, sensitive and reproducible determination of erythrophagocytosis in microtitre plates. This method is based on the photometric detection of haemoglobin of phagocytosed erythrocytes via a peroxidase reaction. Standardised photometric MMA could in future be more widely applied especially in haemolytic disease of newborn for the functional characterisation of erythrocytic antibodies, the incidence of intra-assay and inter-assay errors being low.

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in plasma and urine by use of a chiral cellulose-derivative column

This assay allows simultaneous determination of the enantiomers of both disopyramide and its active metabolite, mono-N-dealkyldisopyramide, in 1 mL of plasma or 0.1 mL of urine within approximately 35 min by HPLC with a chiral cellulose-derivative column and ultraviolet detection. Recoveries for the analytes and the internal standard (racemic verapamil) with an extraction from alkalinized plasma or urine into diethyl ether were greater than 90%. Intra- and interassay CVs for disopyramide enantiomers were less than 5.5% at 2.5 mg/L in plasma and less than 6.5% at 25 mg/L in urine; for mono-N-dealkyldisopyramide enantiomers they were less than 6.3% and less than 8.9%, respectively. Intra- and interassay relative errors for determining these analytes in plasma and urine at 2.5 and 25 mg/L, respectively, ranged from -5.9% to +2.5%. The calibration curves for the respective analytes were linear (r = 0.995 or greater, P less than 0.01) from 0.025 to 5.0 mg/L in plasma and from 0.5 to 10 mg/L in urine. The lower detection limits (signal-to-noise ratio of 3) for S(+)-disopyramide and the other analytes were 0.010 and 0.025 mg/L, respectively. We evaluated clinical applicability of this method by determining steady-state plasma concentrations and urinary excretions of the respective analytes in a pediatric patient being treated with racemic disopyramide.

Age and hormonal dependence of tonin levels in rat submandibular gland as determined by a new direct radioimmunoassay.

A simple and sensitive direct radioimmunoassay for tonin (EC.3.4.99.-) has been developed. This assay incorporates a modified and convenient poly(ethylene glycol) technique for separation of free from bound tonin. A rabbit antiserum in a final dilution of 1:160,000 was used and the purified tonin was labelled with 125I by using a lactoperoxidase method. It detects 20 pg of immunoreactive tonin per tube. Serial dilutions of rat submandibular gland extracts showed complete parallelism with tonin standard curves. No cross-reactivity with rat tissue kallikrein was seen. Intra- and inter-assay errors were 3.2 and 5.6%, respectively. Using this assay, immunoreactive tonin was detected in the rat submandibular gland as early as 3 weeks after birth (body wt. approximately 50-60 g). Tonin levels are shown to be dependent on age and sex with significantly higher levels in male than in female rats. Castration results in decrease of tonin levels and 17 alpha-methyltestosterone replacement reversed the level to higher than the sham-operated control rats. Cortisol treatment increased, but thyroxine or oestradiol had no effect, on tonin levels in the submandibular gland of castrated rats. This newly developed radioimmunoassay can now be used to measure low levels of tonin in various tissues and body fluids to address questions about its regulation and functional significance.

Statistical evaluation of a radioimmunoassay for alpha-melanotropin

Data from a radioimmunoassay for α-melanotropin was analyzed by means of a weighted nonlinear regression procedure that fitted a log-logistic model. The assay was performed with several doses of both standard and unknown samples. This allowed separate curves to be fitted simultaneously to standard and unknown data, and tests for linearity and parallelism to be performed. Errors of potency estimates predicted from within-assay statistics were compared with observed errors, calculated from replicate potency estimates. Significant interassay errors and errors associated with a preliminary extraction of samples were detected.