Double blind study of vitamin E compared to placebo in the prevention of anemia in the low birth weight infant during the 7th week of life: therapeutic study

Sephadex G-25 (Kernlute test) yields satisfactory results for the identification of infants at risk for BE and for estimation of the bilirubin binding capacity of the serum. In the presence of more than 2 mg/100 ml of conjugated bilirubin, the interpretation of the Sephadex G-25 test requires caution. Gel filtration through Sephadex LH-20 and chloroform elution of the free unconjugated bilirubin not bound to albumin seems to be a useful tool for eliminating interference by conjugated bilirubin, which is not chloroform-extractable.     

Microdetermination of unbound bilirubin in icteric newborn sera: an enzymatic method employing peroxidase and glucose oxidase

An enzymatic assay method for the microdetermination of unbound bilirubin in newborn icteric sera is described. Unbound bilirubin is oxidized to colorless compounds by peroxidase in the presence of hydrogen peroxide derived from glucose by the mediation of glucose oxidase. In this method, the bilirubin is not significantly degraded before the addition of peroxidase, in contrast to the method using hydrogen peroxide. The oxidation rate is determined by spectrophotometry and chloroform extraction is eliminated. The unbound bilirubin concentration can be determined from the initial oxidation velocity of total bilirubin. The Michaelis constant, KM, was approximately 20 micrometer. The coefficient of variation for icteric serum determination was 4.4--6.5%. The concentration of unbound bilirubin was reduced after five days of storage at -20 degrees C. The bilirubin-albumin affinity was studied with purified albumin and adult serum. The dissociation constants were 2 x 10(-8) M and 5 x 10(-9) M, respectively, at bilirubin/albuminor molar ratios below 1.0. Clinically, serum samples from 75 icteric newborn infants were analysed, and the sera of premature infants were found to have remarkably high levels of unbound bilirubin compared to those of fullterm infants. The sera of a Rhesus immunization infant and an ABO incompatibility infant were remarkably higher than that of the nonhemolytic icteric sera. The unbound bilirubin concentration was also affected, in an in vitro study, by the addition of hemolysate.     

Effect of nonenzymatic glycosylation on the titers of circulating autoantibodies in pemphigus and pemphigoid

Hyperglycemia is observed in some patients with autoimmune bullous diseases complicated by diabetes mellitus or treated with systemic corticosteroids. High concentrations of glucose can react with various proteins and change their structural and functional properties. We previously reported that nonenzymatic glycosylation of antibody can impair antigen-antibody binding. We ascertained whether glycosylation of autoantibody decreases the autoantibody titer by examining 30 sera from patients with pemphigus and pemphigoid. Nonenzymatic glycosylation in the physiological range was induced by incubation of sera with 1650 mM D-glucose at 4 degrees C for 7 days. The titers of sera were determined by indirect immunofluorescence (IIF). In all cases, the immunofluorescence intensity of glycosylated sera was weaker than that of nonglycosylated sera. Glycosylated sera showed a lower antibody titer by 1 doubling dilution in 18 out of 30 cases, compared with nonglycosylated sera. The ten BP patients' sera were also analyzed by immunoblotting for reactivity with the BP180-GST fusion proteins, S delta 1 and 4575. All BP sera reacted with S delta 1, and 5 out of 10 BP sera reacted with both S delta 1 and 4575. In all the sera that reacted only with S delta 1, the glycosylated sera showed a 1 doubling dilution decrease in autoantibody titer. Interestingly, in 4 out of 5 sera that reacted with both S delta 1 and 4575, there were no differences in the antibody titer between glycosylated and nonglycosylated sera. These results indicate the possibility of a false decrease in autoantibody titers of sera from patients with autoimmune bullous diseases complicated with hyperglycemia. Although the false decrease in titers of autoantibodies induced by nonenzymatic glycosylation is not dramatic, it must be considered in order not to underestimate the disease activity of pemphigus in such cases.     

Icteric plasma suggests Gilbert's syndrome in the blood donor

BACKGROUND: In a recent quality assurance audit of component returns over a 6-month period, 9 of 81 returns were due to icteric plasma. With the sensitive, new methods used to screen donors for anemia and hepatitis, it seemed likely that the icteric discoloration reflected benign unconjugated hyperbilirubinemia (Gilbert's syndrome) in the donor, rather than liver disease or hemolysis. The donors were recalled for repeat blood study to resolve this question. STUDY DESIGN AND METHODS: Seven of the nine donors could be reached, and they submitted blood samples for measurement of serum levels of conjugated (direct-reacting) and total bilirubin and for complete blood and reticulocyte counts. RESULTS: All seven donors had mild unconjugated hyperbilirubinemia, with total bilirubin levels ranging from 1.3 to 2.8 mg per dL. None showed evidence of overt hemolysis. CONCLUSION: All seven donors of the components with icteric plasma have Gilbert's syndrome, a benign genetic anomaly occurring in approximately 3 to 5 percent of the general population. With the sensitive screening tests for viral hepatitis used today, the presence of icteric plasma in a component suggests that the donor has Gilbert's syndrome. Policies about the acceptability of icteric components from blood donors merit reassessment.     

Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type b polysaccharide by enzyme-linked immunosorbent assay

An interlaboratory study was conducted to determine whether an enzyme-linked immunosorbent assay (ELISA) with an antigen preparation composed of various-sized fragments of Haemophilus influenzae type b polysaccharide conjugated to human serum albumin could be standardized across laboratories and whether the ELISA-derived results from different laboratories are equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-H, influenzae type b polysaccharide antibodies. Twenty coded human serum samples were quantitated by ELISA in 11 laboratories and by RABA in 5 laboratories. The mean RABA-derived values served as the basis for all comparisons. While the overall correspondence of antibody values between the two methods was good, significant differences were found among some of the 11 ELISA data sets and among the mean RABA values. Seven laboratories generated higher ELISA antibody values for low-titered sera. Four laboratories generated antibody concentrations that were not statistically different between the two assay methods. The results therefore indicate that the ELISA can tolerate substantial variations in protocol, such as the use of different plates and different antibody reagents, without affecting the quantitation of serum antibodies. However, attention should be focused on low-titered sera, as some assay conditions may yield spurious results. This ELISA is a serologic assay which can serve as an alternative to the RABA for quantitation of antibodies to H. influenzae type h polysaccharide.     

Oral calcium phosphate: a new therapy for Crigler-Najjar disease?

BACKGROUND & AIMS: Calcium phosphate binds unconjugated bilirubin in vitro, and dietary calcium phosphate supplementation reduces the serum bilirubin level in rats with hereditary unconjugated hyperbilirubinemia (Gunn rats). The aim of this study was to evaluate the effect of oral calcium phosphate supplementation on plasma bilirubin levels in patients with Crigler-Najjar disease. METHODS: A placebo-controlled, double-blind, crossover design was used. Eleven patients, 2-42 years of age, participated. The group included 5 patients with type I disease who were all treated with phototherapy and 6 patients with type II disease who were primarily treated with phenobarbital. In addition to plasma bilirubin levels, dietary intake and urinary and fecal excretion of calcium and phosphate were evaluated. RESULTS: A modest but significant decrease in serum bilirubin was observed in patients with type I disease (18% +/- 6%, P = 0.03) but not in patients with type II disease during treatment with calcium phosphate. Urinary output of calcium and phosphate did not change during the treatment period. CONCLUSIONS: Oral calcium phosphate may be a useful adjuvant to photo-therapy in Crigler-Najjar type I disease.     

Analytical and clinical performance characteristics of Tandem-MP Ostase, a new immunoassay for serum bone alkaline phosphatase

The performance characteristics of the Tandem-MP Ostase assay, a new microplate immunoassay for bone-specific alkaline phosphatase (bone ALP; EC 3.1.3.1) in human sera, are described. Bone ALP is bound to streptavidin-coated microwells by a single biotinylated anti-bone ALP monoclonal antibody. Antigen is detected by the addition of p-nitrophenyl phosphate. The assay is performed at room temperature in <90 min. Imprecision was 2.3-6.1% with a detection limit of 0.6 microg/L. Method comparison of bone ALP measurements with the Tandem-MP Ostase assay and the mass-based Tandem-R Ostase assay (n = 285) indicated regression statistics of Tandem-MP Ostase = 1.03 Tandem-R Ostase + 0.22 microg/L, S(y/x) = 4.0 microg/L, r = 0.97. Serum bone ALP values in apparently healthy men and in pre- and postmenopausal women were also similar between the two Ostase assay formats. Liver ALP reactivity determined using the slope and heat inactivation methods was similar in both Ostase assays. Liver ALP reactivity ranged from 3 microg/L (heat inactivation) to 6 microg/L (slope method) per 100 U/L of liver ALP activity, whereas bone ALP reactivity was 37 microg/L per 100 U/L of bone ALP activity, indicating a liver ALP relative reactivity of 8.1-16.2%. Similar results were obtained with the Alkphase-B bone ALP immunoassay. The Tandem-MP Ostase bone ALP assay demonstrated increased concentrations of serum bone ALP in conditions where bone metabolism is increased and showed a rapid, temporal decrease in serum bone ALP in Paget disease patients on bisphosphonate therapy. In conclusion, the Tandem-MP Ostase assay for serum bone ALP is a rapid, simple, robust nonisotopic alternative to the Tandem-R Ostase immunoradiometric assay that provides an accurate and sensitive assessment of bone turnover.     

Results with six "kit" radioimmunoassays for primary bile acids in human serum intercompared

We examined six radioimmunoassay procedures for measuring primary bile acids in human serum (two 3H-labeled and four 125I-labeled). A significant (p < 0.01) correlation was observed between measurements in the assay both for cholic acid and chenodeoxycholic acid, at low and high concentrations of serum bile acids. All kits were acceptable with respect to accuracy, precision, stability, and analytical recovery. All six procedures gave similar results for chenodeoxycholic and cholic acid in sera of 80 healthy subjects; the agreement was also close when the two primary bile acids were compared with their sum in serum. Normal values ranged from 0.4 to 2.5 mumol/L for conjugated chenodeoxycholic acid and from 0.3 to 1.5 mumol/L for conjugated cholic acid. The 125I assays do not require liquid-scintillation equipment but 125I induces a decrease in the affinity constant of antibody. The sensitivity of the assays was still adequate for measuring bile acids in the serum of healthy fasting persons and liver-disease patients.     

Establishment and evaluation of a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen adapted to the fully automated ACCESS system

We have established a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen (CEA), designated ACCESS CEA, which is adapted to the fully automated ACCESS immunoassay analyzer. The assay is based on a one step sandwich-type method using two monoclonal antibodies, one of which is immobilized on micrometer-size paramagnetic particles and the other is conjugated to alkaline phosphatase. Ten microliters of calibrators or sera are incubated for 5 minutes at 37 degrees C with the particles and with the alkaline phosphatase conjugate. The particles are then magnetically separated and washed to remove unbound components. Time needed to obtain the first result is less than 15 minutes. The assay range was 0.04-1000 micrograms/l of CEA, and the possible high-dose hook effect was prevented at CEA concentrations up to 100000 micrograms/l in this working range. The coefficient of variation (CV) for intra-assay precision was 3.0 to 4.7%, and inter-assay CV was 3.4 to 5.6%. The sample carryover was less than 0.001%. The analytical recovery ranged from 98 to 104% and a dilution linearity was demonstrated. No interference was detected in any sample with levels up to 300 mg/l for bilirubin, 12000 mg/l for haemoglobin, 50000 mg/l for human serum albumin, 8500 mg/l for triacylglycerol, and 500000 IU/l for rheumatoid factor. The ACCESS CEA assay also showed very homogeneous reactivity with purified CEA preparations from different tumours and could discriminate CEA from four CEA-related normal antigens tested. Serum samples (n = 362) from patients with malignant or non-malignant disease, as well as from healthy individuals, were analyzed by the ACCESS CEA assay and by the established IMx CEA assay. The CEA values determined by the ACCESS CEA assay were in good agreement with those determined by the IMx CEA assay, and the ACCESS CEA assay significantly increased the sensitivity and specificity of tumour diagnosis as compared with the IMx CEA assay.     

Cholecystectomy with intraperitoneal drain. Presence and significance of conjugated bilirubin in the drainage fluid

The content of conjugated bilirubin in the drainage fluid of 85 patients, operated upon consecutively with cholecystectomy and intraperitoneal drain for nonacute gallbladder pathology was measured by the adapted method of Jendrassik and Grof. The measured amounts varied from 0 to 755 micromol. A weak correlation was found between the concentration of conjugated bilirubin and the total amount of drainage fluid (r = 0.37). In the majority of patients the evacuated amounts of conjugated bilirubin corresponded to the content of bilirubin in a few milliliters of hepatic bile. In 10% of the patients there were however greater amounts of conjugated bilirubin in the drainage fluid. Greater amounts of conjugated bilirubin were significantly more often evacuated from patients operated upon by surgeons with less than 3 years of surgical experience compared to patients operated upon by more experienced surgeons. The amount of conjugated bilirubin in the drainage fluid was not significantly correlated with operative blood loss, dryness of the operative field at the end of the operation or iatrogenic perforation of the gallbladder during operation. Higher (however not significant) temperatures and bilirubin levels in serum were observed in patients with greater amounts of conjugated bilirubin in the drainage fluid. Increased amounts of conjugated bilirubin in the drainage fluid were not significantly associated with increased postoperative morbidity. Two of the patients with large amounts of conjugated bilirubin in the drainage fluid were reoperated because of bile leakage/abscess but the remaining patients had no serious complication, which could be a result of efficiency of the intraperitoneal drain.     

Immune response to synthetic peptides of hepatitis delta antigen

Hepatitis delta antigen (HDAg) is the only viral protein known to be expressed during hepatitis delta virus (HDV) infection. Detection of antibody to HDAg (anti-HD) is the usual method for diagnosis of HDV infection since viremia lasts only a few weeks. In an effort to identify the major epitopes recognized by humans during natural infection, four oligopeptides including residues 2 to 17 (SP1), 155 to 172 (SP2), 168 to 182 (SP3), and 189 to 211 (SP4) of the HDAg molecule were synthesized and probed by enzyme-linked immunosorbent assay with a panel of 80 serum specimens from 45 patients suffering from either HDV-hepatitis B virus coinfections (n = 17) or HDV superinfections (n = 28). Sera from infected patients recognized these relatively short peptides. Peptide SP2 was the most antigenic; 71% of serum specimens reacted. Antibody to SP2 was also the commonest in sera taken early in the course of the disease. Peptide SP2 corresponds to one of the two regions which is highly conserved between different isolates. Among the 63 serum specimens which scored anti-HD positive by a commercial assay, all but 3 reacted to at least one of the peptides (95% agreement). Peptide assays appeared to be significantly more sensitive than the commercial assay with native HDAg early in the course of HDV infection since 14 of 17 (82%) serum specimens which scored anti-HD negative in the commercial assay reacted to one or more peptides. All serum specimens giving one or more positive results with the various peptides were confirmed as being HDV positive by an inhibition assay with free peptide in solution. The immune response to HDAg peptides vared greatly between individuals. No specific reactivity profile could be assigned to those with either HDV-hepatitis B virus coinfections or HDV superinfections. Overall, HDAg peptides appeared to be convenient reagents in addition to native antigen for the development of new and improved diagnostic tests for HDV infection.     

The effectiveness of psychiatric treatment for HIV-infected patients

p-hydroxymethamphetamine (OHMAP) is one of the major metabolites of the widely abused drug methamphetamine (MAP). The demethylation of OHMAP to p-hydroxyamphetamine (OHAP) has been shown in vitro but has never been reported in vivo. The disposition kinetics as well as the metabolism of OHMAP was investigated employing a sensitive HPLC method which can separate the enantiomers of OHMAP and OHAP. Both conjugated and unconjugated forms of these compounds can be quantitated. Male Sprague-Dawley rats were given an iv bolus of racemic OHMAP (20 mg kg-1) and serum and urine samples were collected at selected times. The serum concentration-time data for OHMAP enantiomers could be described by a biexponential equation. The clearance of D-OHMAP (93.5 mL min-1 kg-1) was slightly, but statistically significantly, greater than that of the L-enantiomer (83.9 mL min-1 kg-1). The steady-state volumes of distribution of L- and D-OHMAP were (mean +/- SD) 3.15 +/- 0.84 and 4.23 +/- 1.76 L kg-1, respectively. No significant concentrations or amounts of OHAP enantiomers could be detected in any serum or urine sample. Rats excreted more unchanged L-OHMAP (34%) than D-OHMAP (29%). In contrast, more conjugated D-OHMAP (57%) was recovered compared to the conjugated L-OHMAP (52%). The results suggest that there is slight stereoselectivity in the disposition of OHMAP enantiomers. The N-demethylation product (OHAP) was not produced in vivo.     

Antioxidation of human low density lipoprotein by unconjugated and conjugated bilirubins

We demonstrate here that both unconjugated bilirubin (Bu) and conjugated bilirubin (Bc) can protect human low density lipoprotein(LDL) against oxidation by oxyradicals generated by 2,2'-azo-bis (2 amidinopropane) dihydrochloride at 37 degrees. The oxidation was assessed by agarose gel electrophoresis and was further corroborated by assaying the malondialdehydes and lipid peroxides formed throughout oxidation. On a per mole basis, Bu and less so Bc was more effective than ascorbate in preventing LDL oxidation. Since oxidative modification of human LDL was implicated in plaque formation in blood vessels leading to atherogenesis, the data suggested that either bile pigment may help reduce the risk of atherogenesis.     

Contribution of glutamate decarboxylase antibodies to the reactivity of islet cell cytoplasmic antibodies

The contribution of glutamate decarboxylase (Mr 65000) antibodies to the reactivity of islet cell cytoplasmic antibodies with the 'whole' islet staining pattern from patients with newly diagnosed Type I diabetes was investigated. Diluted sera (n = 10) were preincubated with increasing concentrations of purified recombinant human islet glutamate decarboxylase (Mr 65000) and the change in islet cell cytoplasmic antibody binding was evaluated by quantitative immunocytochemistry. Binding to islet cells was partially blocked by glutamate decarboxylase in 9/10 diluted sera; the maximum blocking obtained at high concentrations of glutamate decarboxylase (5 micrograms/ml) was 36% (median, range 24-61%). In contrast, binding to islet cells in three diluted sera (two polyendocrine patients without Type I diabetes and one patient with newly diagnosed Type I diabetes) with the 'selective' islet staining pattern was totally blocked by glutamate decarboxylase. The concentration of glutamate decarboxylase required to achieve maximum blocking was less for the 'whole' islet (0.4-8.0 micrograms/microliters undiluted serum) compared to the 'selective' islet (20-645 micrograms/microliters undiluted serum) positive sera. All sera were positive for glutamate decarboxylase antibodies in an immunoprecipitation assay using 35S-methionine labelled extract of baby hamster kidney cells transfected with glutamate decarboxylase. However, the binding activity of these antibodies was less in the sera positive for the 'whole' islet compared to the 'selective' islet staining pattern. In conclusion, glutamate decarboxylase antibodies contribute partially to the reactivity of islet cell cytoplasmic antibodies of the 'whole' islet staining pattern in the sera of newly diagnosed patients with Type I diabetes, and totally to reactivity of the 'selective' islet staining pattern. The antigens recognized by the other antibodies contributing to the 'whole' islet reactivity remain to be defined.     

The influence of various aminoglycoside preparations on bilirubin/albumin binding

The effect of antibiotics of aminoglycoside structure on the albumin binding of bilirubin has been tested in homozygous (jaundiced) Gunn rats aged 3-5 days. The following drugs were investigated: different preparations of gentamycin, kanamycin, tobramycin and sisomicin. The animals received 50-75% of the LD50 of heterozygous (non-jaundiced) Gunn rats. Mortality, weight gain and changes in the plasma bilirubin concentration were recorded. It was found that the displacement of bilirubin from albumin is caused by the different stabilizers used and not by the antibiotic itself. With the exception of lyophilized preparations of gentamycin for intrathecal application all vials contain different amounts of these preservatives. Special preparations used during the newborn period contain relatively more of these stabilizers. The toxicity of the additives has already a negative influence on the LD50 for heterozygous Gunn rats when the low dosed Refobacin and Sulmicin vials are given. For Refobacin (production 1973/74) the tolerance is reduced by nearly 50%. The toxicity caused by the stabilizer alone is even more marked when given to homozygous (jaundiced) Gunn rats. It becomes evident that benzylalcohol is the substance responsible for the displacement of bilirubin from albumin. The serum concentration of bilirubin decreases for 3-24 hrs depending on the doses given to the animal. This offers the opportunity to measure the competitive displacement of bilirubin easily and exactly. The free unbound, unconjugated bilirubin tends to diffuse into the lipid of the brain with resultant kernicterus. This was shown in histochemical preparations of the cerebellum of young homozygous Gunn rats. Using enzyme reactions for lactic acid dehydrogenase and NADH2-tetrazolium reductase the cytotoxic effect of bilirubin on PURKINJE cells could be demonstrated. The effect of the stabilizers used in the other antibiotic drugs tested can be neglected under clinical conditions. Finally the steepness and duration of the decrease of plasma bilirubin after injection of the dangerous stabilizers was studied in animals of different age (3-5 days; 3-4 weeks). Different results observed can be explained by the more rapid metabolism of benzoates in older animals. However, it remains an open question at what age Gunn rats reflect most precisely the human situation in premature and newborn babies.     

Stress on the wrist joint in semilunar bone necrosis--a morphologic study in vivo

Sn-mesoporphyrin (SnMP), a potent inhibitor of heme oxygenase (HO), controlled hyperbilirubinemia in rats of the mutant strain EHBR/Eis. This mutant strain displays pronounced conjugated hyperbilirubinuria and dark pigmentation of hepatocytes, characteristics of the Dubin-Johnson syndrome. SnMP administered at a dose of 10 mumol/kg body weight produced an immediate decrease in plasma bilirubin concentrations which could be maintained by weekly injections of this synthetic heme analogue. Marked inhibition of HO activity was demonstrated in liver, kidney and spleen but not in brain. These results demonstrate that SnMP can lower plasma bilirubin concentrations for extended periods in a new mutant rat model of Dubin-Johnson syndrome.     

High-performance liquid chromatographic determination of phenylephrine in human serum using column switching with fluorescence detection

A method for the determination of total phenylephrine (free plus conjugated) in human serum was developed using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. After serum was deproteinized with acetonitrile, the conjugated phenylephrine was hydrolyzed with diluted hydrochloric acid. The solution was evaporated to dryness. The reconstituted residue was analyzed with HPLC using a heart-cut technique. Good recoveries of the analytes from spiked human serum samples were obtained with small coefficients of variation. A good linear response was obtained for the concentration range 5-500 ng/ml. The lower limit of quantitation for phenylephrine in human serum was 5 ng/ml. The method was applied to the determination of phenylephrine in human serum after oral administration of phenylephrine hydrochloride.     

Adverse events and discomfort in studies on healthy subjects: the volunteer''s perspective. A survey conducted by the German Association for Applied Human Pharmacology

The aim of this work was to adapt the Western blot method to analyse the humoral response to proteins of rotavirus strains having various antigens. 10 serum samples from animals immunized with SA11, 18 serum samples from hospitalized children with rotavirus infections and 17 serum samples from healthy adults were examined for rotavirus antibodies using Western blot. Antibodies against all structural rotavirus proteins were detected. The examined sera reacted differently with rotavirus proteins depending on the strain used. The highest IgG reactivity was observed for Wa and SA11, while the lowest was noted for DS-1. At the same time a different level of cross-reactivity of human sera with specific proteins of antigenically varying rotaviruses was observed. The sera taken from adults showed a lower reactivity with proteins of antigens used compared with sera from children. The exception was VP6 of the Ito strain for which the reactivity of adult sera was statistically higher. Essential differences in the reactivity of children's and adult sera were found mainly in the case of VP2 and VP4 belonging to the SA11 strain and VP4 and VP7 of the Ito strain. From these investigations we can conclude that the Western blot method may be useful in assessing immune response caused by rotaviruses. The results of reactivity of some human sera with proteins of strains used indicates that the persons had previously been infected by antigenically varied rotaviruses.     

Immunoglobulin G and complement C3 levels in pregnancy induced hypertension

A specific assay for the quantitative determination of the new antibiotic azithromycin in a low volume of human serum is described. The assay uses on-line high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI). Deuterium-labeled azithromycin was synthesized and used as the internal standard of the assay. The drug and the internal standard are extracted from 50 microliters of serum, and aliquots are injected onto a standard reverse-phase HPLC column. The effluent from the HPLC column at 1 ml/min is introduced into the atmospheric pressure source of a SCIEX API III mass spectrometer. Azithromycin concentrations in serum are determined by the selected monitoring of the protonated molecular ions of the drug and the internal standard. Our assay yields accurate and precise results over the range 10 ng/ml to 250 ng/ml. The correlation between the assay and a standard HPLC-electrochemical method, requiring a larger volume of serum, has been determined. The two methods showed excellent agreement. Because of its low volume requirement, our HPLC-APCI assay can be substituted for the standard assay for the investigation of azithromycin pharmacokinetics in children.     

Direct determination of benzoylecgonine in serum by EMIT d.a.u. cocaine metabolite immunoassay

An EMIT d.a.u. immunoassay for urine testing was applied on the Syva ETS Plus analyzer for the detection of the cocaine metabolite, benzoylecgonine (BE), in human serum. Serum was analyzed without prior extraction, concentration, or matrix modification. Calibrators and serum controls were prepared from EMIT d.a.u. calibrators that were reconstituted and diluted with EMIT Tox serum calibrator. The assay cutoff concentration for BE was 50 ng/mL. The within-run and between-run precisions of the assay were both less than 5%. Analysis of 162 patient serums yielded 43 BE positive results. All EMIT positive serum BE results were confirmed by gas chromatography-mass spectrometry. All patients with positive BE serums also had BE positive urine samples. Serum bilirubin and triglycerides as high as 38 mg/dL and 319 mg/dL, respectively, did not interfere with the assay. Modification of the EMIT urine assay allowed for a simple, rapid, and reliable method for the detection of BE in serum.