Steven R. Binder

Automated liquid chromatographic analysis of drugs in urine by on-line sample cleanup and isocratic multi-column separation

A multi-column system has been developed for automated analysis of basic drugs in urine. Two polymeric pre-columns, containing PRP-1 and Aminex A-28, were used to isolate the drugs. A short reversed-phase column, coupled to a 150 x 4.6 mm I.D. silica column, produced the analytical separation. Sample preparation consisted of dilution and centrifugation. The entire procedure required less than 30 min. Careful optimization of mobile phase conditions led to retention of benzoylecgonine and barbiturates. For most drugs, levels of 0.3 mg/l were sufficient to produce peaks that could be matched against stored spectra with a computerized library search program.

Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 ‘shared epitope’ alleles

Introduction A hallmark of rheumatoid arthritis (RA) is the development of autoantibodies targeting proteins that contain citrulline. Anticitrullinated protein antibodies (ACPAs) are currently detected by the commercial cyclic citrullinated peptide (CCP) assay, which uses a mix of cyclised citrullinated peptides as an artificial mimic of the true antigen(s). To increase the sensitivity of ACPA detection and dissect ACPA specificities, we developed a multiplex assay that profiles ACPAs by measuring their reactivity to the citrullinated peptides and proteins derived from RA joint tissue. Methods We created a bead-based, citrullinated antigen array to profile ACPAs. This custom array contains 16 citrullinated peptides and proteins detected in RA synovial tissues. We used the array to profile ACPAs in sera from a cohort of patients with RA and other non-inflammatory arthritides, as well as sera from an independent cohort of RA patients for whom data were available on carriage of HLA-DRB1 ‘shared epitope’ (SE) alleles and history of cigarette smoking. Results Our multiplex assay showed that at least 10% of RA patients who tested negative in the commercial CCP assay possessed ACPAs. Carriage of HLA-DRB1 SE alleles and a history of cigarette smoking were associated with an increase in ACPA reactivity—in anti-CCP+ RA and in a subset of anti-CCP− RA. Conclusions Our multiplex assay can identify ACPA-positive RA patients missed by the commercial CCP assay, thus enabling greater diagnostic sensitivity. Further, our findings suggest that cigarette smoking and possession of HLA-DRB1 SE alleles contribute to the development of ACPAs in anti-CCP− RA.

Analysis of benzodiazepines and tricyclic antidepressants in serum using a common solid-phase clean-up and a common mobile phase

A single, rapid and specific solid-phase clean-up procedure was developed for the analysis of benzodiazepines and tricyclic antidepressants using a carefully selected wash step and specific sequential elution. Benzodiazepines were eluted from the solid-phase column using as mixture of water-methanol-acetonitrile (2:3:3) followed by the elution of tricyclic antidepressants with methanol containing 0.6% diethylamine. A 30% solution of acetonitrile in phosphate buffer containing dimethyloctylamine was used as a common isocratic mobile phase for the analysis of benzodiazepines and tricyclic antidepressants on a reversed-phase column and detection was carried out at 242 nm. The sensitivity limit of the assay for benzodiazepines and tricyclic antidepressants was 25 ng/ml in serum with recoveries of 95-105% for benzodiazepines and 76-95% for tricyclic antidepressants. The results were linear for benzodiazepines over the range 50-2000 ng/ml and for tricyclic antidepressants over the range 25-500 ng/ml. Analysis for benzodiazepines and tricyclic antidepressants gave good precision, with a coefficient of variation of less than 5.0%. The method described here will be suitable for use in a clinical setting, where there is a concomitant use of benzodiazepines and tricyclic antidepressants.

Computer-Assisted Pattern Recognition of Autoantibody Results

ABSTRACT Immunoassay-based anti-nuclear antibody (ANA) screens are increasingly used in the initial evaluation of autoimmune disorders, but these tests offer no “pattern information” comparable to the information from indirect fluorescence assay-based screens. Thus, there is no indication of “next steps” when a positive result is obtained. To improve the utility of immunoassay-based ANA screening, we evaluated a new method that combines a multiplex immunoassay with a k nearest neighbor (kNN) algorithm for computer-assisted pattern recognition. We assembled a training set, consisting of 1,152 sera from patients with various rheumatic diseases and nondiseased patients. The clinical sensitivity and specificity of the multiplex method and algorithm were evaluated with a test set that consisted of 173 sera collected at a rheumatology clinic from patients diagnosed by using standard criteria, as well as 152 age- and sex-matched sera from presumably healthy individuals (sera collected at a blood bank). The test set was also evaluated with a HEp-2 cell-based enzyme-linked immunosorbent assay (ELISA). Both the ELISA and multiplex immunoassay results were positive for 94% of the systemic lupus erythematosus (SLE) patients. The kNN algorithm correctly proposed an SLE pattern for 84% of the antibody-positive SLE patients. For patients with no connective tissue disease, the multiplex method found fewer positive results than the ELISA screen, and no disease was proposed by the kNN algorithm for most of these patients. In conclusion, the automated algorithm could identify SLE patterns and may be useful in the identification of patients who would benefit from early referral to a specialist, as well as patients who do not require further evaluation.

Detection of Immunoglobulin M Antibodies Specific for Toxoplasma gondii with Increased Selectivity for Recently Acquired Infections

ABSTRACT Toxoplasma gondii infections can cause serious complications in pregnant women, leading to miscarriage, stillbirth, and birth defects. Definitive diagnosis of T. gondii acute infection is therefore critical for the clinical management of a mother and her fetus. Positive immunoglobulin M (IgM) results are not sufficient as evidence of recent infection, as these antibodies are often present for many months. Further, IgG avidity and differential agglutination tests, two tests used by reference laboratories to distinguish between recent and past infections, are not always in agreement, and both methods yield a significant number of indeterminate results. We report the development of a new toxoplasma IgM immunoassay that is performed by using a bead-based immunoassay on an automated analyzer (BioPlex 2200). Initial validation included 204 samples from pregnant women and 198 samples from asymptomatic healthy adults. An overall specificity of 99% was observed. Further, 100% sensitivity for acute infections was observed for 10 well-characterized seroconversion panels. We then examined 50 samples from pregnant women, all of which were IgM positive by ELISA, which had been fully evaluated in a reference laboratory. Of the 50 samples, 34 (68%) tested positive in the BioPlex 2200 toxoplasma IgM assay, of which 32 of 34 (94%) exhibited an acute or equivocal pattern by differential agglutination. Of the 16 negative samples, 15 (94%) showed high-IgG-avidity antibodies. Collectively, these results suggest that this new toxoplasma assay shows a preferential response to IgM antibodies produced by recent infections, reducing the number of positive results for pregnant women that will require extensive additional clinical evaluation.

Use of REMEDi HS in Emergency Toxicology for a Rapid Estimate of Drug Concentrations in Urine, Serum, and Gastric Samples

The REMEDi HS is a broad spectrum drug identification system, designed for emergency toxicology screening and forensic applications. The total analysis time is about 20 min. The current library has 555 drugs and metabolites. The system has a software routine that uses an internal standard (IS) to perform quantitative analysis for target compounds when calibrators are available; further, response factors (RF) are supplied for a rapid estimate of drug concentrations when calibrators are unavailable. In the present study, The concentrations of six drugs (bromisovalum, ephedrine, hydroxyzine, diphenhydramine, ranitidine, and lidocaine) and a metabolite of lidocaine (glycinexylidide) were determined using both methods. The slopes of the regression lines between the rapid estimate method and the IS method were generally within 20% of unity, in agreement with the manufacturers claim. Semiquantitative estimates based on RF also showed good agreement with results obtained using multipoint calibration. These estimates were sufficient for clinical differentiation of routine and toxic levels. Our study demonstrated that the REMEDi HS is particularly useful for a rapid estimate of drug concentrations in the samples from emergency cases when calibrators are not readily available. Our study also showed that this system can be used for the therapeutic monitoring of ranitidine, bromisovalum, lidocaine, and diphenhydrmine.

The Development of a Broad-Spectrum Toxicology Screening Program in Taiwan

Our institute serves as a centralized clinical laboratory for municipal and private hospitals in Taipei, a major international metropolis in the Asian region. Two key considerations leading to the development of our toxicology program are: a large number of foreign visitors and local residents returning from overseas trips may bring in chemicals which are less commonly seen in this region; and the lack of readily available assays for a large percentage of commonly used medicines, including prescription and over-the-counter drugs. Our toxicology screening program addresses the needs of both the Emergency Department Drug Screening and Drug of Abuse Screening. In Emergency Department Drug Screening, REMEDi HS is used as the general screening method. In Drug of Abuse Screening, the TDx is used for the initial screening of amphetamine-like substances and opiates, followed by REMEDi HS for the confirmation of positive samples. Emergency Department data collected at our institute over one year (September 1992 to August 1993) identified 57 different drugs in 713 samples. Opiates, narcotics and central stimulants accounted for 24% of the encountered drugs. Presently, there is no extensive reporting of misuse of benzodiazepines in this region. The detection of herbal ingredients like ephedrine and methylephedrine (from the Ma-Huang plant) in patient samples illustrates a large area often overlooked by western toxicology.

Measurement of urinary vanilmandelic acid and homovanillic acid by high-performance liquid chromatography with electrochemical detection following extraction by ion-exchange and ion-moderated partition

An improved protocol has been developed to isolate homovanillic acid (HVA) and vanilmandelic acid (VMA) from urine with strong anion-exchange resin. The sample is diluted with acetate buffer and passed through a disposable column. HVA, uric acid, and many hydrophobic organic acids are removed with 1.0 M acetic acid--ethanol. Then VMA is eluted with 0.5 M phosphoric acid. Two isocratic mobile phases allow rapid high-performance liquid chromatographic measurement of VMA (5 min) and HVA (8 mins) on a 5-micron ODS column. Selective conditions were developed with dual-electrode coulometric detection to permit specific measurement of VMA, HVA, and internal standards, with less than 5% between-run variation.

Use of direct-probe mass spectrometry as a toxicology confirmation method for demoxepam in urine following high-performance liquid chromatography

The identification of the metabolite demoxepam in human urine establishes that chlordiazepoxide, a common benzodiazepine, has been administered. Like N-oxide metabolites of other drugs, demoxepam cannot be detected by gas chromatography-mass spectrometry (GC-MS), due to thermal decomposition, and the product, nordiazepam, is a metabolite common to many benzodiazepines. Demoxepam can be readily screened using a high-performance liquid chromatography (HPLC) system such as REMEDi HS; at 35 degrees C, no thermal decomposition will occur. Currently, there is no confirmation method available for the detection of demoxepam in urine samples. In this study, we demonstrated that following collection of the HPLC fraction, demoxepam can be confirmed using the technique of direct-probe MS. The mass spectra of demoxepam and nordiazepam differ and are easily distinguishable from each other. Ten urine samples that were analyzed by HPLC and determined to contain demoxepam were evaluated; demoxepam was confirmed in each case by direct-probe MS.

Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease.

ABSTRACT The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease.