Does Labetalol Trigger False Positive Drug Testing Results?

OBJECTIVES: Labetalol, an α- and ß-adrenergic antagonist used to treat hypertension in pregnancy has been blamed for causing false-positive amphetamine and methamphetamine results. In this study, we tested 3 concentrations of labetalol prepared with 4 specimen types (urine, plasma, meconium, and umbilical cord tissue), for amphetamine, methamphetamine, and several other drugs with screen and confirmation tests. METHODS: Residual drug-free specimens were pooled. Labetalol hydrochloride dissolved in methanol was used to prepare spikes in triplicate per specimen type (2.7, 50, and 100 µM), which were tested with 41 previously validated drug tests performed by immunoassay or liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Labetalol triggered false-positive amphetamine and methamphetamine results by immunoassay in meconium but did not trigger positive results for any of the targeted drugs or drug metabolites tested by LC-MS/MS. No positive results were generated by any immunoassay or LC-MS/MS test included in the study, when challenged with high concentrations of labetalol in urine, plasma, or umbilical cord tissue. CONCLUSIONS: In summary, false-positive results can be generated by labetalol when tested by immunoassay, but false-positive results are not expected when testing is performed by highly specific analytical approaches such as LC-MS/MS.

Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device.

BACKGROUND: Therapeutic drug monitoring (TDM) for immunosuppressive (ISP) drugs is an important component of organ and tissue transplantation and chemotherapy management. Whole blood is the specimen type for the quantitative analysis of cyclosporine A, everolimus, sirolimus, and tacrolimus. Some alternatives to venous whole blood samples have the potential to reduce blood volume requirements and simplify sample collection and transport. METHODS: The feasibility of ISP drug (cyclosporine A, everolimus, sirolimus, and tacrolimus) monitoring via microsampling device (MitraTM, Neoteryx) was assessed by comparing venous samples collected and extracted using microsampling device to conventional extraction procedure. Analysis was performed by LC-MS/MS. RESULTS: All analytes were found to be linear across the measurement range of 22.7-937.0 ng/mL (18.9-779.1 nmol/L) for cyclosporine A, 2.3-44.2 ng/mL (2.4-46.1 nmol/L) for everolimus, 2.2-47.2 ng/mL (2.4-51.6 nmol/L) for sirolimus, and 2.2-41.3 ng/mL (2.7-51.4 nmol/L) for tacrolimus. Imprecision was evaluated at concentrations within the therapeutic range and was found to be 10.1% and 5.8% for cyclosporine A, 10.0% and 10.0% for everolimus, 15.0% and 11.9% for sirolimus, and 6.8% and 8.5% for tacrolimus. Method comparison (n = 30 for each analyte, using Deming regression) indicated slopes of 1.08, 1.02, 0.90, and 1.15 and intercepts of -12.8 ng/mL (-10.7 nmol/L), 0.8 ng/mL (0.8 nmol/L), 1.5 ng/mL (1.7 nmol/L), and -0.3 ng/mL (-0.3 nmol/L) for cyclosporine A, everolimus, sirolimus, and tacrolimus, respectively. CONCLUSIONS: This feasibility study demonstrates that precision and bias of ≤15% can be achieved for microsampling-based ISP monitoring.

A 6 Second Analytical Method for Quantitation of Tacrolimus in Whole Blood by Use of Laser Diode Thermal Desorption Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring of immunosuppressive drugs is imperative for organ transplant recipients. High-performance LC-MS/MS is considered gold standard; however, immunoassays provide rapid turnaround time. New technology was developed to reduce mass spectrometry analytical run-time. The laser diode thermal desorption source coupled with tandem mass spectrometry (LDTD-MS/MS) eliminates chromatographic separation to increase analytical throughput. METHODS: A rapid, 6 second, LDTD-MS/MS analytical method was developed for the quantification tacrolimus in whole blood. Whole blood samples were lysed, followed by protein precipitation and solid-phase extraction. Extracted samples with desorption solution were spotted onto a LazWell plate then dried and loaded into the LDTD source for analysis with an AB SCIEX 5500 mass spectrometer in positive multiple reaction monitoring mode. The LDTD laser profile ramps from 0% to 65% of full power over 3 s and is held at 65% for 1 s before returning to initial conditions for 2 s. RESULTS: Data presented include tacrolimus by LDTD-MS/MS comparison to LC-MS/MS, sensitivity, imprecision, interference, linearity, and stability. Method comparison between LDTD-MS/MS and a validated in-house LC-MS/MS assay yielded the following: (LDTD-MS/MS) = 1.119 (LC-MS/MS) + 0.23 ng/mL, Sy/x = 1.26, r = 0.9871 (n = 122). The limit of quantification by LDTD-MS/MS for tacrolimus was <0.3 ng/mL and total imprecision was <10%. CONCLUSIONS: Laser diode thermal desorption tandem mass spectrometry technology can provide rapid turnaround time to result for tacrolimus. The analytical time for LDTD-MS/MS was 6 s compared to 135 s by LC-MS/MS, a >95% decrease in analytical time.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method to Quantify Gabapentin and Pregabalin in Urine.

Gabapentin and pregabalin are anticonvulsant drugs that are also utilized for pain management. A mass spectrometry method was developed and validated to quantify gabapentin and pregabalin in urine to support testing for adherence.

Selecting a Structural Analog as an Internal Standard for the Quantification of 6-Methylmercaptopurine by LC-MS/MS.

BACKGROUND: When choosing an analog internal standard (IS) in a quantitative LC-MS/MS assay, careful selection and thorough verification are important for developing an accurate quantitative assay. The IS is a critical component in quantitative mass spectrometry because it is used to normalize results by compensating for variations in sample preparation and instrument performance. Here we present the results of our investigation in the selection process for a structural analog IS (SA-IS) to be used in the quantification of 6-methylmercaptopurine (6-MMP) in cytolysed red blood cell (RBC). METHODS: A cocktail solution of 9 SA-ISs including the isotopically labeled structural isomer and the 6-MMP stable isotope-labeled IS (SIL-IS) was spiked into cytolysed RBC controls and patient samples. Linearity, accuracy, sensitivity, precision, run stability, method comparison, and reinjection reproducibility experiments were performed. Ion suppression was also assessed by T-infusing the cocktail solution. RESULTS: All analogs were linear from 100 to 1200 ng/mL 6-MMP with acceptable precision and sensitivity by use of a spiked blank lysate. Method comparison plots of 6-MMP concentrations in patient samples had excellent agreement for 2 of the SA-ISs (i.e., the isotopically labeled structural isomer and an SA-IS with an added methyl group) when compared to the SIL-IS. Halogen-substituted analogs (i.e., Cl and Br) also met the criteria as an acceptable IS. However, 2 of the selected SA-ISs having substituted amine moieties showed unacceptable performance, with ≥15% bias when compared to the SIL-IS. CONCLUSION: There are many parameters to consider when determining if an analog will be a good IS choice, and the approaches highlighted in this article can be applied to the selection of SA-IS in the development of other LC-MS/MS assays.

LC-MS/MS method for quantitation of mycophenolic acid, mycophenolic acid acyl-glucuronide, and 7-O-mycophenolic acid glucuronide in serum.

Mycophenolic acid (MPA) is the active metabolite of the immunosuppressant drug mycophenolate mofetil (MMF), which is commonly prescribed after organ transplantation in conjunction with other immunosuppressants. MMF therapy is monitored to balance therapeutic efficacy with minimizing adverse effects associated with high serum concentrations. A LC-MS/MS method was developed for the quantification of MPA and two additional metabolites, 7-O-mycophenolic acid glucuronide (MPAG) and mycophenolic acid acyl-glucuronide (AcMPAG), in serum using reverse-phase chromatography and multiple reaction monitoring (MRM) in positive electrospray ionization mode. Analytes were chromatographically resolved and the method was linear from 0.5 to 30.0 µg/ml MPA, 4.7 to 300 µg/ml MPAG, and from 0.5 to 30.0 µg/ml AcMPAG. Calibration curves for all analytes had r ≥ 0.990. Intra- and inter-assay imprecision coefficients of variation (CVs) were ≤6.9% and ≤14.5%, respectively. No ion suppression or interferences were observed. The method compared favorably with an unaffiliated reference laboratory. Retrospective data analyses indicate interpatient differences in drug metabolism.

Performance characteristics of the ARCHITECT Active-B12 (Holotranscobalamin) assay.

BACKGROUND: Vitamin B12 (cobalamin) is a necessary cofactor in methionine and succinyl-CoA metabolism. Studies estimate the deficiency prevalence as high as 30% in the elderly population. Ten to thirty percent of circulating cobalamin is bound to transcobalamin (holotranscobalamin, holoTC) which can readily enter cells and is therefore considered the bioactive form. The objective of our study was to evaluate the analytical performance of a high-throughput, automated holoTC assay (ARCHITECT i2000(SR) Active-B12 (Holotranscobalamin)) and compare it to other available methods. METHODS: Manufacturer-specified limits of blank (LoB), detection (LoD), and quantitation (LoQ), imprecision, interference, and linearity were evaluated for the ARCHITECT HoloTC assay. Residual de-identified serum samples were used to compare the ARCHITECT HoloTC assay with the automated AxSYM Active-B12 (Holotranscobalamin) assay (Abbott Diagnostics) and the manual Active-B12 (Holotranscobalamin) Enzyme Immunoassay (EIA) (Axis-Shield Diagnostics, Dundee, Scotland, UK). RESULTS: Manufacturer's claims of LoB, LoD, LoQ, imprecision, interference, and linearity to the highest point tested (113.4 pmol/L) were verified for the ARCHITECT HoloTC assay. Method comparison of the ARCHITECT HoloTC to the AxSYM HoloTC produced the following Deming regression statistics: (ARCHITECT(HoloTc)) = 0.941 (AxSYM(HoloTC)) + 1.2 pmol/L, S(y/x) = 6.4, r = 0.947 (n = 98). Comparison to the Active-B12 EIA produced: (ARCHITECT(HoloTC)) = 1.105 (EIA(Active-B12)) - 6.8 pmol/L, S(y/x) = 11.0, r = 0.950 (n = 221). CONCLUSIONS: This assay performed acceptably for LoB, LoD, LoQ, imprecision, interference, linearity and method comparison to the predicate device (AxSYM). An additional comparison to a manual Active-B12 EIA method performed similarly, with minor exceptions. This study determined that the ARCHITECT HoloTC assay is suitable for routine clinical use, which provides a high-throughput alternative for automated testing of this emerging marker of cobalamin deficiency.

Intrinsic factor blocking antibody interference is not detected in five automated cobalamin immunoassays.

OBJECTIVES: To systematically assess five automated cobalamin assays for intrinsic factor binding antibody (IFBA)-induced interference using pooled serum. METHODS: Six pools created from IFBA-negative and IFBA-positive serum representing low, normal, and high cobalamin concentrations were analyzed before and after polyethylene glycol (PEG) precipitation of immunoglobulins on five cobalamin assays: the Centaur XP (Siemens Healthcare Diagnostics, Tarrytown, NY), IMMULITE 2000 (Siemens Healthcare Diagnostics), ARCHITECT i2000SR (Abbott Diagnostics, Abbott Park, IL), UniCel Dxl 800 (Beckman Coulter, Chaska, MN), and Modular E170 (Roche Diagnostics, Indianapolis, IN). RESULTS: Cobalamin concentrations before and after PEG treatment were similar, almost all within a 30% total allowable error, with no difference in pattern between the IFBA-negative and IFBA-positive pools regardless of the cobalamin concentration. CONCLUSIONS: Our results suggest that, under optimal conditions, the five automated cobalamin assays assessed are not susceptible to IFBA-mediated interference.

Comparison of three immunoassays for detection of antibodies to Strongyloides stercoralis.

Due to the limited sensitivities of stool-based microscopy and/or culture techniques for Strongyloides stercoralis, the detection of antibodies to this intestinal nematode is relied upon as a surrogate for determining exposure status or making a diagnosis of S. stercoralis infection. Here, we evaluated three immunoassays, including the recently released InBios Strongy Detect IgG enzyme-linked immunosorbent assay (ELISA) (InBios International, Inc., Seattle, WA), the SciMedx Strongyloides serology microwell ELISA (SciMedx Corporation, Denville, NJ), and the luciferase immunoprecipitation system (LIPS) assay performed at the National Institutes of Health (NIH), for their detection of IgG antibodies to S. stercoralis. A total of 101 retrospective serum samples, previously submitted for routine S. stercoralis antibody detection using the SciMedx assay, were also evaluated by the InBios and LIPS assays. The qualitative results from each assay were compared using a Venn diagram analysis, to the consensus result among the three assays, and each ELISA was also evaluated using the LIPS assay as the reference standard. By Venn diagram analysis, 65% (66/101) of the samples demonstrated perfect agreement by all three assays. Also, the numbers of samples considered positive or negative by a single method were similar. Compared to the consensus result, the overall percent agreement of the InBios, SciMedx, and LIPS assays were comparable at 87.1%, 84.2%, and 89.1%, respectively. Finally, the two ELISAs performed analogously but demonstrated only moderate agreement (kappa coefficient for the two assays, 0.53) with the LIPS assay. Collectively, while the two commercially available ELISAs perform equivalently, neither should be used independently of clinical evaluation to diagnose strongyloidiasis.

A spectrophotometric method to precisely determine endpoint titers in complement fixation assays.

Since the early 20th century, complement fixation (CF) testing has been used to quantify the humoral response to various pathogens. The qualification of a positive result is based on a subjective determination of 30% lysis of sheep red blood cells, which can lead to variability in the analysis. A spectrophotometric reading of a standard with a known 30% lysis was used to standardize the currently used CF method and tested with controls and patient sera for various fungal assays. By utilizing this method a precise and non-subjective determination of endpoint titers was achieved.

Comparison of Western immunobloting to an enzyme-linked immunosorbent assay for the determination of anti-Bordetella pertussis antibodies.

During Bordetella pertussis infection, it has been established that an increase of anti-pertussis toxin (PT) and anti-filamentous hemagglutinin (FHA) antibodies occurs. Immunoblots from two manufacturers using FHA and PT antigens were compared with an enzyme-linked immunosorbent assay (ELISA) that used both FHA and PT. One manufacturer used two concentrations of PT bands for the IgG immunoblot, calibrated to the World Health Organization standard for PT in international units (IU/ml), 100 IU/ml (PT-100) and 8 IU/ml (PT). The second immunoblot kit measured antibodies to a single calibrated PT band. Both kits measured IgA antibodies, and one additionally measured IgM antibodies. Two of 41 (5%) ELISA IgM positives were confirmed positive by IgM immunoblotting, suggesting poor specificity of the IgM ELISA. The agreements of the IgG and IgA immunoblots with the ELISA ranged from 72.5% to 85.3%, with only 38 to 51% of IgA positives confirmed by immunoblotting and only 61 to 68% of IgG positives confirmed by immunoblotting. The two immunoblots correlated well with each other, with 91.7% and 94.3% agreement for IgG and IgA, respectively. When the FHA band was used with the PT band as the criterion for positivity, significant differences existed in specificity compared to the ELISA (IgG, 84.1% versus 33.3%; IgA, 82.4% versus 71.0%). When the positive IgA immunoblots (evidence of natural recent infection) were compared to the positive PT-100 IgG immunoblots (evidence of recent infection or vaccination), the PT-100 blot showed a 71% sensitivity in detecting natural recent infection. B. pertussis immunoblots, alone or in combination with ELISAs, can aid in the diagnosis of B. pertussis infection.