Development and Validation of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Concurrent Measurement of Gabapentin, Lamotrigine, Levetiracetam, Monohydroxy Derivative of Oxcarbazepine, and Zonisamide Concentrations in Serum in a Clinical Setting.

BACKGROUND: Therapeutic drug monitoring of antiepileptic drugs (AEDs) is often necessary to prevent associated destructive toxicities. Tandem mass spectrometry (MS/MS) with stable-isotope-labeled internal standards is considered the gold standard for the measurement of AEDs. This study presents the development and validation of a clinical ultra-performance liquid chromatography-MS/MS method for the concurrent measurement of gabapentin, lamotrigine, levetiracetam, monohydroxy derivative of oxcarbazepine, and zonisamide in human serum. METHODS: To determine the optimal assay analyte range, one year of AED therapeutic drug monitoring results (n = 1825) were evaluated. Simple protein precipitation with acetonitrile containing isotopically labeled internal standards was used. Reverse-phase ultra-performance liquid chromatography chromatographic separation was used, having a total run time of 3 minutes. Quantification of analytes was accomplished using electrospray ionization in positive ion mode and collision-induced dissociation MS. Assay parameters were evaluated per Food and Drug Administration bioanalytical guidelines. RESULTS: After evaluating internal patient data, the analytical measuring range (AMR) of the assay was established as 0.1-100 mcg/mL. All AEDs were linear across the AMR, with R values ranging from 0.9988 to 0.9999. Imprecision (% coefficient of variation) and inaccuracy (% difference) were calculated to be <20% for the lower limit of quantitation and <15% for the low, mid, and high levels of quality controls across the AMR. All AEDs demonstrated acceptable assay parameters for carryover, stability under relevant storage conditions, matrix effects, recovery, and extraction and processing efficiency. In addition, the assay displayed acceptable concordance to results obtained from a national reference laboratory, with Deming regression R of 0.99 and slope values ranging from 0.89 to 1.17. CONCLUSIONS: A simple, cost-effective, and robust ultra-performance liquid chromatography-tandem mass spectrometry method for monitoring multiple AEDs was developed and validated to address the clinical needs of patients at our institution.

A rapid UPLC-MS/MS assay for the simultaneous measurement of fluconazole, voriconazole, posaconazole, itraconazole, and hydroxyitraconazole concentrations in serum.

BACKGROUND: Triazole antifungals are essential to the treatment and prophylaxis of fungal infections. Significant pharmacokinetic variability combined with a clinical need for faster turnaround times has increased demand for in-house therapeutic drug monitoring of these drugs, which is best performed using mass spectrometry-based platforms. However, technical and logistical obstacles to implementing these platforms in hospital laboratories have limited their widespread utilization. Here, we present the development and validation of a fast and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to measure fluconazole, voriconazole, posaconazole, itraconazole, and hydroxyitraconazole in human serum suitable for incorporation into a hospital clinical laboratory. METHODS: Serum samples (20 µL) were prepared using protein precipitation in the presence of deuterated internal standards. Chromatographic separation was accomplished using reversed phase UPLC and analysis was performed using positive-mode electrospray ionization and collision-induced dissociation MS. RESULTS: Total analytical run time was 3 min. All analytes demonstrated linearity (r2>0.998) from 0.1 to 10 µg/mL (1-100 µg/mL for fluconazole), acceptable accuracy and precision (%DEV<15% and %CV<15% at all levels tested), suitable stability under relevant storage conditions, and correlated well with reference laboratory results. CONCLUSIONS: A simple and rapid UPLC-MS/MS method for monitoring multiple triazole antifungals was developed with a focus on the needs of hospital laboratories. The assay is suitable for clinical utilization and management of patients on these medications.

Evaluation of the Waters MassTrak LC-MS/MS Assay for Tacrolimus and a Comparison to the Abbott Architect Immunoassay.

BACKGROUND: Tacrolimus (Prograf, Advagraf, and FK-506) is the most commonly prescribed calcineurin inhibitor after kidney and liver transplantation. The use of tacrolimus (in conjunction with other drugs) has successfully contributed to the maintenance of solid organ allografts; however, it also exhibits toxic side effects. Therapeutic drug monitoring of tacrolimus is used as an aid to achieve drug concentrations within a narrow therapeutic window. METHODS: The Waters MassTrak Immunosuppressants assay (LC-MS/MS) for the quantification of tacrolimus in whole blood was evaluated for precision, linearity, lower limit of quantification, matrix effects, and accuracy. A method comparison with the Abbott Architect Tacrolimus immunoassay was also performed. RESULTS: The mean concentration (nanograms per milliliter) and coefficient of variation for low, mid, and high patient pools were 0.6% ± 19.9%, 16.0% ± 5.4%, and 31.2% ± 5.8%, respectively. The MassTrak assay was linear from 0.5 to 30.0 ng/mL. Although the MassTrak and Architect assays correlated well (R = 0.97) for patient samples, the MassTrak assay displayed an average negative bias of 18.5% versus Architect (range of 0.0%-36.7%). Analysis of a certified tacrolimus reference material in human whole blood [European Reference Materials (ERM)-DA110a, LGC Standards] on both platforms failed to completely explain the observed difference for patient samples. CONCLUSIONS: Two widely used assays for therapeutic drug monitoring of tacrolimus are not in agreement with one another. Care should be exercised when interpreting results generated on these 2 assay platforms.

Standardization of LC-MS for therapeutic drug monitoring of tacrolimus.

BACKGROUND: LC-MS is increasingly used for therapeutic drug monitoring of tacrolimus. A recent summary from an international proficiency-testing scheme demonstrated that the mass spectrometry respondents were the largest method group. However, these methods lack standardization, which may explain the relatively poor interlaboratory agreement for such methods. This study aimed to provide one path toward the standardization of tacrolimus quantification by use of LC-MS. METHODS: A 40-member whole blood tacrolimus proficiency panel was circulated to 7 laboratories, 4 in the US and 3 in Europe, offering routine LC-MS-based quantification of tacrolimus. All laboratories used a common LC-MS platform and followed the manufacturer's instructions that accompanied an LC-MS reagent kit intended for tacrolimus quantification in whole blood samples. Four patient pools were prepared that had sufficient volume to allow comparison with a tacrolimus reference measurement procedure. RESULTS: For the 40-member panel, the standardized MassTrak LC-MS assay demonstrated excellent agreement with a validated LC-MS method used by Analytical Services International (y = 1.02x - 0.02; r = 0.99). The CVs for the pooled patient samples ranged from 2.0% to 5.4%. The mean difference from the reference measurement procedure ranged from 0.4% to 4.4%. CONCLUSIONS: Tacrolimus assay standardization, which must include all facets of the analysis, is necessary to compare patient results between laboratories and to interpret consensus guidelines. LC-MS can provide accurate and precise measurement of tacrolimus between laboratories.

A rapid ultra performance liquid chromatography tandem mass spectrometric method for measuring amino acids associated with maple syrup urine disease, tyrosinaemia and phenylketonuria.

BACKGROUND: Patients with inherited disorders of amino acid metabolism including maple syrup urine disease, tyrosinaemia and phenylketonuria on dietary management require frequent monitoring of disease-relevant plasma amino acids in order to optimize therapeutic benefit. Poorly controlled maple syrup urine disease in particular may result in catastrophic metabolic decompensation. Most methods for monitoring amino acid concentrations are time-consuming and have clinically impractical turnaround times, particularly when the required time to run standards and control samples is taken into account. METHODS: We have analysed plasma amino acids using standard ion-exchange chromatography with ninhydrin detection in an amino acid analyser and compared the data with that obtained for the same samples using ultra-performance liquid chromatography (UPLC) separation with detection by tandem mass spectrometry. RESULTS: The two methodologies compared very well for the measurement of six important amino acids with correlation coefficients greater than 0.96 for all. The time for sample preparation was longer for the UPLC methodology as batched derivatization and evaporation is required but UPLC-tandem mass spectrometry generated sample results every 8 min while conventional ion-exchange chromatography took almost 1 h per sample. CONCLUSION: UPLC-tandem mass spectrometry generates data that compares well with existing 'gold standard' methodologies but significantly reduces sample turnaround time. Decreasing the turnaround time for amino acid analyses is very likely to improve clinical care for patients with amino acid disorders as dietary adjustments can be made sooner.