Analytical Considerations When Developing an LC-MS/MS Method for More than 30 Analytes

Abstract

Background: While validation of analytical (LC-MS/MS) methods has been documented in any number of articles and reference texts, the optimal design and subsequent validation of a method for over 30 analytes presents special challenges. Conventional approaches to calibration curves, controls, and run time are not tenable in such methods. This report details the practical aspects of designing and implementing such a method in accordance with College of American Pathologists validation criteria. Methods: Conventional criteria were followed in the design and validation of a method for 34 analytes and 15 internal standards by LC-MS/MS. These criteria are laid out in a standard operating procedure, which is followed without exception and is consistent with College of American Pathologists criteria. Results: The method presented herein provides quality results and accurate medication monitoring. The method was optimized to negate interferences (both from within the method and from potential concomitant compounds), increase throughput, and provide reproducible quality quantification over relevant analyte concentrations ranges. Conclusions: The method was designed primarily with quality and accurate medication monitoring in mind. The method achieves these goals by use of novel approaches to calibration curves and controls that both improve performance and minimize risk (financial and operational). As automation and LC-MS/MS equipment continue to improve, it is expected that more methods like this one will be developed. IMPACT STATEMENT This method is used for therapeutic drug monitoring of commonly prescribed pain management medications (opiates, opioids, gabapentin, pregabalin, and benzodiazepines) and some illicit drugs (amphetamines and cocaine). An in-depth analysis of the development, validation, and retrospective quality control data of this method shows that large drug assays can be robust and efficient compared to smaller, class-based assays. Maximum throughput of the assay is highly dependent on automation throughout the process. This report is unique in that it describes a 30+ analyte quantitative method as well as the development and considerations required for efficient and cost-effective validation and implementation.

Dose Dependent Urine Concentrations of Gabapentin (Neurontin®)

“…An enzyme solution was prepared by diluting IMCSzyme ® β-glucuronidase solution (IMCS, Irmo, SC) to 10,000 units/mL in 0.02 M sodium phosphate buffer, pH 7.5. Inasmuch as the analysis of gabapentin was part of a larger method, a hydrolysis control was prepared by making a stock of 2.5 ng/mL of morphine-3β-D glucuronide in normal human urine [11]. This control is run with every batch to verify proper hydrolysis of all samples.…”

“…This control is run with every batch to verify proper hydrolysis of all samples. Further details about this method and validation thereof are available in an earlier report [11].…”

“…The LC-MS/MS method detailed herein was originally performed on an Agilent LC/MSMS 6460 system [11]. The current version of this method runs on a Thermo HPLC/MSMS system using an Ultra MSMS unit.…”

“…The injection volume was set to 15μL and column temperature was set to 30°C. Specific analyte transitions and internal standard assignments are given in the earlier report [11].…”

“…As mentioned, the LC/MSMS method is a much larger method testing for 34 analytes using 15 internal standard compounds in a single injection. The specifics of this method and the validation data are summarized in an earlier report [11]. Validation results for gabapentin are summarized as followed.…”