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Abstract Introduction
Leukotriene E4 (LTE4) is a urinary biomarker for systemic mastocytosis and inflammatory response within the pulmonary and cardiac pathways. LTE4 guides clinical care in asthma patients as LTE4 concentrations correlate with disease severity. While LTE4 production inhibitors decrease LTE4 concentration, it is of utmost importance to measure as low as possible for an accurate clinical evaluation and diagnosis. Therefore, there is a need for an assay capable of accurate and precise measurements of LTE4 at low concentrations (≤20 pg/mL). Historically, LTE4 has been quantitatively measured by enzyme-linked immunoassays (ELISA) and LC-MS/MS; however, historical methods using both technologies have demonstrated suboptimal performance, with specificity and interferences being major challenges due to the high prevalence of molecules with similar chemical structures and characteristics. Mass spectrometry has the potential to provide highly accurate, precise, and specific results. Despite advantages of LC-MS/MS, the aforementioned challenges with interferences and specificity must still be overcome with careful development of sample preparation techniques and chromatographic conditions. Therefore, we have developed and validated a highly specific anion exchange solid-phase extraction and a chromatographic method utilizing a high-efficiency analytical column with unique selectivity.
Methods
250 mcL of patient urine was pipetted into a 96-well plate, followed by 50 mcL each of deuterium labeled internal standard (10007858, Cayman Chemicals) and 1N NaOH. Lastly, 1 mL of water was added, and the plate mixed thoroughly. The SPE plate (A3967010, Agilent) was conditioned with methanol and water, with positive pressure applied between each reagent addition. The samples were transferred into the SPE plate and ~1 psi pressure was applied for 7 minutes. The SPE plate was washed with water and methanol. The elution (methanol with 1% acetic acid and 1mcg/mL estriol) was collected and 50 mcL of water was added and mixed. The plate was injected and analyzed on the HPLC-MS/MS (Thermo Scientific TLX-4 coupled to Sciex 6500+). The LC separation was performed with a RP-MS analytical column (17626-053030, Thermo Scientific). The mobile phases were 0.02% acetic acid in both water (MPA) and methanol (MPB). The mobile phase gradient was adjusted to 55% organic for 30 seconds, with a ramping increase to 80% for 300 seconds and then to 98% for 90 seconds, at a constant flow rate of 0.6 mL/min. To confirm the validity of the assay; accuracy, precision, and sensitivity studies were performed using previously tested patient urine samples and pooled patient urine samples. The analytical measuring range (AMR) was 10 – 1280 pg/mL, by setting up a linear calibration curve using a LTE4 stock standard (20410, Cayman Chemicals) prepared in methanol and diluted with 0.1% BSA. The quality control materials comprised of pooled sample aliquots, spanning the AMR, with established means and acceptance criteria within 2 SD.
Results
When compared to methods with minimal sample preparation (“dilute and shoot”) and more traditional reverse phase analytical columns, the developed method had a tremendous effect on the chromatograms, greatly reducing the intensity of the baseline and the number of potentially interfering signals in the chromatograms. Therefore, the developed method increased the signal-to-noise ratio of the target signals, improved the reproducibility of chromatogram integration, and improved the specificity of the assay, which enabled validation of the test with a lower limit of quantitation (LLOQ) of 10 pg/mL, compared to 40 or 50 pg/mL in most cases. Assay validation studies were well within the established acceptance criteria and exceeded expectations. For intra-day precision, of four pools: 11.2, 20.1, 96, and 240 pg/mL, the %CVs are all less than 10%. For inter-day precision, of five pools, 16.4, 9.7, 58.7, 294, 652 pg/mL, the %CVs are less than 12%. Of the 59 previously tested patient samples evaluated for accuracy, the linear regression comparison gives a slope of 1.044 and a R-squared of 0.992. These strong results demonstrate that this method is a viable option for the measurement of urinary LTE4 with an improved LLOQ and reduction of interferences.
Discussion
The most significant improvement of our LTE4 assay is the exceptional lower limit of quantitation, which can be attributed to the reduction of baseline and interferences. This enhances peak picking and quantitation of low LTE4 concentrations. During evaluation of the limit of detection, we were able to measure sample concentration as low as 10 pg/mL with sufficient accuracy and precision. The novel SPE, analytical column, and LC parameter improvements of our LTE4 assay benefit patient care by providing highly sensitive and specific LTE4 measurements. |
= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
