MSACL 2024 Abstract

Introduction

LC-MS/MS has established a critical role in clinical laboratory testing for measurements of challenging small molecules such as steroids, drugs, and metabolites largely due to the exceptional specificity and sensitivity of the technique. However, even with carefully designed chromatographic methods and tandem mass spectrometry analysis, reducing interferences and increasing the signal-to-noise is an ever-present challenge. Differential mobility spectrometry (DMS) has the potential to improve LC-MS/MS measurements by providing an orthogonal and complementary chemical separation. DMS is an ion mobility technique that filters ions based on mobility in the presence of an electric field and a buffer gas, and is ideally suited for use with the widely utilized triple quadrupole mass spectrometers due to its small size, short residence times, and availability of commercial devices. Through this abstract, we hope to demonstrate the benefits of using DMS when performing LC-MS/MS measurements of steroids.

Methods

Liquid chromatography separation was performed using a Thermo Scientific TLX-2 system, which was coupled to a Sciex 6500+ mass spectrometer equipped with a Sciex SelexION DMS device. This system was used for measurements of androstenedione and 17-hydroxyprogesterone in serum prepared using protein precipitation with acetonitrile, 24, 25 dihydroxyvitamin D2 and D3 in serum enriched using solid phase extraction, and cortisol and cortisone in urine diluted prior to LC-MS/MS analysis. All analytes were quantitated using isotopically labeled internal standards and external multi-point calibration curves. Residual clinical samples were analyzed with and without the SelexION installed to assess the benefits of DMS technology for these analytes. After promising initial studies, a full validation of cortisol and cortisone measurements in urine was performed to assess the analytical and clinical implications of using the technology.

Results

Visual comparison of chromatograms without the use of DMS to those with DMS clearly indicates a reduction of interferences and chemical noise when using DMS technology. This has significant clinical implications, as results are not reported for patient samples that exhibit apparent chromatographic interferences. In addition to checking for the presence of chromatographic interferences, clinical laboratories typically analyze at least two fragment ions for each target compounds and the ion ratios or calculated concentration results for these fragment ions must meet predetermined acceptance criteria prior to reporting results, such as results for both fragments must be within 20%. When these criteria cannot be met, no results are reported. In our study, DMS improved the agreement between quantifier/qualifier fragment ion results for cortisol and cortisone as indicated by the increase in R2 from approximately 0.81 to 0.98. Signal-to-noise ratio (S/N) across all samples was also increased, with an average increase across all samples ranging from 1.6 to 13.8 fold. The results from the validation studies of cortisol and cortisone in urine were also encouraging, as all studies met acceptance criteria. Of note, the interassay imprecision study yielded %CVs of less than 8% across all levels and the agreement with the reference method was satisfactory.

Conclusions

Our investigations have shown that DMS technology provides significant benefits when conducting LC-MS/MS measurements of steroids. DMS can eliminate interferences that are difficult or impossible to separate chromatographically, while also improving S/N and agreement between fragment ions. Additionally, all acceptance criteria were met for the validation studies performed. Therefore, these benefits do not come at the expense of other aspects of analytical performance. The results of this investigation provide further evidence that DMS has the potential to greatly benefit not just clinical measurements of challenging analytes, but most clinical LC-MS/MS analyses.