High-Throughput Quantitative Analysis of Tricyclic Antidepressants and Selective Serotonin Re-uptake Inhibitors in Human SerumUsing Ultrafast SPE/MS/MS

Mohamed Youssef1, NikunjR. Parikh1, Michelle V. Romm1, Vaughn P. Miller1and William A. LaMarr1

1. Agilent Technologies, Inc., Wakefield, MA

Introduction

Steady increases in the need for greater analytical capacity and throughput have placed demands on common analytical technologies like HPLC or LC/MS/MS. In the present study, we evaluated the ability of an ultrafast SPE/MS/MS system to measure different analytes oftricyclic antidepressant (TCA) and serotonin re-uptake inhibitor (SSRI) classesin human serum with sample cycle times under 13 seconds per sample.Comparable accuracy, precision, and linearity results were achieved at rates 20-30 fold faster than traditional LC/MS/MS methods.

Methods

TCApanel: amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, and norclomipramine. SSRI panel:citalopram, N-desmethyl citalopram, fluoxetine, norfluoxetine, sertraline and paroxetine.Calibration standards and quality controls were prepared by spiking blank human serum with the analytes in a range of (10-500 ng/mL). Sample preparation included a simple protein precipitation using zinc sulfate and methanol for TCAs or zinc sulfate and acetonitrile for SSRIs containing the deuterated internal standard mix. The resulting supernatants were then diluted with water.

Analysis of all samples was performed at a rate of <13 seconds per sample using a RapidFire High-throughput Mass Spectrometry system coupled to a triple quadrupole mass spectrometer operating in positive ion mode with short (15ms) dwell times. Online SPE methods were optimized for each analyte class using a C18 cartridge.

Results

Both analyte panels had excellent linearity within its measured range of 10-500 ng/ml with an R^2 value greater than 0.99. Intra and interday accuracies for all concentrations were within 10% and intra and interday precision was within 10%. A robustness test comprising of 2000 sequential injections of the same analyte panelsproduced a CV of less than 5% with no changes to peak signal or SPE cartridge pressure. Carry-over was found to be insignificant (figure 1).

Conclusions

Anaytes in the TCA and SSRI class can be quantified accurately and precisely with thishigh-throughput SPE/MS/MS system.This analytical method employs protein precipitation followed by dilute and shoot enabling analysis of 8 TCAsor 6 SSRIs in less than 13 seconds per sample. This method is capable of throughputs greater than 240 samples per hour producing significant savings in analysis time and solvent consumption compared to traditional HPLC andLC/MS/MS methods.

Figure 1. Representative calibration curve data for each of the eight TCA analytes showing the injection to injection interval of 13 seconds. Carryover assessment using a matrix blank immediately after the highest calibrator for all analytes shows no significant carryover was observed for any of the analytes.