Development and application of a high-throughput sample cleanup process based on 96-well plate for simultaneous determination of 16 steroids in biological matrices using liquid chromatography–triple quadrupole mass spectrometry

Abstract

A novel high-throughput sample pretreatment system was developed by the integration of protein precipitation (PP), phospholipid removal (PPR), and hollow fiber liquid-phase microextraction (HF-LPME) into two simple 96-well plates and a matching 96-grid lid. With this system, 16 steroids were separated from biological matrices of plasma, milk, and urine and analyzed by liquid chromatography-triple quadrupole mass spectrometry. In the tandem sample cleanup process, the prepositive PP and PPR step preliminarily removed some of the interferences from the biological matrices. The following HF-LPME step kept the residual interference out of the hollow fiber and enriched the steroids in the hollow fiber to achieve high sensitivity. By a series of method optimizations, acetonitrile was chosen as the crash solvent for PP and PPR. A mixture of octanol and toluene (1:1 v/v) was used as the acceptor phase for HF-LPME. The extraction was conducted at 80 rpm for 50 min in a donor phase containing 1 mL 20% sodium chloride at 25 °C. Under these conditions, the limits of detection for the 16 steroids were 3.6-300.0 pg(.)mL(-1) in plasma, 3.0-270.0 pg·mL(-1) in milk, and 2.2-210.0 pg(.)mL(-1) in urine. The recoveries of the 16 steroids were 81.9-97.9% in plasma (relative standard deviation 1.0-8.0%), 80.6-97.7% in milk (relative standard deviation 0.8-5.4%), and 87.3-98.7% in urine (relative standard deviation 1.0-4.9%). Further, the integrated 96-well platform of PP, PPR, and HF-LPME enabled us to run this assay in an automatic and high-throughput fashion. The reliability of the method was further corroborated by evaluation of its applicability in plasma and urine samples from volunteers and fresh bovine milk from local dairy enterprises.

A Fast Quantitative Multi-analyte Method for Growth Promoters in Bovine Meat Using Bead-Disruption, 96-well SPE Clean-up and Narrow-Bore UHPLC-MS/MS Analysis

“…Also, the use of 96-wells format increase the efficiency of sample clean-up (O'Brien et al, 2009;Pinel et al, 2008). In a recent paper of Luo et al (Luo et al, 2016), the use of hollow-fibre liquid-phase microextraction (HF-LPME) was combined in a 96-wells format to extract 16 steroids from urine, milk and plasma in only a few hours. Another fast method is based on a modified Quick Easy Cheap Effective Rugged Safe (QuEChERS) approach (Attalah et al, 2016) to extract four steroids from meat.…”

An analytical study to determine prohibited anabolic residues in red meat tissue using LC-MS/MS system

“…In a study conducted by Luo et al (2016), 16 hormones were analyzed together and the RSD% ranged between 1-8% and the recovery was between 81.9-97.9% in the validation. In another study by Yang et al (2015) for the determination of 26 endocrine chemicals in fish and water, the modified QuEChERS method combined with solid phase extraction and UHPLC-MS/MS was used and recovery was achieved between 69.1% and 120.5%.…”

“…In the preparation step of the sample, the QuEChERS, which is a common method usually performed in pesticide detection was used for anabolic detection (Stubbings & Bigwood, 2009;Tripathy et al, 2019;Yang et al, 2015). Accordingly, the samples were treated with more acetonitrile and the extracts were cleaned by dispersive solid-phase extraction method instead of conventional solid-phase extractions (Chiesa et al, 2016;Fan et al, 2014;Luo et al, 2016;Schmidt et al, 2008;Lehotay et al, 2007;Yang et al, 2009).…”

Methodology and validation of a new tandem mass spectrometer method for the quantification of inorganic and organic 18O-phosphate species

“…This is essential as the Hedley sequential extraction matrices have a negative effect on the LLOQs due to high salt concentrations, where measured limits varied from 0.075 mg/L for 0.5 M HCl to 0.3 mg/L for 0.5 M bicarbonate. Newer triple quadrupole instruments have the capacity to achieve greater sensitivity and selectivity into picogram/mL range [51], which will aid to decrease the LLOQ. Furthermore, the use of MS/MS allows for improved accuracy and reproducibility at the lower end of the calibration curve [50], permitting examination of P-deficient soils.…”