To fully automate a 96-well plate sample preparation technique using liquid handling robotics for the purpose of significantly increasing sample throughput for whole blood samples taken under the UK Road Traffic Act (RTA).

Introduction: demand for analysis of whole blood samples taken under the Road Traffic Act 1988 has increased in recent years due to UK Policing and Government policy on tackling driving under the influence of drugs (DUID). Previous extraction techniques utilised by our laboratory include manual or semi-automated approaches that are lengthy and labour intensive. Both approaches require time consuming human input, to precipitate blood and prepare calibrators and quality controls. Recent developments have sped up chromatographic methods through use of techniques such as ultra-high performance liquid chromatography. However, there has been little progress with speeding up sample preparation and extraction processes, particularly for whole blood to compliment increased LC-MS/MS capacity. Use of 96-well plate formats for protein precipitation and extraction provide a platform that can be readily coupled to automated liquid handling (ALH) instruments. This provides a solution to the increase in demand by allowing for extraction of more samples, with minimal human input, in a much shorter amount of time. Here we present development, validation and implementation of an automated robotic sample preparation method for whole blood for analysis of drugs in RTA cases.

Aliquots (200μL) of blank blood and casework samples were dispensed into a 96-well plate using a robotic pipetting system before being loaded onto a Hamilton ALH. Casework samples and blank blood were spiked with internal standard. Additional blank blood samples were further spiked to produce calibrants and quality controls. The plate was mixed with the addition of a cell lysis solution and the aliquots transferred to a protein/lipid depletion (PPT) plate. A positive pressure manifold was used to draw the solution through the PPT plate into a collection plate where the precipitate was dried. The precipitate was reconstituted and loaded by the Hamilton onto a microelution SPE plate. Following extraction, the samples were reconstituted and the collection plate placed in the LC-MS/MS autosampler for analysis. The LC-MS/MS method consists of a 2.1mm×30mm, 1.7μm, C18 column running a 4minute gradient of water and methanol with 0.1% formic acid.

Quantitation of 34 compounds (including common drugs of abuse and their metabolites) was successfully validated with CVs ranging from 3.0%–9.0% and accuracies of 85.8%–111.7%. Within batch precision had CVs of<10.0%. The robotic platform was able to undertake sample pre-treatment and extraction of 40 case samples in 3.5hours. The method was implemented into casework following UKAS accreditation and in 2021, 12,400 samples were analysed using the method.

Introduction of this method to our laboratory has resulted in a significant increase in speed for a sample to be processed and results obtained. Each ALH is capable of extracting two batches per day; equating to 160 case sample versus 28 cases on semi-automated methods. This, along with a reduced sample volume requirement and reduced solvent consumption has had a very beneficial impact in terms of cost, turnaround times and ability to use low sample volume cases.

Full automation of sample preparation of whole blood samples coupled with high speed chromatographic techniques can significantly increase throughput, decrease turnaround times and reduce solvent consumption.