Quantitative determination of phosphatidylethanol, or PEth, by LC-MS/MS requires reliable and reproducible preparation of whole blood samples. Critical workflow steps include the transfer of whole blood, the addition of precipitation solution containing an internal standard and the subsequent transfer of the processed supernatant.

As sample volumes increase, manual preparation can require considerable laboratory resources. Automated PEth sample preparation can reduce repetitive pipetting, support standardization between analytical runs and decrease the active workload of laboratory personnel.

Automation on the Hamilton Microlab Prep

A new application note evaluates the automated preparation of the ARCHImedline “PEth from whole blood” kit on the Hamilton Microlab Prep.

The automated workflow follows the same analytical principle as the established manual procedure. Whole blood samples, calibrators and controls are processed using precipitation solution containing an internal standard.

The Hamilton Microlab Prep performs the transfer of whole blood samples, addition of the precipitation solution, mixing and transfer of the processed sample to the measurement plate. PEth 16:0/18:1 and PEth 16:0/18:2 are subsequently determined by LC-MS/MS.

Excellent agreement with manual sample preparation

For the method comparison, 20 whole blood samples were prepared using both the manual and automated procedures. The results demonstrated excellent agreement between the two sample preparation methods for both PEth analytes.

Correlation coefficients of 0.997 for PEth 16:0/18:1 and 0.994 for PEth 16:0/18:2 were obtained. Passing-Bablok regression slopes were close to 1, confirming the high comparability of automated and manual sample preparation.

Precision was also assessed across low, medium and high concentration levels. The results demonstrated reliable repeatability and reproducibility of the automated workflow across the evaluated measuring range.

External quality control materials and proficiency testing samples were processed alongside the routine samples. The measured concentrations showed good agreement with their respective assigned target values. No relevant carryover or cross-contamination was observed after processing samples with high PEth concentrations.

Substantially reduced hands-on time

In addition to analytical comparability, workflow efficiency was evaluated.

For a batch of 47 samples, automation reduced hands-on time from 48 minutes to 4 minutes. Total sample preparation time decreased from 58 minutes to 23 minutes.

Automated sample preparation therefore substantially reduces repetitive manual activities and allows laboratory personnel to focus on other tasks while samples are being processed.

Standardized PEth sample preparation for LC-MS/MS

The results demonstrate that the ARCHImedline “PEth from whole blood” kit can be successfully integrated into an automated workflow on the Hamilton Microlab Prep.

Automated sample preparation provided excellent comparability with the manual procedure, reliable precision and a substantial reduction in active hands-on time.

The complete results, performance data and details of the automated workflow are available in the full application note: