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Abstract INTRODUCTION: Typical gradient LC methods include two segments: the gradient in which the separation occurs, and the regeneration step where the column is washed with strong solvent and re-equilibrated to initial conditions, ready for subsequent sample injections. Washing with 3-5 column volumes of a suitable strong solvent is usually recommended, as this prevents accumulation of phospholipids and potentially extends the useful life of the LC column. Allowing sufficient re-equilibration time is important for method robustness. Reducing the time allowed for regeneration may produce acceptably performing high-throughput methods in some cases, but it comes with the risk of poor-quality data and limiting column longevity.
There is an alternative hardware configuration, parallel column regeneration, which splits the elution gradient and regeneration phases over two consecutive injections, allowing sufficient washing and re-equilibration to be performed on the ‘passive’ column, while the gradient separation occurs on the ‘active’ column. This has the potential to dramatically shorten the analytical run, increase sample throughput, without compromising UPLC best practices.
OBJECTIVES: To demonstrate time savings and reduction in residual sample matrix of the parallel column regeneration system using various analytes in biological matrices (whole blood, serum, and dried blood spots).
METHODS: An ACQUITY UPLC system was configured with two Binary Solvent Manager pumps, a single Sample Manager, a two-position, heated, Column Manager with fluidics valves, and a tandem mass spectrometer. The configured hardware was controlled with MassLynx instrument control software, making use of the dual-pump configuration capability of the instrument driver software. Serum-based steroid hormone calibrator and control samples were precipitated and subjected to solid phase extraction; lysophosphatidylcholine species were eluted from dried blood spot punches, and immunosuppressant drugs were analyzed in whole blood calibrator and control sample supernatant. All test matrices were extracted using stable isotope-labelled internal standard, allowing a cursory estimation of instrument-associated repeatability, by replicate injections of pooled, extracted sample (n=10 on each column, by each injection mode). The extracted samples were separated by gradient elution UPLC and analyzed by multiple reaction monitoring (MRM) with electrospray ionization, with and without parallel column regeneration. Chromatographic retention time and analyte: internal standard peak area response ratios were monitored.
The effect of changing the regeneration conditions on phospholipid accumulation was monitored in the UPLC column eluate of columns by detection of precursors of the m/z 184 fragment ion, representing the phosphocholine head group of phospholipids.
RESULTS:
Retention times and response ratios were equivalent for all analyzed samples (p<0.05), with or without parallel column regeneration. The analyte: internal standard response ratio showed <9%, <4.5% and <15.5% within-batch imprecision for immunosuppressant drugs, steroid hormones, and lysophosphatidylcholines, respectively, in either serial or parallel regeneration mode of analysis.
Using parallel regeneration for the analysis of lysophosphatidylcholines, the number of samples analyzed per hour increased from 10.3 to 18.1, representing a 75% improvement in throughput. For the measurement of steroid hormones, the throughput was improved by 14% (from 8.3 with serial, to 9.5 samples/hr with parallel regeneration). For the analysis of immunosuppressant drugs, which is already a rapid 2.1 min method by conventional serial analytical methods, the number of samples analyzed per hour increased from 28.4 to 32.1, representing a 13% improvement in throughput for this clinical research method.
For the simplest extracted sample (immunosuppressants in whole blood) the average phospholipid ion intensity in the wash/hold eluate of columns using parallel column regeneration was 71% less than with serial regeneration (n=10 injections of a pooled, extracted matrix sample). The volumes of washing and re-equilibration were more than doubled with parallel regeneration, whilst still achieving a time saving of 24 minutes per 96 samples. An even greater impact on phospholipid ion intensity was seen in the steroid method, with a 99% reduction compared with serial regeneration. In this instance, the column volumes of washing and equilibration were increased more than 5-fold using parallel regeneration, whilst also saving 1 hour 33 minutes per 96 samples analyzed. Phospholipid removal in the serial analysis of lysophosphatidylcholines was already extensive, due to a generous 2.3 and 4.4 column volumes already allowed for washing and re-equilibration, respectively. Using parallel column regeneration and dividing the method almost equally between gradient separation and reconditioning phases, a notable shortening of the analytical method was possible (4 hours saved per 96 samples), and a modest increase to 3.2 wash volumes was allowed. This was accompanied by a 99% reduction in phospholipid ion intensity compared with serial regeneration.
CONCLUSION: Parallel column regeneration can be easily configured by adding an additional pump and valves to any conventional LC-MS/MS system commonly encountered in clinical research. The greatest relative improvements in throughput will be found for analytical methods where the wash and equilibration periods are similar in length to the elution period. Even when the methods to be transferred to parallel column regeneration do not meet this ideal gradient to regeneration ratio, it is still possible to make modest gains in throughput, whilst harnessing the opportunity to improve phospholipid removal by optionally increasing the extent of column regeneration.
For Research Use Only, not for use in diagnostic procedures.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
