Elimination of autosampler carryover in a bioanalytical HPLC-MS/MS method: a case study

doi:10.1016/j.jpba.2004.09.010

Journal of Pharmaceutical and Biomedical Analysis

Volume 36, Issue 5, 4 January 2005, Pages 1073-1078

https://doi.org/10.1016/j.jpba.2004.09.010 Get rights and content

Abstract

A case study in identifying and eliminating the source of autosampler carryover in a bioanalytical HPLC-MS/MS assay is described. Through a series of systematic experiments, the carryover was traced to the injection valve and was eliminated by switching from a partial loop to a full loop injection, which provided more effective flushing of the sample flow path. The susceptibility of the HPLC system to carryover was demonstrated to depend on the absolute sensitivity of the detection method and the mass of analyte injected at the assay lower limit of quantitation (LLOQ).

Introduction

Autosampler carryover is a common, often stubborn problem that can compromise the accuracy of HPLC assays. This is particularly the case for HPLC-MS/MS based bioanalytical methods used to support pharmacokinetics (PK) studies, in which carryover problems can be exacerbated by a number of factors, including low limits of quantitation (LLOQs), complex sample matrices, and wide dynamic ranges necessary for unknown samples that vary widely in analyte concentrations. Although some researchers argue that the effects of carryover in these assays can be ameliorated by arranging the run sequence based on expected analyte concentrations, this represents bias that could potentially be a concern from a regulatory perspective. This issue of bias has been recognized and mechanisms to eliminate or reduce such bias have been suggested [1] in which analysts are blinded as to the identity of the unknowns. Under blinded conditions, autosampler carryover could severely diminish the accuracy of an assay.

One approach to circumventing carryover is to narrow the dynamic range of the assay. This is undesirable, especially during dose escalation studies conducted early in phase I clinical trials, and/or for drug candidates that exhibit high peak to trough ratios, because many samples would require reanalysis after dilution, creating additional work and increasing the time required to generate data. Another potential problem with this approach is that analyte concentrations in post-dose samples often far exceed the upper limit of quantitation of the assay, in which case significant carryover might persist for numerous injections, potentially affecting subsequent unknowns or quality control samples. In light of these concerns, the preferred approach with respect to carryover is to identify its source and to eliminate it while maintaining as wide as possible a dynamic range.

This report describes a case study from our laboratories in identifying and eliminating autosampler carryover in an HPLC-MS/MS assay of a drug candidate (I) in human plasma. I is a weakly acidic compound with a pK_a of 5 and a molecular weight of 388. A partial chemical structure of I is provided in Fig. 1 showing the important phenolic hydroxyl group present in the molecule.

Section snippets

Experimental

Plasma samples were prepared by liquid–liquid extraction and the extracts were dissolved in mobile phase prior to injection onto the HPLC-MS/MS system. The standard curve range for I in human plasma was 0.2–120 ng/mL.

The HPLC system consisted of a Varian (Walnut Creek, CA, USA) ProStar 430 autosampler and a Perkin–Elmer (Norwalk, CT, USA) Series 200 Micropump. Separations were performed on a Thermo-Hypersil-Keystone (Bellefonte, PA, USA) BDS C-18 HPLC column (3.0 mm × 50 mm, 3 μm), which was used in

Initial assessment of carryover

Initially, the autosampler was programmed for an injection volume of 5 μL (partial loopfill) and a needle wash volume of 1 mL between injections. The needle wash solvent was 0.1% (v/v) acetic acid in 20:80 (v/v%) water:acetonitrile. When a control plasma double blank was injected immediately following the upper limit of quantitation plasma standard (ULOQ = 120 ng/mL), a severe carryover peak was present that corresponded to approximately 0.1% of the ULOQ. The severity of this carryover peak became

Conclusion

In this report, a case study in the elimination of autosampler carryover in a bioanalytical HPLC-MS/MS method was presented. Through a systematic set of experiments and a thorough understanding of the operation of the autosampler, a relatively straightforward solution was implemented by switching from a partial loop to full loop injection. Since these experiments were conducted, this approach has been successfully utilized in other assays in our laboratories that were prone to autosampler

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