Ghost Peak Investigation in a Reversed-Phase Gradient LC System

May 01, 2011
Volume 29, Issue 5, pg 394–400

Ghost peaks can be difficult to eliminate when minor peaks are important in an LC separation. This case study shows how to isolate the source of such peaks.

Ghost peaks can be referred to as artifact (erroneous) peaks, system peaks, pseudo peaks, vacancy peaks, eigenpeaks, induced peaks, or spurious peaks (1). These are often observed unexpectedly in a chromatogram and can pose a challenge to analytical scientists. These peaks may arise from unknown impurities or artifacts within the liquid chromatography (LC) system (such as contaminants from a dirty injector needle, an air bubble in the pump, or a trapped contaminant in the guard column), from the mobile phase, from an extractable contaminant, from autosampler vials and caps, or from a contaminant carried over from a previous injection.

The occurrence of ghost peaks is seen more frequently in the gradient mode. The presence of a ghost peak in a sample chromatogram, especially at the elution time of a known impurity, may trigger an out-of-trend (OOT) or out-of-specification (OOS) investigation. An OOT or OOS investigation will lead to efforts to identify an assignable cause, assess the impact of the result, and propose further corrective or preventive action, if required. Because the contamination or artifact causing the ghost peak may come from many sources or may be transient in nature, the investigation can be time-consuming. In certain cases, the cause of the ghost peak may remain unresolved.

Furthermore, in some company standard operating procedures (SOPs) in a good manufacturing practice (GMP) environment, if the cause of a random ghost peak cannot be identified and the peak is ruled out as an impurity unrelated to the sample, the data may need to be reported "as is." In some situations a blank subtraction can be justified when reporting the data with ghost peak problems; however, this is usually not preferred in routine quality control (QC) laboratories. The biggest problem with significant ghost peaks is that they make automatic integration of chromatograms difficult. Hence, when manual integration or blank subtraction is used, ghost peaks can cause inconsistent and inaccurate impurity results. The problem eventually intensifies if the inconsistent impurity result has been used for trending purposes in stability studies.

Many publications (including past installments of this column) have cited ghost peak problems and perspectives (2). Here, we outline a step-by-step approach we have used to investigate the source of ghost peaks in a gradient LC method and share our empirical perspectives as quick approaches for solving ghost peak problems in LC. As a case study, we'll offer our experience with a reversed-phase LC method that was used to analyze a nonpolar, small-molecule active pharmaceutical ingredient (API) in an early development stage.

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