Does Your Lc/ms/ms Data Look Better Than It Really Is? #2447

S. Sitaraman , A. Witkowski , J. Plassard , B. Schrader , K. Szczap , and B. Beato
BASi Analytics/ Bioanalytical Systems, Inc. West Lafayette IN, USA

ABSTRACT

Purpose

The selectivity of LC/MS/MS yields chromatograms appearing deceptively clean. Interferences such as plasticizers and matrix components can adversely affect quantitation via ion suppression. Co-eluting and/or isomeric metabolites in incurred samples can also interfere with quantitation. An LC/MS/MS assay for a drug and one metabolite (M1) provides a case study demonstrating ways to locate and handle various potential interferences.

Methods

A protein precipitation method was used to prepare plasma samples from dosed and non-dosed rats and monkeys. Extracts were collected in plastic 96-well plates, with the drug, metabolite, and various interferences monitored by LC/MS/MS.

Results

Full scan mass spectra revealed a compound that leaches from plastic 96-well plates into extracts. A late-eluting matrix component in rat plasma was indirectly found via ion suppression in the subsequent injection. Both of these potential interference problems were solved chromatographically. A third type of potential interference, an additional metabolite (M2) appearing only in incurred monkey plasma, co-eluted with the metabolite of interest (M1). Resolving M2 and M1 chromatographically proved impractical. Further experiments comparing incurred samples and spiked standards verified that the presence of M2 did not affect quantitation of M1 in this case.

Conclusions

Potential interferences arise from many sources, and although often difficult to observe, must be sought during method development to ensure that they do not impact quantitation. Separating potential interferences from analytes during sample preparation or via chromatography is ideal. If this is impractical, however, additional tests are required to demonstrate that these compounds do not compromise data integrity.

Purpose

• Identify and resolve chromatographic interferences such as matrix components and plasticizers that adversely affect quantitation via ion suppression
• Ensure data integrity when using LC/MS/MS by weeding out non-selective co-eluting peaks

Assay Specifics

Sample Volume:	50 µL
Sample Preparation:	Protein Precipitation
Validated Range:	10 - 1000 ng/mL
Column:	Zorbax Eclipse XDB-C8
Mobile Phase:	Step gradient, 30% ® 50% ACN, aqueous portion contains 1% IPA and 1% formic acid
Quantitation:	Quadratic regression with 1/concentration2 weighting. Quantitation by peak area ratio
Detection:	Sciex API 3000 LC/MS/MS with TurboIonspray ionization source in the positive ion mode

Effect of Increasing Run Time on Ion Suppression

• Observation: A late- eluting matrix component was found to cause ion suppression in the n + 1 injection
• Solution: Addition of 2 minutes to the run time ensured that the late eluter did not cause any ion suppression
• Drug: 387 → 240
• MI: 403 → 147
• ISID: 381 → 216

Investigating Unexpected Peak Observed in Q1MS of Drug Standard

• Q1MS over a narrow mass range around the parent ion of interest (m/z 387.3) was run by injecting a system check
• Two major peaks were observed for m/z 387.3

Investigate Origin and Ramifications of Unexpected Peak

• Daughter ion mass spectra were obtained for both m/z 387 peaks and yielded very different spectra, indicating the two peaks arose from chemically different species
• Comparison was done using neat solutions injected from various 96-well plates and discrete autosampler vials
• The origin of the unexpected peak was traced back to the plastic 96-well plate (confirmed to be a plasticizer)
• Extraction of the contaminant/interference from the 96-well plate into the reconstitution solution takes about 30 minutes

Extraction of Plasticizer From Plate Over Time

Eliminating the Plasticizer Peak

Figure shows the amount of plasticizer in reconstitution solution (incubation period of one hour) using a non-treated plate (left pane) versus a plate sonicated with methanol(right pane)

Additional Metabolite

• Additional peak appeared in incurred monkey samples (left pane)
• Additional peak coeluted with metabolite of interest (right pane)
• Experiments comparing incurred samples and spiked standards verified that the additional peak did not affect the quantitation of metabolite of interest

RESULTS

• Addition of 2 minutes to the run time eliminated the matrix suppression effects arising from a late eluter
• Plasticizer peak originating from the 96-well plate eliminated using methanol sonication

CONCLUSIONS

Interferences that can affect data can come from a variety of sources including blank matrix, equipment and incurred samples. Ion suppression from coeluting compounds can cause inconsistent data and needs to be identified and resolved. Slight changes in chromatography can cause interference peaks to shift resulting in ion suppression. Therefore, the identification and elimination of interference peaks is a huge plus in ensuring data integrity.