Investigation of dried blood spot card-induced interferences in liquid chromatography/mass spectrometry

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Abstract

Unique and remarkable interferences were observed when dried blood spot (DBS) sampling was used in conjunction with liquid chromatography/mass spectrometry (LC/MS) assays. In particular, chromatographic retention time shifting and chromatographic peak shape distortion were observed, along with a severe suppression of MS signal intensity. The type of DBS cards, and chromatographic conditions were investigated using the same set of test compounds to gain insight into these interferences. It was determined that a constituent of the DBS cards, primarily sodium dodecyl sulfate (SDS), was responsible for the interferences by means of an ion-pairing mechanism. SDS formed ion pairs with compounds containing basic amine groups, which resulted in increased retention on a C18 stationary phase, peak shape distortion and ion suppression. These interferences were greatly alleviated and/or completely overcome with non-acidic mobile phases and/or DBS cards with no SDS coating. To the best of the authors’ knowledge, this is the first in-depth report of interferences induced by DBS cards.

Highlights

► Severe interferences were observed when DBS sampling was used in LC/MS/MS analysis. ► It was determined that SDS coated on DMPK-A cards formed ion pairs with compounds containing basic amine groups. ► Non-acidic mobile phases and DBS cards with no SDS coating alleviated the interferences. ► DBS users should always be aware of possible analyte interactions with DBS card constituents.

Introduction

Dried blood spotting (DBS) as a sample collection technique has been used to screen newborns for errors of metabolism since Dr. Robert Guthrie first collected blood samples on filter paper in 1963 [1]. In spite of this, the technique was limited in qualitative analysis because analytical instruments did not have the required sensitivity. Improvements in the sensitivity of analytical instrumentation, and in particular mass spectrometers, have recently given a new life to DBS, allowing the pharmaceutical industry to leverage its many benefits.

DBS is an alternative sample collection method with advantages over conventional sampling such as lower sample volumes, simpler sample collection and handling, as well as easier storage and transportation logistics [2]. Small sample volumes require less blood from humans during clinical trials, fewer animals for preclinical studies and serial bleeding sampling can be performed for small animals such as mouse which improves the quality of pharmacokinetic (PK) data. These advantages allow for significant savings in the cost of drug research and development. In addition, regulatory authorities have acknowledged that blood is an acceptable biological matrix for drug exposure measurements [3]. Therefore, DBS coupled with LC/MS/MS has been gaining momentum in the pharmaceutical industry, and has been recently applied in many areas such as PK, toxicokinetic, drug metabolism and clinical studies [2], [4], [5], [6], [7].

In spite of the potential benefits of a new technique, its implementation into a bioanalytical workflow demands careful and rigorous characterization to ensure reliable and robust assay performance. Over the years, bioanalytical scientists have come to recognize matrix effects and interferences as a potentially significant source of error in quantitative LC/MS assays, and appropriate strategies have been reported to deal with the most common of these, e.g., ionization suppression, chromatographic/isobaric interferences, etc. [8], [9]. Herein, an interference unique to DBS was discovered and thoroughly examined as part of a larger effort in the authors’ lab to characterize DBS in conjunction with quantitative LC/MS assays for support of PK studies in drug discovery. A group of compounds was chosen which covered a wide range in physical chemical properties to study this interference. It was discovered that certain compounds exhibited matrix effects/interferences, some of which were expected, e.g., severe ionization suppression, and others were not, e.g., chromatographic retention time (RT) shifting and chromatographic peak shape distortion. The interferences were observed only when a certain type of DBS card was used. This manuscript describes the investigation of these unique DBS-induced interferences to better understand the cause of the interferences and RT shifting, and the impact on the application of DBS for PK quantification.

Section snippets

Reagents and materials

Lidocaine, erythromycin, dexamethasone (>98% purity), reserpine, cetirizine and glyburide were purchased from Sigma–Aldrich (St. Louis, MO). Fluoxetine hydrochloride and fexofenadine hydrochloride were purchased from ToCirs (Bristol, UK). Amitriptyline (98–102% purity) was purchased from Spectrum (Gardena, CA). Ethacrynic acid, cephalexin and taurocholic acid sodium salt were obtained from MP Biomedicals, LLC (Solon, Ohio). Mycophenolic acid was obtained from Calbiochem (Gibbstown, NJ). Sodium

Recovery/matrix effect characterization

The evaluation of DBS in the authors’ laboratory began with a routine assessment of extraction recovery and interferences (Section 2.5) using DMPK-A cards. Table 2 shows the matrix effect, recovery, process efficiency and the retention time from Experiment A, B (in matrix) and C (in neat solution) for all test compounds on FTA® DMPK-A cards with acidic mobile phase conditions.

The results in Table 2 indicated that all test compounds had recovery greater than 70% with acetonitrile–methanol–water

Conclusion

It is currently common practice that the choice of DBS cards is mainly determined by experiments and performance criteria, e.g., recovery and analyte stability improvement. There is minimum up-front guidance and prediction on which card to use without trial and error. Moreover, there is a paucity of knowledge and awareness of the ingredients on each type of card and what impact DBS cards may have in a bioanalytical assay. The significant and unique interferences caused by the ingredients of

Acknowledgements

The authors would like to thank James Robbins and Julie Hilton from GE Healthcare for their support during our DBS evaluation and Upendra Argikar for helpful discussions.

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