mass spectrometry

Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 30–37

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Investigation of dried blood spot card-induced interferences in liquid chromatography/mass spectrometry Xiaohui Chen ∗ , Hongjuan Zhao, Panos Hatsis, Jakal Amin Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research Inc., 250 Massachusetts Avenue, Cambridge, MA 02139 USA

a r t i c l e

i n f o

Article history: Received 13 October 2011 Received in revised form 11 November 2011 Accepted 12 November 2011 Available online 23 November 2011 Keywords: Liquid chromatography/mass spectrometry Dried blood spotting Interference Ion-pairing FTA® DMPK-A card

a b s t r a c t 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. © 2011 Elsevier B.V. All rights reserved.

1. 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

Abbreviations: DBS, dried blood spotting; PK, pharmacokinetics; RT, retention time; WB, whole blood; SDS, sodium dodecyl sulfate; IS, internal standard; DMSO, dimethyl sulfoxide. ∗ Corresponding author. Tel.: +1 617 871 3818; fax: +1 617 871 4081. E-mail address: [email protected] (X. Chen). 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.11.015

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 [2,4–7]. 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.

X. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 30–37

2. Experimental 2.1. 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 dodecyl sulfate was obtained from Thermo Scientific (Rockford, IL). HPLC grade acetonitrile, methanol, formic acid (88% purity), and dimethyl sulfoxide were purchased from Fisher Scientific (Fair Lawn, NJ). Ammonium formate (99% purity) was supplied by Acros (NJ, USA). Ultra-high purity nitrogen was supplied by an in-house nitrogen system. Water was in-house Milli-Q water generated by a Millipore lab water purification system (Billerica, MA). The blank Sprague Dawley rat whole blood was supplied by Bioreclamation, Inc. (Hicksville, NY). FTA® DMPK-A, FTA® DMPK-B and FTA® DMPKC cards were supplied by Whatman plc (part of GE Healthcare) (Springfield Mill, UK). FTA® DMPK-A and DMPK-B cards are coated with different chemicals to lyse cells, denature proteins and/or inactivate pathogens. FTA® DMPK-C cards are uncoated. 2.2. Apparatus Harris punches and cutting mats were obtained from Ted Pella, Inc. (Redding, CA). Microman positive displacement pipettes and tips used for blood spotting on cards were obtained from Gilson (Middleton, WI). A Tecan liquid handling system (Model Freedom EVO® ) was obtained from Tecan Schweiz AG (Mannedorf, Switzerland). All experiments were performed on a HPLC-MS/MS system, which consisted of a CTC HTC PAL autosampler (Zwingen, Switzerland), an Agilent 1100 LC system with an integrated divert valve, and a Sciex API4000 (AB/Sciex, Canada) equipped with an electrospray ionization source. Post column infusion and infusion optimization of analyte MS/MS parameters were performed with a Harvard Pump 11 Plus syringe pump (Holliston, MA). 2.3. Sample preparation Primary stock solutions for all test compounds and glyburide as internal standard (IS) were prepared at 1 mg/mL in dimethyl sulfoxide (DMSO). Working solutions for each compound were diluted from the primary stocks with DMSO to 50 ␮g/mL. All the solutions were stored at −20 ◦ C and brought to room temperature before use. Test solutions were prepared fresh on the day of analysis by diluting the appropriate amount of the working solutions with blank Sprague-Dawley rat whole blood or the appropriate solvent to give a concentration of 500 ng/mL. Unless otherwise mentioned, this is the concentration of all test analytes used in this work. Extraction solution was prepared by mixing acetonitrile, methanol and water at the ratio of 1:1:1 (v/v/v) with or without 100 ng/mL glyburide. 2.4. LC/MS/MS analysis Chromatographic separations employed an ACE C18 , 3 ␮m, 30 mm × 2.1 mm i.d. HPLC column (MAC-MOD Analytical, Inc. Chadds Ford, PA), and a solvent gradient employing two separate solvent systems. The default solvent system used acidic mobile phases consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The second solvent system used non-acidic mobile phases consisting of 10 mM ammonium formate in water (A) and 10 mM ammonium formate in methanol (B). Following

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sample injection of 10 ␮L, the mobile phase composition was held at 2% B for 0.5 min. It was then increased linearly to 98% B over 1.5 min and then held at 98% B for another 0.5 min. This was followed by re-equilibration of the column at the initial mobile phase conditions. The flow rate was 0.7 mL/min, and the column was maintained at 40 ◦ C. The post column flow was diverted to waste for the first 0.5 min of each injection. LC/MS/MS was performed in either positive (5500 V) or negative polarity (−4500 V) depending on the test compound. The source temperature was 550 ◦ C. Gas 1 and Gas 2 settings for nitrogen were set to 60. The curtain gas and collision gas were also nitrogen and were set to 25 and 10 (arbitrary units). All test compounds were acquired with selected reaction monitoring (SRM) transitions with dwell times of 75 ms. Table 1 contains the chemical structure, molecular weight (MW), experimental pKa value [10–14], SRM transition (Q1 → Q3), declustering potential (DP, V) and collision energy (CE, V) of all test compounds. All data were acquired and quantified (peak integration) using Analyst® 1.4.2 software (AB/Sciex). Further data analysis and plotting were performed in Excel (Microsoft Corporation, Redmond, WA). All results are reported as the mean of three replicate analyses. 2.5. Recovery evaluation The DBS recovery evaluation consisted of three parts, Experiment A, B and C as described in detail in Fig. 1. Experiment A was a complete DBS workup with analyte extraction from dried blood spots on DBS cards. An aliquot of 7 ␮L of a 500 ng/mLwhole blood (WB) sample was applied in triplicate to FTA® cards and allowed to dry at room temperature for at least 1 h. The blood spot was cut with a 6 mm diameter puncher and placed into a 96-deep well plate. The use of a small whole blood sample volume and a 6 mm puncher ensured that the applied samples were completely punched out of the DBS cards. Automated analyte extraction was performed on a Tecan by adding 300 ␮L of extraction solution with IS to the plate, sealing the plate and sonicating for 5 min without heat. The samples were centrifuged for 10 min at 2844 × g and 4 ◦ C, and 125 ␮L of the supernatant was transferred to a new plate for LC/MS/MS analysis with the default acidic mobile phases. Experiment B was similar to Experiment A, however 500 ng/mL analyte was added to the sample after extraction. Experiment C of the evaluation used analytes prepared at the same 500 ng/mL concentration, but in neat solution, and served as a reference for Experiment A and B. Experiments B and C used 293 ␮L of extraction solution to compensate for the 7 ␮L of analyte added as a final step. The analytes’ matrix effect, recovery and process efficiency were calculated using the following equations: Matrix effect =



1−

Peak area of analyte from Experiment B Peak area of analyte from Experiment C



× 100 Recovery =

Peak area of analyte from Experiment A × 100 Peak area of analyte from Experiment B

Process efficiency =

Peak area of analyte from Experiment A Peak area of analyte from Experiment C × 100

FTA® DMPK-A cards were the primary DBS cards used in the recovery evaluation based on a previous investigation into the stability of a set of proprietary test compounds in the authors’ lab (data not shown). Surprisingly, RT shifting, ionization suppression and peak distortion were observed during the evaluation,

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X. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 30–37

Table 1 All test compounds’ structure, molecular weight (MW), experimental pKa , SRM transition (Q1 → Q3), declustering potential (DP) and collision energy (CE). Compound

Structure

Amitriptyline

MW

pKa

Q1 → Q3

277.2

9.4

347.1

DP (V)

CE (V)

278.2 → 105.2

81

33

4.5

348.0 → 158.0

125

20

388.2

8.3

389.0 → 201.0

125

40

392.2

NA

393.2 → 373.1

86

13

302.0

3.5

301.0 → 243.0

−100

−30

733.5

8.9

734.5 → 158.2

81

41

309.1

8.7

310.2 → 148.2

61

11

N NH2

H N

S

O

Cephalexin

N O O

Cetirizine

OH

N

OH

O O

N

Cl

OH

O OH

HO H

Dexa-methasone

F

H

O Cl

Cl

Ethacrynic acid

O

HO

O

O

Erythromycin

H N

O F

Fluoxetine

F

F

X. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 30–37

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Table 1 (Continued) Compound

Structure

MW

pKa

Q1 → Q3

501.3

9.5

501.8 → 466.2

234.2

8.0

235.1 → 86.2

320.1

4.5

608.3

515.3

DP (V)

CE (V)

125

40

61

21

319.2 → 275.1

−55

−22

6.6

608.8 → 195.0

100

60

1.4

514.4 → 80.2

−160

−84

O HO OH Fexofenadine

N OH

H N

N

Lidocaine

O OH

O

HO Mycophenolic acid

O

O

O

N

MeO

N

H

H

Reserpine

MeO

O

OMe

O

H O

OMe

OMe

OMe O

OH NH H

Taurocholic acid

H HO

H

O

H OH

S

O

OH

Fig. 1. Scheme of DBS recovery evaluation experiment.

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X. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 30–37

Table 2 Matrix effect, recovery, process efficiency and retention time in solution and in matrix for all test compounds on FTA® DMPK-A cards with acidic mobile phase condition. Compound

FTA® DMPK-A cards Matrix effect (%)

Amitriptyline Cephalexin Cetirizine Dexamethasone Ethacrynic acid Erythromycin Fluoxetine Fexofenadine Lidocaine Mycophenolic acid Reserpine Taurocholic acid a

66 18 80 2 97 99.6 58 40 65 99.6 68 61

RT (min) Recovery (%) 87 89 104 88 a

95 96 74 118 74 116 71

Process efficiency (%)

In solution

In matrix

30 73 20 86

1.77 1.46 1.78 1.77 1.94 1.69 1.80 1.76 1.43 1.85 1.80 1.75

1.91 1.46 1.89 1.77 1.94 1.86 1.92 1.89 1.84 1.85 1.91 1.72

a

0.4 41 44 41 0.3 37 28

Below detection limit of 1 ng/mL.

which triggered an in-depth investigation. Control recovery experiments were performed using the identical sample preparation and instrument conditions but with different types of DBS cards: DMPKB and DMPK-C cards. A control experiment was also performed using the identical sample preparation and instrument conditions but with non-acidic mobile phases containing 10 mM ammonium formate. 2.6. Effect of sodium dodecyl sulfate (SDS) concentrations on lidocaine The DBS interference was further investigated by examining the effect of SDS concentration on the retention and peak shape of lidocaine in two separate experiments. The first experiment was to vary the concentration of SDS using blank DMPK-A discs. Lidocaine samples were prepared in triplicate as described in Experiment A of Section 2.5. Prior to extraction, a pre-punched DMPK-A disc was placed in one sample and two discs in the second sample. The third sample had no additional blank DMPK-A discs added. All three samples were extracted, centrifuged and analyzed by LC/MS/MS with acidic mobile phases as described in Section 2.5. The second experiment was to titrate lidocaine solution with increasing amounts of pure SDS and analyze the samples by LC/MS/MS. The DBS interference was characterized in terms of RT shifting and peak shape. 2.7. SDS elution monitoring Certain samples from Experiment A of Section 2.5 were reanalyzed using two mobile phase systems, consisting of either 0.1% formic acid or 10 mM ammonium formate, to evaluate the elution of SDS off the chromatographic column. The SRM transition m/z 265.0 → 79.9 was monitored for SDS in negative ion mode. The problematic analytes were also simultaneously monitored to identify any co-elution with SDS, and thus explain the observed ionization suppression. 2.8. Post-column infusion A post-column infusion experiment was performed with erythromycin to further investigate the observed ionization suppression with 3 types of DBS cards. Erythromycin and glyburide (IS) were infused post-column into the mass spectrometer, and the following five samples were injected for LC/MS/MS analysis: (1) 50:50 0.1% formic acid/acetonitrile; (2) protein precipitated blank rat plasma; (3) a blank DMPK-C card extraction; (4) a blank DMPKB card extraction, and (5) a blank DMPK-A card extraction. The SRM transition of erythromycin was recorded to evaluate changes in MS response.

3. Results and discussion 3.1. 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 (1:1:1, v/v/v) as extraction solution except ethacrynic acid. Most of the compounds showed greater than 85% of recovery. However, there was a marked difference between test compounds in terms of DBS matrix effect. Some compounds such as cephalexin and dexamethasone demonstrated small matrix effects (18% and 2%), while others showed a significant drop in MS signal intensity compared with neat solution. The matrix effect for these compounds was greater than 40% of the intensity in a neat solution. For extreme instances such as erythromycin and mycophenolic acid, signal intensity in matrix was suppressed more than 1000 times. The process efficiencies were consequentially very poor for these compounds. Furthermore, retention time shifting was observed for certain compounds. Most of these compounds were retained on column longer in matrix than their neat solutions. Chromatographic peak distortion and splitting were also observed. The first peak was of lower intensity with the same RT as in neat solution, and the other was of higher intensity eluting at a later RT. In order to understand the cause of the observed RT shifting and peak distortion, control experiments were conducted with different types of DBS cards. Table 3 shows the matrix effect, recovery and process efficiency on FTA® DMPK-B cards (with different chemical coating) and FTA® DMPK-C cards (uncoated) respectively, using the

Table 3 Matrix effect, recovery and process efficiency with FTA® DMPK-B and DMPK-C cards and acidic mobile phase condition for the problematic compounds. Compound

Amitriptyline Cetirizine Erythromycin Fluoxetine Fexofenadine Lidocaine Reserpine

FTA® DMPK-B/DMPK-C cards Matrix effect (%)

Recovery (%)

Process efficiency (%)

10/21 60/8 19/19 25/21 76/12 8/−1 51/38

93/98 96/90 92/80 89/81 97/79 92/70 90/81

83/78 38/83 74/64 67/64 23/69 86/70 44/50

X. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 30–37

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Fig. 2. The effect of SDS concentration on the chromatography of lidocaine. (A) Neat solution; (B) lidocaine spotted on a DMPK-A card; (C) lidocaine spotted on a DMPK-A card with one extra blank DMPK-A disc added before extraction; (D) lidocaine spotted on DMPK-A card with two extra discs added before extraction.

compounds that had the most pronounced RT shift. Interestingly, RT shifting and peak distortion were not observed with DMPK-B and -C cards (data not shown). Ionization suppression was also greatly improved. For example, the matrix effect for erythromycin was significantly decreased to 19% in B- and C-cards from 99.6% in A-cards. The results confirmed that the constituents of FTA® DMPKA cards definitely played a role in the observed RT shifting and MS intensity loss. 3.2. Cause of retention time shifting with FTA® DMPK-A cards An effort was undertaken to look for the chemical composition of DBS cards provided by GE Healthcare. Unfortunately, this was complicated by the fact that the chemistry of these cards is of a proprietary nature. Inspection of the corresponding MSDS sheets revealed that the ingredients on FTA® DMPK-A cards are sodium dodecyl sulfate (<5%) and tris (hydroxymethyl)aminomethane (<5%) [15]. The ingredient on FTA® DMPK-B cards is guanidinium thiocyanate (30–50%) [16]. The vendor did not specify the basis for the percentage of ingredients, i.e. (w/w) or (v/v). The chemical structures and physical-chemical properties of all the test compounds were also carefully re-visited and evaluated. All the compounds that exhibited RT shifting with DMPK-A cards contained amine groups, and their pKa values ranged from 6.6 to 9.5 (Table 1). The default mobile phase used in the recovery experiment consisted of 0.1% formic acid, and the pH* was measured 2.6. It was also noticed that the larger the difference between the analyte pKa and the mobile phase pH*, the more RT shifting % was observed. The slope of a correlation plot of the test compounds’ RT shifting % vs. the compounds’ pKa was 1.4. The linear correlation coefficient was 0.9 (data not shown). The retention time shifting with DMPK-A cards could be best explained as a result of an ion-paring effect. Sodium dodecyl sulfate (SDS) coated on DMPK-A cards is a well known ion-pairing agent widely used in HPLC separation for poorly retained, positivelycharged compounds. When the pH of the mobile phase was at least

2 units lower than the pKa of the problematic compounds, the compounds’ amine groups became protonated to produce cations. The cationic compounds could then ion pair with SDS anions extracted from DMPK-A cards, resulting in reversed phase and ion pair retention mechanisms. This hypothesis was tested with the use of HPLC mobile phases modified with 10 mM ammonium formate. The pH* was measured 7.1. Basic compounds are expected to ionize to a smaller degree at this mobile phase pH although the exact amount depends on the particular test compound’s pKa . Given that the strength of association within ion pairs is a function of the degree of analyte protonation [17], this alteration in mobile phase pH had a dramatic reduction on ion-pair formation. As result, RT shifting was greatly alleviated for compounds such as amitriptyline and erythromycin. No RT shifting was observed for all other compounds (data not shown). Neutral molecules, such as dexamethasone, or acidic molecules, such as ethacrynic acid, will never become positively charged in acidic mobile phase conditions, which results in retention being dominated by reversed phase interaction with the C18 stationary phase. Moreover, the presence of SDS may reduce the magnitude of reversed-phase interaction for acidic compounds due to competition for the stationary phase and an ion exclusion effect [17]. Taurocholic acid, for example, eluted slightly earlier when spotted on DMPK-A cards compared to neat solution. This hypothesis was further tested with two experiments (Section 2.6) that demonstrated the effect of SDS concentration on lidocaine. The results of the first experiment using blank DMPK-A discs are shown in Fig. 2. Chromatogram A of Fig. 2 shows a retention time of lidocaine in a neat solution when there was no SDS in the sample. Retention followed conventional reversed-phase chromatography. Chromatogram B shows a broad and partially split chromatographic peak resulting from the presence of SDS in the form of one DMPK-A card disc. Addition of one and two more blank DMPK-A disc(s), further increased the amount of SDS in the lidocaine sample and resulted in chromatograms C and D,

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X. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 30–37

Fig. 3. SDS background from a blank injection (A) and SDS elution profile with mobile phases containing 0.1% formic acid (B) and containing 10 mM ammonium formate (C).

respectively. In going from chromatogram A to chromatogram B and C, the amount of SDS increased causing a decrease in the amount of retention due to reversed-phase interactions and an increase in ion pairing interactions. The addition of two discs of DMPK-A cards in chromatogram D showed that ion pairing became the dominant retention process. The results of this experiment are supported by the SDS titration experiment. Once again, lidocaine appeared as a single peak when no SDS was present. This peak diminished and a second peak appeared with increasing intensity and retention on column when pure SDS was added. When the amount of SDS continued to increase, the lidocaine peaks kept shifting to higher retention time in a similar pattern. Eventually, a mole ratio of 2.7 × 106 was reached and lidocaine appeared as a single peak at a higher and constant retention time (data not shown).

3.3. Ion suppression caused by SDS Retention time shifting was not the only manifestation of the SDS from DMPK-A cards. Significant ionization suppression of analyte was also observed, and was attributed to the presence of SDS, by means of SDS elution monitoring (Section 2.7 and Fig. 3) and a post-column infusion experiment (Section 2.8). Chromatogram A of Fig. 3 indicated SDS background from a blank injection. When mobile phases containing 0.1% formic acid were used, SDS began to elute as a front at approximately 1.79 min and lasted for more than a half minute due to the high concentration present in DMPK-A cards (Chromatogram B of Fig. 3). Aminecontaining compounds interacted with the SDS, which resulted in increased retention, and chromatographic peaks shifted into the SDS elution region. Analyte intensity was therefore suppressed due to the interference of the nonvolatile SDS in the electrospray process [18]. Furthermore, ion suppression may occur to any analytes if they elute within the SDS elution region even if they do not exhibit retention time shifting, e.g., ethacrynic acid and mycophenolic acid. When mobile phases containing 10 mM ammonium formate were used, SDS was observed to elute at 2.3 min as a much narrower peak (Chromatogram C of Fig. 3) and the test compounds eluted before SDS, thus mitigating ionization suppression.

The post-column infusion experiment (Section 2.8) performed with erythromycin further illustrated the ionization suppression caused by DMPK-A cards. There was a pronounced drop in erythromycin response during its peak elution window when a blank DMPK-A card extraction was injected onto the column. This clearly indicates co-elution of erythromycin with DMPK-A card components, thus resulting in the suppression in analyte response. In contrast, there was no interference with DMPK-B and -C card extractions for the compound (data not shown). 4. 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 upfront 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 coated DBS cards demonstrated by our work may serve as a guide to fill in the blank. It is quite common that drug candidates contain amine functionalities in their structures. We have now obtained a better understanding and prediction on how amine-containing analytes will behave chromatographically with DMPK-A cards and acidic mobile phases. RT shifting may be used as an indicator of ion pairing formation and an alert for consequential interferences caused by the ion pairing formation. Our work also demonstrates the need for an extensive assay validation and careful assessment of DBS interaction and interferences. In our case, it was ion-pairing of analytes containing basic amine groups with SDS, but different cards with different chemistries can produce unique interferences. Therefore, DBS users should always be on the lookout for analyte interactions with DBS card constituents. 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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