New Microfluidic-Based Sampling Procedure for Overcoming the

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Technical Note

A new microfluidic-based sampling procedure for overcoming the hematocrit problem associated with dried blood spot analysis Luc Alexis Leuthold, Olivier Heudi, Julien Déglon, Marc Raccuglia, Marc Augsburger, Franck Picard, Olivier Kretz, and Aurélien Thomas Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503931g • Publication Date (Web): 21 Jan 2015 Downloaded from http://pubs.acs.org on January 26, 2015

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Analytical Chemistry

A new microfluidic-based sampling procedure for overcoming the hematocrit problem associated with dried blood spot analysis

Luc Alexis Leuthold1, Olivier Heudi1*, Julien Déglon2, Marc Raccuglia1, Marc Augsburger3, Franck Picard1, Olivier Kretz1 and Aurélien Thomas3

1

Novartis Institutes for Biomedical Research, DMPK / Bioanalytics, Novartis Campus, 4056 Basel, Switzerland 2 DBS System, Route des Avouillons 2, 1196 Gland, Switzerland 3 University Center of Legal Medicine, Lausanne-Geneva, Rue du Bugnon 21, 1011 Lausanne, Switzerland

*Corresponding author: Olivier Heudi Tel.: (+41) 79 53 59 611 Fax: (+41) 61 696 85 84 E-mail: [email protected]

1 Environment ACS Paragon Plus


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ABSTRACT

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidicbased sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 µL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (n=3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The inter-spot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipette. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis.

Keywords: quantitative analysis; LC-MS/MS; dried blood spot (DBS); hematocrit effect; microfluidic.


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INTRODUCTION

Over the past decade, there has been an increasing interest in the use of dried blood spots (DBS) for quantitative bioanalysis

1-3

. DBS offers numerous advantages over

conventional blood sample collection. In addition, the procedure is clearly less invasive and is cost effective in terms of sample collection, shipment and storage 3. Since its introduction for phenylketonuria screening in newborns 4, DBS sampling has been successfully applied to multiple neonatal metabolic disorder screening programs and, more recently, to the monitoring of therapeutic agents pharmacokinetic

8,9

and toxicokinetic studies

10,11

5-7

and

. Although DBS is an interesting

sampling technique, certain issues are associated with its widespread application. It is well known that hematocrit (Hct) is one of the challenges linked with the use of DBS, especially concerning sample analysis

12-14

. As noted by De Kesel et al.,

several approaches have been proposed to address the Hct issue

15

. The different

proposed approaches have required additional analyses that are often difficult to implement in a routine laboratory or clinical setting. Interestingly, Fan and Lee have suggested to simply analyze the entire volumetrically applied spots instead of using fixed-size partial punches

16

. The entire DBS will only provide accurate data if a

controlled volume of blood is applied on the paper disc used for the analysis. Spotting an accurate volume on a card is straightforward with a manual pipette in an equipped laboratory with trained personnel. However, this method represents a daunting challenge when applying a fixed volume of blood onto the card within the course of clinical studies or at home in the case of unsupervised sampling. Adapted solutions for DBS or microsampling, including the development of multi-dispensers or a dried matrix on other materials

17

, are emerging to overcome this issue. DBS would



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significantly benefit from the development of a simple low-cost micro-sampling device that could be used for any biological fluid collection 18. In this study, we describe a novel and simple microfluidic-based device for blood collection on a DBS card. The device incorporates fixed-volume channels and utilizes a commercially available DBS card. A predefined volume of 5 or 10 µL can be directly sampled from a capillary droplet by the microfluidic device. The performance of the present sampling device was examined using two methods. The first method was to evaluate the reproducibility of the microfluidic sampling volume by quantifying the in vivo caffeine and paraxanthine levels from five extracted spots collected at two different time points (i.e., 4 h and 24 h) and by measuring, in vitro, Mavoglurant in spiked samples (at two concentrations of 2,000 and 15.0 ng/mL) at two different Hct levels, 26% and 62%. Mavoglurant was chosen as the test compound due to prior studies performed on its quantitative analysis from DBS samples (unpublished data). The second method was to investigate the effects of four blood Hct values (26%, 37%, 47% and 62%) on Mavoglurant determination in spiked blood (at three different concentrations) sampled on DBS cards. The results of the proposed experiments were compared to those obtained using the manual blood spotting on the card with a volumetric micropipette.

MATERIALS AND METHODS

Chemicals, reagents and stock solutions Caffeine

and

paraxanthine

were

purchased

from

Sigma–Aldrich

(Buchs,

Switzerland), and midazolam-d4 (used as the internal standard for paraxanthine and caffeine measurements) was purchased from Cerilliant (Round Rock, TX, USA).


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Mavoglurant and its internal standard [13CD3]-Mavoglurant were synthesized by Novartis (Basel, Switzerland). Acetonitrile and methanol (MeOH) were of HPLC grade from Merck (Darmstadt, Germany). Water was deionized and purified using a Milli-Q gradient system from Millipore (Bedford, MA, USA). The FTA DMPK-A and DMPK-C cards were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).

Microfluidic-based sampling The current microfluidic device allows the complete process required to ensure the microsampling of controlled volumes of blood and their transfer to predefined spot locations on commercially available DBS cards (Fig. 1). As displayed, a flexible DBS sampling card is inserted into a holding element, which includes a microfluidic plate (Fig. 1A). The latter contains sized capillaries, which are set in-line to allow multiple and independent samplings according to the number of spot locations (i.e., outlets) of the sampling card. Each channel is designed to induce a capillary effect, i.e., a driving force, on the blood sample, which enters the channel and produces a blood spot on the card. In operation, a fingertip blood drop for analysis is brought into contact with the inlet(s) (Fig. 1B). The blood then instantly travels through the channel towards the outlets (forming a right angle with an aperture oriented toward the card). In this study, the microfluidic channel is calibrated to accept the predetermined volume of blood fluid of 5 or 10 µL for the in vitro and in vivo assays, respectively. The standard use is to fill the four channels sequentially and without interruption with the same drop of capillary blood, i.e., at the same time point. We did not observe any drying during the time required to fill the first and fourth channels (typically less than two minutes) that would have affected the later transfer of the



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blood volume on the card. After the channels are filled, the cover is manually closed and clipped on itself to orient the paper card towards the outlet (Fig. 1C). The contact between the card and the blood at the outlets generates the blood spots on the card typically within 5 seconds. Once closed, the holder secures the paper card for drying (air drying at room temperature), handling, shipment and storage. Finally, the DBS card is removed from the holder slot, and DBS extraction can be performed (Fig. 1D). As intended for bioanalytical work, three additional spots are available for repeat analysis.

Blood sampling volume reproducibility The reproducibility of the in vivo blood sampling volume was evaluated by determining the precision data for the measurement of caffeine and its metabolite paraxanthine. After drinking coffee, finger pricks of blood from two volunteers were collected on five spots on a DMPK-C paper card with the 10-µL microfluidic sampling device, as described above; the samples were collected at 4 h and 24 h for the first and second volunteers, respectively. All of the DBS samples were allowed to dry at room temperature for at least 1 h prior to analysis. The entire spots were punched and analyzed as previously described by Bosilkovska et al. 19. For comparison, 10 µL of capillary blood was simultaneously collected with a micropipette and, spotted on a DMPK-C card, and the DBS samples were analyzed as described above. The in vitro accuracy and precision were determined on blood spiked with Mavoglurant at two different concentrations and Hct levels. A drop (i.e., ≥ 20 µL) of the sample to be analyzed was deposited on Parafilm M® with a volumetric pipette. The blood was sampled as described above using the 5-µL microfluidic device with a DMPK-A card. The entire DBS spot was punched and extracted by adding


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MeOH/water (1:1) (200 µL) containing the internal standard ([13CD3]-Mavoglurant (50 ng/mL)) to each Eppendorf tube. After mixing for 30 min, the supernatant was transferred to a 96-well-plate, and 50 µL was injected into the chromatographic system for Mavoglurant analysis (see Supporting Information for details).

In vitro Hct effect on Mavoglurant analysis Fresh human blood (containing the anticoagulant EDTA) was centrifuged at 4°C for 10 min at approximatively 1,500 g, and plasma was separated from the erythrocyte pellet. The latter was gently mixed with the collected plasma to obtain blood batches with Hct levels of 26%, 37%, 62% and 82%. Each blood sample was spiked in triplicate with Mavoglurant at three different concentrations (2,000, 800 and 15.0 ng/mL), and the resulting blood was sampled and handled as described above (see the in vitro portion). The Hct for the blood used for the calibration standards (Cs) and quality control samples (QCs) (reference) was 47%.

Preparation of the Cs and QC samples and Mavoglurant analysis The Cs and QC samples for Mavoglurant were freshly prepared. The Cs were prepared from the same pooled human blood batch, yielding Cs concentrations of 2,500 (ULOQ), 1,000, 100, 50.0, 10.0 and 5.00 (LLOQ) ng/mL. QC samples were prepared from pooled fresh human blood batches. The Cs and QC samples (5 µL) were spotted on FTA DMPK-A paper cards using a volumetric micropipette. The analyses were performed using a LC–MS/MS system (see Supporting Information for details).



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RESULTS

Blood sampling volume reproducibility The precision data, expressed as CV (%) (n=5), of the extracted punch from the DBS samples submitted to the in vivo microfluidic collection procedure were below 9.0% for caffeine and its metabolite (Table 1). In addition, a good correlation was observed between the microfluidic device and the volumetric micropipette (ρs=0.966, P

New Microfluidic-Based Sampling Procedure for Overcoming the

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