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A Book-type Dried Plasma Spot Card For Automated Flow-through Elution Coupled With On-line SPE-LCMS/MS Bioanalysis of Opioids and Stimulants in blood Imelda Ryona, and Jack Henion Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03691 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

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Analytical Chemistry

A Book-type Dried Plasma Spot Card For Automated Flow-through Elution Coupled With On-line SPE-LC-MS/MS Bioanalysis of Opioids and Stimulants in blood Imelda Ryona and Jack Henion* Q2 Solutions, 19 Brown Road, Ithaca, NY 14850 Despite many benefits including simple point-of-care sample collection, reduced costs, and simplified shipping and storage, dried blood spot (DBS) techniques have faced adoption resistance due to factors such as the hematocrit (Hct) effects and the established preference for bioanalysis of plasma rather than whole blood. One way to potentially circumvent these challenges is to adopt the concept of dried plasma spot (DPS) techniques. One approach to accomplishing this is through an on-card red blood cell (RBC) filtration to generate plasma from whole blood without the need for centrifugation. In this report, a book-type DPS card has been developed and validated by employing fully automated flow-through elution coupled with on-line SPE-LC-ESI-MS/MS for the quantitative determination of four representative opioids (Morphine, Codeine, Oxycodone, Hydrocodone) and five stimulants (Amphetamine, Methamphetamine, 3,4-Methylenedioxymethamphetamine (MDMA), Phentermine, and Mephedrone) in one method using their corresponding deuterium labeled analogues as internal standards. Method validation results showed good linearity (R2≥0.9963) ranging from 5 to 1000 ng/mL Intra-day and inter-day precision and accuracy were within the acceptable limits at four quality control (QC) levels. Extraction recovery was ≥87.9% at both the lower limit of quantitation (LLOQ) and the upper limit of quantitation (ULOQ) along with acceptable selectivity and sensitivity. DPS on-card short-term stability was compound-dependent and storage-dependent. The additional benefits of the validated book-type DPS card include a wider applicability range of Hct (30% to 60%), automated on-line analysis compatibility, and a higher plasma volume yield.

INTRODUCTION In bioanalysis, microsampling typically refers to sampling techniques that utilize low microliter volumes such as 10 to 100 µL biological fluid1. Sampling at such reduced volumes is less invasive and offers a plethora of benefits such as minimization of biological and chemical hazards in sample handling and processes, simplified sample collection and processing thus facilitating the feasibility for point of care service. All of these benefits result in significant cost savings. Dried blood spot (DBS) techniques were initially introduced for the application of newborn screening back in 19632 In recent years, the techniques, which encompass the benefits of microsampling features, have garnered considerable interest not only in pharmaceutical industries but also in the field of anti-doping 3,4. Despite its many benefits, one of the limitations in DBS is the use of whole blood as the matrix. Historically, plasma or serum has been the preferred matrix over whole blood thus leading to clinical reference data established from either plasma or serum. Hence, bridge experiments using plasma/serum matrices are often required to compare and validate any results generated in whole blood matrix, which incurs additional cost and work for the analytical laboratory. Plasma/serum is also the preferred matrix over whole blood in spectrophotometric immunoassays where hemoglobin may cause interferences in assays 5,6. Another limitation in DBS analysis is the hematocrit

(Hct) issue where different Hct level in blood resulting in different spot sizes. To overcome Hct issues, several strategies have been proposed which include whole spot analysis 7, dried plasma spots (DPS) analysis 8-10, the use of a non cellulosebased collection material made of layers of woven polyester 11 , and prediction of Hct level in blood by measuring endogenous compounds in blood such as potassium and hemoglobin 12-14 . Capillary microsampling (CMS) 15 techniques may also be a way to resolve Hct issues. This approach uses centrifugation to fractionate plasma from whole blood followed by conventional sample preparation and LC-MS/MS analysis of the resulting micro samples. Alternatively, a non DBS device which is the volumetric absorbance microsamping (VAMS), also known as a MitraTM tip, can also be adopted to overcome Hct-bias issues 16,17 although this technique also faces the concern of sampling whole blood as the matrix. Of the proposed approaches, a dried plasma spot (DPS) analysis could potentially provide a practical alternative since it offers similar microsampling benefits of DBS techniques but from plasma. Previous studies have reported the application of a DPS card where fractionation of plasma from whole blood does not require an external device such as a centrifuge. This was accomplished by filtering whole blood through an asymmetric RBC membrane filter that captures RBC from the whole blood while allowing plasma to flow through to a membrane filter onto a cellulose based paper substrate 8-10. Unfortunately, limitations were noted on its applicability beyond a

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narrow range of Hct (40% to 55%) 8, the lack of fully automated analysis 9,10, and low yield of plasma volume 8-10. In DBS or DPS, extraction of dried spots can be very laborious and impractical if performed by manually punching spots followed by off-line solid-phase extraction (SPE) or liquidliquid extraction (LLE). To improve efficiency, a fully automated on-line analysis employing flow-through elution of the dried sample spots coupled with on-line SPE and LC-MS/MS analysis can be adopted and is proposed in this study and reported by others 7,8,18-21. In this work, a book-type DPS card has been developed providing some benefits beyond those previously reported. These include simplicity of sample application, fully automated on line analysis, successful applicability to a wider range of Hct (30% to 60%), and higher plasma volume yield. The functionality of the card was evaluated and validated by employing fully automated flow-through elution coupled with on-line SPE-LC-ESI-MS/MS analysis for determination of four opioids (Morphine, Codeine, Oxycodone, Hydrocodone) and five stimulants (Amphetamine, Methamphetamine, 3,4Methylenedioxymethamphetamine (MDMA), Phentermine, and Mephedrone) using their corresponding deuterium labeled analogues as internal standards. These drugs-of-abuse were selected for this study for introducing the potential application of DPS analysis in the field of anti-doping where the simplicity of point-of-care collection can be of benefit for any unscheduled in and out of competition drug testing.

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Eppendorf tubes. Fortified samples were subsequently incubated at 37 ᵒC with 200 rpm agitation for 30 min. Eight-point calibrators were 5, 10, 25, 50, 100, 250, 500, and 1000 ng/mL while the quality control (QC) samples were 5 ng/mL at the lower limit of quantitation (LLOQ) QC, 15 ng/mL at Low QC (LQC), 300 ng/mL at Medium QC (MQC), and 900 ng/mL at High QC (HQC). After incubation, samples were set aside at room temperature for 30 min prior to DPS preparation using the book-type DPS cards as shown in Figure 2. Briefly, with the card in a closed position, an aliquot of blood (10 to 50 µL) was applied to the upper membrane disk and the card remains in the closed position for 3 min to complete the filtration process. Testing a range of 1 to 4 min filtration time, a minimum of 3 min was required to yield good analytical precision. When the card was closed for 6 min, no hemolysis was observed. Next, four paper clips were removed to retrieve the paper substrate containing filtered plasma. The paper substrate was then affixed to an appropriate paper card stock which was a Perkin Elmer 226 card with the sampling window removed. Any card stock that meets the system automated configuration criteria can be used.

EXPERIMENTAL SECTION Chemicals, reagents, and materials. Detailed information can be found in the supporting document.

Figure 1 – The construction process of the book-type DPS card. The numbers indicated the assembly order no. 1 to 6 which were described in details in the ‘construction of the DPS card’ section.

Construction of DPS card. Adobe Illustrator program was used to design and set the dimensions of the materials. To prepare the RBC filter disks, the iPOCDX membrane filter sheet was punched using Harris Uni-Core punch (Amazon, USA). The bottom support disks, which used the card stock material, were prepared using an Osborne arch punch (Amazon, USA). As shown in Figure 1, after preparing each layer of the materials, the card was assembled by first placing the polyester layer on the inner side of the upper card stock (1) followed by punching the equally spaced four holes on the upper card stock through the polyester layer (2). The bottom elevated supports were made of two round disks of the card stock material affixed on the inner surface of the bottom card stock using an adhesive tape (3). The cellulose-based paper substrate was then placed directly on top of the elevated bottom supports (4). This book-type DPS card utilized two slightly different RBC filter membrane disks: one larger and one smaller and the final step was to place these two disks layered on the card. The larger disks were inserted in between the inner side of the upper card stock and the polyester layer and aligned with the opening holes (5). The book-type card was then held closed using four paper clips. Finally, the smaller membrane disks were placed on the outer side of the upper card stock where the opening holes are located (6).

Hematocrit effects. A hematocrit measuring device - the StatSpinTM CritSpinTM from Thermo Fisher Scientific (Waltham, MA, USA), was used to measure the Hct level of whole blood samples. Blood was placed into a capillary tube and spun in the device at 13,700xg for 2 min. After centrifugation, the Hct level was measured using the device. To prepare blood with 30%, 45%, and 60% Hct, 1 mL of blood was placed into a 2 mL LoBind Eppendorf tube and spun at 3000xg for 3 min. With the measured level of the initial Hct, calculation was carried out to determine how much plasma was to be added to or removed from the 1 mL centrifuged blood in order to achieve the desired Hct levels, which were confirmed by the Hct device. After adjusting the plasma volume, sample were then gently mixed on a vortex mixer and 500 µL were transferred to 1.5 LoBind Eppendorf tubes for standards fortification according to the sample preparation method described above. Evaluation of Hct effect was performed at LLOQ QC and HQC. The calibration curves were prepared using blood with 42% Hct. Variable sampling volume. As described above, the construction of the book-type DPS card utilizes two RBC filtration disks (iPOCDX X and S/G membrane filters) to sequentially and efficiently filter out RBC for up a 60% Hct level. The size of the disks can be determined depending on the application volume. The critical point is to avoid over fill or under fill of the disk with blood. Over fill will over saturate filtration capacity resulting in whole blood overflows through the edge of the disk. If under filled, a fraction

Preparation of working solutions. Detailed information can be found in the supporting document. Sample preparation. All samples were prepared by fortifying 500 µL of blood containing Na2EDTA with 10 µL of the working solution (a mixture of all studied analytes) in 1.5 mL LoBind

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of plasma will be retained in the disk resulting in less available

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plasma

Figure 2 – Photographs of the book-type DPS card and an on-line card stock along with its application steps to generate hemolysis free dried plasma spots from blood with 30%, 45%, and 60% Hct. Step 9 depicts the 3 clamp-head washing positions performed in between samples to minimize carry-over. and may result in incomplete saturation of plasma collection substrate which will then result in analytical imprecision. To evaluate the range of application volume per disk size, the combinations of 4 mm and 6 mm, 5 mm and 7 mm, 5 mm and 9 mm, and 5 mm and 11 mm were evaluated for 10, 12.5, and 15 µL, 15, 17.5 and 20 µL, 20, 27.5, and 35 µL, and 35, 42.5, 50 µL blood, respectively. Three replicates (three dried plasma spots) per applied volume were prepared using volunteers’ blood with 45% Hct fortified at the HQC level. Calibration curves (n=2) were prepared using the 5 mm and 7 mm combination for 20 µL applied volume.

plasma was prepared using the book-type card with 20 µL whole blood. Method Validation. Adopting the US FDA guidelines 22, linearity, precision, accuracy, carry-over, selectivity, recovery, and stability were investigated to validate the functionality of the developed DPS card for a fully automated on-line analysis. Experimental information can be found in the supporting document. The Automated Flow-through Spot Elution and On-line SPE. Detailed information can be found in the supporting document.

Plasma volume yield and centrifuged and filtered plasma comparison. Plasma spots were generated using the DPS card built with the above four combinations for 15, 20, 30, and 50 µL blood respectively. Weight was measured before and immediately after application. Calibration curves (n=2) were prepared by spotting centrifuged plasma (3, 5, 10, 15, 20, and 30 µL) onto a small piece of paper substrate which was weighed prior to and immediately after spotting. The curve of plasma volume versus weight was plotted. To evaluate if analyte concentration is affected by the elution position, center and peripheral elution positions in a spot were compared. Centrifuged DPS was obtained by pipetting 5 µL centrifuged plasma directly to the paper substrate while filtered

LC-MS/MS. Detailed information can be found in the supporting document. RESULTS & DISCUSSION Schematic illustration of the developed DPS card. Figure 3 depicts the construction details of the book-type DPS card. As shown in Figure 1, the card consists of a folded card stock (0.350 mm thickness x 76.2 mm W x 50.8 mm L) and has a function to support layers of different materials. For the purpose of illustration, the upper and lower layers of the folded card stock are shown in detached form in Figure 3. Viewed from the upper surface, the two iPOCDX filter disks (layers 1 and 3) feature asym-

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metric pore dimensions. The upper disk (layer 1) was the thinner iPOCDX X membrane (35 µm top, 5 µm bottom) and positioned in close contact directly above the lower disk (layer 3) which was iPOCDX S/G membrane disk (35 µm top, 2.5 µm bottom). When combined, the two disks can sequentially and effectively filter out the RBC from whole blood with Hct up to 60% without showing any evidence of hemolysis. The sizes of the two disks (upper - 4 to 5 mm diameter and bottom - 6 to 11 mm diameter) were determined accordingly to accommodate variable applied volumes.

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S/G membrane. The cellulose-based paper substrate (layer 5) was an Ahlstrom grade 601 paper (0.190 mm thickness X 76.2 mm W x 25.4 mm L) made from cotton linter pulp. The bottom support (layer 6) was made of a stack of two round disks obtained from the card stock (11 mm diameter bottom disk and 5 mm diameter top disk) held in place and in alignment with the X and S/G membrane filter disks by an Avery 8665 adhesive tape (76.2 mm W x 25.4 mm L). The function of the bottom support is to ensure a close physical contact between the paper substrate and the S/G membrane. This book type DPS card has the capacity to produce up to 4 plasma spots per card as shown in Figure 3. In this work, the card was constructed to produce 3 plasma spots because the employed 4 mm clamp size has the clamp-head washing positions configured at spot position 4 as shown in Figure 2 step 9.

A non-uniform filtration rate was observed at different Hct levels. To circumvent this issue, a pinhole was punctured through the upper iPOCDX X membrane to create a small through-hole which provides a more uniform flow. Alternatively, the iPOCDX X membrane bottom pore size (5 µm) and density (vendor proprietary information) could potentially be modified and optimized. This alternative may be investigated to provide future improvements in the card through a collaborative effort with a manufacturer. The upper card stock (layer 2) was punched to create cardstock openings which match the diameter of the iPOCDX X membrane. The function of these openings was to position the iPOCDX X membrane securely to be centered above the iPOCDX S/G membrane. An Ahlstrom 3256 (layer 4) polyester layer (0.058 mm thickness x 76.2 mm W x 25.4 mm L) was punched to create open holes of 5 mm diameter and held in place by an Avery 5667 easy-peel adhesive tape. The punched holes were intentionally set to be smaller than the S/G disk (6 mm to 11 mm) for holding the membrane disk in place allowing a direct contact between the disk and paper substrate through the 5-mm opening area.

Method development. Major challenges for developing this type of fully automated on-line analysis have been discussed elsewhere 7,23 . Despite the benefit of full automation, such a setting poses a few challenges. For example, higher organic solvent composition in the flow-through elution solvent may provide a cleaner extract and better extraction efficiency especially for non-polar analytes; however, the loading of higher percentages of organic solvent onto the SPE cartridge will likely decrease the retention of the analytes on the SPE cartridge, which may compromise subsequent LC/MS/MS results. Since an LC gradient is used to elute analytes from the SPE cartridge, the elution solvent is limited to the applied LC gradient. These issues related to automated on-line setting need to be addressed during method development. Recently, fully automated on-line analysis has been developed for quantitation of opioids 7 and stimulants 23 in DBS samples. In this work, the two independent DBS methods were transferred into a single DPS method for quantitation of both opioids and stimulants. Unlike DBS analysis where determination of stimulants could not be performed using ESI source due to signal suppression 23, transferring the method to a DPS analysis enables quantitation of stimulants in ESI without encountering signal suppression issue. This may be due to a cleaner matrix of DPS compared to DBS samples. When compared to DBS analysis 23,DPS flow-through elution required less organic composition and less volume to attain comparable recovery. The C2 and C8 SPE cartridges were compared and C8 cartridge was selected because it showed better overall detection sensitivity (especially for Morphine) and chromatographic performances.

When a drop of blood was applied to the filter disks, visual inspection showed rapid diffusion and absorption of blood through the disk in less than 30 seconds. It was also noted that plasma diffused more rapidly than RBC at the horizontal plane resulting in plasma overflow through the edge of the iPOCDX S/G membrane disk and onto the paper substrate. Without the polyester layer, the initiation of plasma absorption and spreading started from the edge of the filtration disk onto the paper substrate and thus resulted in inhomogeneous shape of plasma spot such as a semi-full circle or horseshoe-shaped spots. With the polyester layer designed to have opening holes of 5 mm, this prevented direct contact between the edge of the 7-mm S/G disk and paper substrate. The thinness of the polyester plays an important role by maximizing the close contact between the paper substrate and the

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Figure 3 – Schematic illustration of the DPS card with each layer of material separated from its neighbor showing the thickness, the type of the material, and its function. This card can produce up to 4 plasma spots on a card and generate 0.303±0.007 µL plasma per µL blood. Therefore, analysis can be run in the order of low to high concentrations. If concentration of a sample cannot be predicted, a blank should be incorporated before and after the sample (≥20% of the LLOQ signal intensity) carry-over signals were observed for amphetamine, methamphetamine, MDMA, and phentermine.

Method validation. Regulatory guidelines define the acceptance criteria as within ±15.0% relative error (RE) for accuracy and ≤15% coefficient of variation (CV) for precision for all QC levels except the LLOQ QC which has ±20.0% RE and ≤20% CV 22. %RE was calculated by (measured mean / nominal value) - 1 x 100) and %CV was (standard deviation/mean) x 100.

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Linearity, Recovery, Precision, and Accuracy. Calibration curves (n=2) were plotted using analyte/IS peak area ratio using 1/x2 weighted linear regression over the quantitative range (Table 1). The curve range covers both the therapeutic and toxic ranges for the studied compounds 24,25. In DBS and DPS analyses, introduction of IS can be performed in various ways as described previously 21,26. In this study, the IS was introduced to the flowthrough elution solvent; hence, the IS could not compensate for any on card extraction discrepancies such as analyte recovery bias and Hct related recovery bias. One way to circumvent this issue is to optimize assay recovery as noted by Abu-Rabie et al. reporting no observable Hct-related recovery bias for assay recovery of over 90% 21. Recovery for this assay was ≥90.0% for all except for amphetamine which was 87.9% as shown in Table 1. Intra-day precision and accuracy results also showed acceptable values (Table S2). Inter-day precision and accuracy were calculated using the average intra-day values (n=3) and results showed passing the acceptable criteria at all QC levels for all nine analytes except for codeine at the LLOQ QC level which was 23% (Table S3).

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min Selectivity and carry over. Selectivity was assessed by evaluating the double blank (matrix blank without IS), blank (matrix Figure 4 – SRM LC/MS chromatograms from fortified blood blank with IS), and fortified samples at the LLOQ and ULOQ samples containing morphine (1), codeine (2), oxycodone (3), levels from six individual matrix lots (six different human whole amphetamine (4), hydrocodone (5), methamphetamine (6), bloods). As shown in Figure 4, the double blank sample showed MDMA (7), phentermine (8), and mephedrone (9) for A) blank negligible carry-over IS signals (about 1% of the total IS intensisample (matrix blank without IS), B) zero sample (matrix blank ty) while blank sample showed non-detectable analyte signals. with IS, showing only analyte signals), C) LLOQ sample (matrix Good chromatographic resolution and detection were observed at fortified with 5 ng/mL standards) and D) their deuterated IS. Septhe LLOQ level for all nine analytes. Inter-lot precision and accuaration of isomers codeine and hydrocodone and isomers methracy at LLOQ and ULOQ were within the acceptance criteria for amphetamine and phentermine may be observed. With the wash all analytes (Table S3). Carry-over was evaluated by running a procedure, the LC MS/MS cycle time per run increased from 4.3 blank spot (a blank card with no sample spot) after the ULOQ to 6.2 min. calibrator. Unacceptable A variety of solvent washes (acidic and basic H2O:MeOH ACN:IPA 2:4:3:1 v/v and 100% MeOH) and Hematocrit effects. As shown in Figure 5, the precision and acprocedures (different volumes, speed, and order of the consecucuracy of various Hct samples were comparable although there tive washes) were tried and results showed improvement but were a trend of negative bias for 60% Hct at LLOQ QC and a failed to reduce the carry-over to the acceptable levels. Thus, the positive bias for 60% Hct at HQC level. carry-over issue was mitigated by employing two sequential blanks (no plasma spot) after the ULOQ calibrator. In pharmacokinetic (PK) studies, relative concentrations of the samples can be predicted such as lower concentrations at the end of the PK curve. Table 1 – Linearity and Recovery of Four Opioids and Five Stimulants Therapeutic Toxic Calibration range range Range R2 %Recovery (± CV) Analyte (ng/mL (ng/mL (ng/mL blood) LLOQ ULOQ plasma)24,25 plasma)24,25 (LLOQ – ULOQ) Opioids Morphine 10 - 100 100 5 - 1000 0.9968 97.6±0.3 97.0±0.0 Codeine 30 - 250 >500 5 - 1000 0.9988 97.8±2.0 97.0±0.0 Oxycodone 5 - 100 200 5 - 1000 0.9963 93.2±2.7 97.3±0.0 Hydrocodone 10 - 50 100 5 - 1000 0.9978 96.6±2.3 97.5±0.2 Stimulants 20-150 200 5 - 1000 0.9982 92.4±4.7 94.6±0.1 Methamphetamine 10-50

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100-350 >500 5 - 1000 MDMA 30-100 5 - 1000 Phentermine 900 50-100 >100 5 - 1000 Mephedrone Blood to plasma ratios range from 0.6 to 1.5 27 and log P values range from 0.3 to 2.4. Compared to a previously developed card 8, this book-type DPS card appears to provide a wider range of Hct applicability which is accomplished through a design incorporating two RBC filter

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93.6±3.8 92.2±4.7 96.6±0.9

96.9±0.0 95.7±0.1 97.5±0.0

accuracy was independent of spotting volumes at 20, 25 and 30 µL centrifuged plasma using the SAFECAP® capillary tube 28. At 10 µL, there was a negative bias 28. The type of cellulose paper substrate used in that study was the DMPK C card which is nearly 2.5 times thicker than the paper substrate (Ahlstrom Grade 601) used in the book-type DPS card. When a thicker paper substrate is employed, the spreading and penetration of the plasma throughout the paper may undergo incomplete penetration described by Henion et al. 29. If so, inaccuracy will be more of an issue at a smaller volume than a larger volume. Center versus peripheral spot positions were also evaluated at 4 different application volumes (15, 20, 35, and 50 µL) producing different spot sizes. Results in Table S4 showed no differences in the measured levels for all analytes between the center and peripheral positions at four different applied blood volumes or various plasma spot sizes. Noticeably, results reported in Table 2 and Table S4 support the rationale that plasma consistency is independent of both the Hct level in blood and the applied volume. Although the card provides the option of variable sampling volume, it may have limitations for direct finger-prick sampling where volume cannot be measured. To evaluate this, we tested the card equipped with 5 mm X membrane and 9 mm S/G membrane for a direct finger prick sampling. Results showed that the card can be successfully used for direct finger-prick application as hemolysis-free plasma spots were observed as shown in Figure 6. Our observations suggest that the direct finger-prick sampling does not produce overfilling of the membrane. However, when sampling position no. 3, the drop of blood was significantly smaller than the previous ones. As a result, a smaller plasma spot (ca. 6 mm) was noted as shown in Figure 6C. The spots were not analyzed in this case as it was not an incurred subject. However, based on the results observed in Table 2, there should not be inaccuracy when analyzing smaller plasma spots.

disks. Figure 5 – Precision and accuracy for DPS analysis using blood with 30%, 45%, and 60% Hct (n = 3) at the LLOQ (A and B) and high QC (C and D). The red line indicates the maximum acceptable criteria of ≤20.0% RE and CV at the LLOQ and ≤ ±15.0% RE and CV at high QC. No hematocrit bias was observed at the Hct range of 30 to 60%. Variable sampling volume. In the application of the book-type DPS card described in this report, accurate and precise quantitation can be obtained through variable sampling volume. Unlike whole blood, plasma consistency is independent to the Hct level. Hence, plasma generated from 30% or 60% Hct blood will have the same spreading consistency on the paper substrate and thus produce homogenous spots. Homogenous spots do not equate to equal spot dimension. It refers to homogenous saturation of plasma within a spot regardless of the spot size. In the current study, a 4-mm partial spot analysis was employed. With a partial spot analysis, a 4-mm spot area is sampled within a dried plasma spot. So, if a plasma spot is homogenous, accuracy of the results should not be affected by whether the 4-mm sampling area was taken partially from an 8 mm or a 14 mm plasma spot and whether the 4-mm sampling area was acquired from the center or the peripheral region within a dried plasma spot. To support this postulate, variable blood volumes ranging from 10 to 50 µL were evaluated using the book-type DPS card generating plasma spot sizes of ca. 8 to 14 mm in diameter. As indicated in Table 2, the book type DPS cards were customized to have different membrane sizes of the X and S/G disks to accommodate variable applied volumes. Quantitation was performed with a calibration curve constructed from 20 µL of blood using DPS cards with 5 and 7 mm X and S/G disks respectively. Comparable accuracy at HQC was observed among variable volumes as shown in Table 2. Although a majority of %RE were within the acceptance limit (≤ ±15%), trends of negative biases were observed from spots produced with 20 µL blood as shown in Table 2. When a pipette is used to apply blood to the card, these biases can be corrected by preparing the calibration curve using volume that is comparable to the applied volume. A previous study has reported similar results where quantitative

Figure 6 – Demonstration of a direct finger-prick sampling using the book-type DPS card where A) a drop of blood was applied to the sampling spot, B) the card remained in closed position for 3 min, and C) the card was opened after 3 min showing hemolysisfree filtration of plasma.

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The book-type DPS card versus the existing DPS cards. The two closest existing DPS cards to that described in this report are the Noviplex card, which is commercially available from Novilytic LLC 10 and the ‘auto DPS card’ previously reported by our laboratory 8. In general, the conceptual design of these two cards and our DPS card is similar as each of them employs an on-card

asymmetrical membrane filtration technique to separate RBC from plasma. However, the card structures and production of plasma in each card format are different. The Noviplex card uses a proprietary membrane material while the auto DPS card uses Pall Corporation Vivid GR

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Table 2 – Flexible Sampling Volume: Different Range of Blood Application Volume Using Different Size of RBC Membrane Filters

Membrane sizes referred to the X and S/G membrane disks in diameter. For quantitation, calibration curve was built using book-type DPS card with 5 and 7 mm combination and 20 µL whole blood.

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which is very similar to the iPOCDX membranes. With the two-membrane design, iPOCDX membranes were selected due to the availability of a thinner X membrane. To produce plasma spots, auto DPS card requires an external filtration device 8 while Noviplex card does not. However, the sample handling for Noviplex card is tedious as it requires a pair of tweezers to remove the small 2-mm disk and manually transfer it for further sample extraction as it is not compatible for automated analysis. While the yield of plasma volume by auto DPS was not determined, the Noviplex card requires a minimum of 25 µL blood to produce about 2.5 µL plasma 10. That is 0.100 µL plasma per µL blood. The book-type DPS card produces a range of plasma volume (4.6 to 14.7 µL depending on the applied blood volume averaging 0.303±0.007 µL plasma per µL blood (Table S5). Finally, to evaluate if the quality of the book-type filtered plasma is comparable to centrifuged plasma, DPS samples obtained from these two techniques were compared at 2 QC levels. Results in Table S6 show comparable precision and accuracy of the two different techniques although centrifuged DPS showed slightly higher measured values especially for Hydrocodone. This may be due to differences in paper substrate saturation where one is through a direct pipette application while the other is through filtration via 2.5 µm pore sizes.

forward. However, future studies on the application of incurred samples obtained via a finger prick sampling option is warranted. Bridge experiments to compare results from DPS and conventional plasma sampling, expansion of applications to a wider variety of drug classes, and inter-laboratory testing are necessary for future studies and will help demonstrate the potential robustness and practicality of the book-type DPS card described herein. ACKNOWLEDGEMENTS This project was funded by the Partnership for Clean Competition and was conducted using the consignment of an LC-MS/MS system by Shimadzu and a DBS autosampler coupled to an online SPE system provided by Spark Holland. The authors would like to acknowledge Ahlstrom Corporation and International Point of Care Inc. for donating their materials. The authors would also like to acknowledge Dr. Ruth Verplaetse for valuable discussions and technical support. SUPPORTING INFORMATION. Supporting materials include some standard information such as preparation of working solution and a list of tables and figures covering characteristics of the studied analytes, precision and accuracy data, on-card stability, hematocrit effect, center versus peripheral spot comparison, and determination of plasma volume generated from the book-type DPS card.

CONCLUSIONS Although DPS addresses some of the challenges faced by DBS, it is not intended to replace the use of DBS. For analytes that have high affinity for RBC, DBS will be the preferred form of analysis over DPS or vice versa. The two techniques are linked by the other microsampling techniques which are complementary. The book-type DPS card includes some improvements from previous reports and offers some functional and practical benefits moving

Corresponding Author *Dr. Jack Henion. Q Squared Solutions 19 Brown Road Ithaca NY 14850 t: 607.330.9801 email: [email protected]

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