13
R eseaRch a Rticle s pecial Focus: D RieD B looD s pots Dried blood spot (DBS) sampling is gaining momentum as an alternative to plasma in sup- port of drug development to evaluate toxico- kinetics and pharmacokinetics [1,2] . Tremendous advantages offered by DBS can be fundamen- tally attributed to its nature of microsampling. As such, satellite animal groups in preclinical studies can either be reduced or completely eliminated, and a reduction in stress for ani- mals during sample collection has been noted when less blood is sampled [2] . This translates into huge ethical benefits with fewer animals used and higher data quality from serial sam- pling in safety assessment. The primary merits offered by DBS for clinical studies include ease of sampling (e.g., finger or heel pricking) and the feasibility of conducting studies in remote areas and in the pediatric and juvenile population [3] . The dried format facilitates sample storage and shipping at room temperature [4,5] , which can provide significant cost savings [6] . Quantitative and qualitative determination of analytes of interest and/or their metabolite(s) in the absorption, distribution, metabolism and excretion is an integral part of preclinical and clinical studies. LC–MS/MS, a traditional gold standard for quantitative bioanalysis [7,8] , contin- ues to become the platform of choice for quanti- tative DBS bioanalysis for small molecules [9–12] . One key challenge encountered in DBS bioana- lysis is its small blood volume available for analysis, which could be 25- to 100-fold smaller compared with a regular plasma assay [13] . Several strategies can be implemented to enhance DBS LC–MS/MS assay sensitivity, including increas- ing punch sizes/use of multiple punches [9] , employment of advanced LC–MS technology [14–16] , column switching [17] and efficient utiliza- tion of sample extracts [18] . Evidently, the hurdles in DBS assay sensitivity can often be overcome with LC–MS/MS bioanalysis. New concepts and technologies are being constantly introduced for direct DBS analyses, including paper spray [19] , the use of thin-layer chromatography [20] and online sample desorption [21] . The DBS concept has been extended to other dried matrices such as dried plasma spots in clinical studies [22] and dried cerebrospinal fluids [23] . These new and emerging technologies abundantly demonstrated that there will be as many analytical choices for qualitative and quantitative analysis of DBS samples as for plasma samples. Nevertheless, there are improvements to be made from current DBS practices. First, there is wastage of DBS samples in bioanalysis. An aliquot of approximately 20 �l of blood sam- approximately 20 �l of blood sam- 20 �l of blood sam- ple is typically spotted to form a DBS sample. A fixed-size blood spot, for example 3 mm, is punched upon sample analysis. Provided that blood spreads uniformly on the cellulose paper, only approximately 2–3 �l blood is utilized for analysis whilst the majority is wasted. Second, Perforated dried blood spots: a novel format for accurate microsampling Dried blood spots (DBS) in their current format encounter challenges in bioanalysis using fixed areas, including but not limited to, waste of DBS samples (only a fraction is used for analysis), the need for sample punching leading to concerns of sample carryover, uncertainty for accurate recovery assessments and hematocrit (HCT) effects. Here we describe a novel concept, namely perforated dried blood spots (PDBS), for accurate microsampling that addresses previous challenges. PDBS discs were prepared from regular filter paper, with a diameter of 6.35 mm and a thickness of 0.83 mm. An accurate amount of blood sample (5–10 µl), was deposited, dried and stored on the PDBS discs. Upon sample analysis, PDBS samples are simply pushed by single-use pipette tips into 96-well plates. The proof- of-concept study was carried out on a PDBS LC–MS/MS assay development and validation under GLP criteria for the quantitation of lansoprazole in human whole-blood (K 3 EDTA). Particularly, the effect of HCT on the accuracy of quantitation was found to be related to recovery from PDBS samples. In all, PDBS was proved to be a viable alternative to conventional DBS, offering additional advantages of complete sample utilization, no requirement for punching, ease of recovery assessments, and elimination of sampling influence due to HCT levels. Fumin Li †1 , John Zulkoski 1 , Douglas Fast 1 & Steve Michael 1 1 Covance Laboratories, Inc., Bioanalytical Chemistry, 3301 Kinsman Boulevard, Madison, WI 53704, USA Author for correspondence: E-mail: [email protected] 2321 ISSN 1757-6180 Bioanalysis (2011) 3(20), 2321–2333 10.4155/BIO.11.219 © 2011 Future Science Ltd

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ReseaRch aRticle

special Focus: DRieD BlooD spots

Dried blood spot (DBS) sampling is gaining momentum as an alternative to plasma in sup­port of drug development to evaluate toxico­kinetics and pharmacokinetics [1,2]. Tremendous advantages offered by DBS can be fundamen­tally attributed to its nature of microsampling. As such, satellite animal groups in preclinical studies can either be reduced or completely eliminated, and a reduction in stress for ani­mals during sample collection has been noted when less blood is sampled [2]. This translates into huge ethical benefits with fewer animals used and higher data quality from serial sam­pling in safety assessment. The primary merits offered by DBS for clinical studies include ease of sampling (e.g., finger or heel pricking) and the feasibility of conducting studies in remote areas and in the pediatric and juvenile population [3]. The dried format facilitates sample storage and shipping at room temperature [4,5], which can provide significant cost savings [6].

Quantitative and qualitative determination of analytes of interest and/or their metabolite(s) in the absorption, distribution, metabolism and excretion is an integral part of preclinical and clinical studies. LC–MS/MS, a traditional gold standard for quantitative bioana lysis [7,8], contin­ues to become the platform of choice for quanti­tative DBS bioana lysis for small molecules [9–12]. One key challenge encountered in DBS bioana­lysis is its small blood volume available for

ana lysis, which could be 25­ to 100­fold smaller compared with a regular plasma assay [13]. Several strategies can be implemented to enhance DBS LC–MS/MS assay sensitivity, including increas­ing punch sizes/use of multiple punches [9], employment of advanced LC–MS technology [14–16], column switching [17] and efficient utiliza­tion of sample extracts [18]. Evidently, the hurdles in DBS assay sensitivity can often be overcome with LC–MS/MS bioana lysis. New concepts and technologies are being constantly introduced for direct DBS analyses, including paper spray [19], the use of thin­layer chromatography [20] and online sample desorption [21]. The DBS concept has been extended to other dried matrices such as dried plasma spots in clinical studies [22] and dried cerebrospinal fluids [23]. These new and emerging technologies abundantly demonstrated that there will be as many analytical choices for qualitative and quantitative ana lysis of DBS samples as for plasma samples.

Nevertheless, there are improvements to be made from current DBS practices. First, there is wastage of DBS samples in bioana lysis. An aliquot of approximately 20 �l of blood sam­approximately 20 �l of blood sam­ 20 �l of blood sam­ple is typically spotted to form a DBS sample. A fixed­size blood spot, for example 3 mm, is punched upon sample ana lysis. Provided that blood spreads uniformly on the cellulose paper, only approximately 2–3 �l blood is utilized for ana lysis whilst the majority is wasted. Second,

Perforated dried blood spots: a novel format for accurate microsampling

Dried blood spots (DBS) in their current format encounter challenges in bioana lysis using fixed areas, including but not limited to, waste of DBS samples (only a fraction is used for ana lysis), the need for sample punching leading to concerns of sample carryover, uncertainty for accurate recovery assessments and hematocrit (HCT) effects. Here we describe a novel concept, namely perforated dried blood spots (PDBS), for accurate microsampling that addresses previous challenges. PDBS discs were prepared from regular filter paper, with a diameter of 6.35 mm and a thickness of 0.83 mm. An accurate amount of blood sample (5–10 µl), was deposited, dried and stored on the PDBS discs. Upon sample ana lysis, PDBS samples are simply pushed by single-use pipette tips into 96-well plates. The proof-of-concept study was carried out on a PDBS LC–MS/MS assay development and validation under GLP criteria for the quantitation of lansoprazole in human whole-blood (K3EDTA). Particularly, the effect of HCT on the accuracy of quantitation was found to be related to recovery from PDBS samples. In all, PDBS was proved to be a viable alternative to conventional DBS, offering additional advantages of complete sample utilization, no requirement for punching, ease of recovery assessments, and elimination of sampling influence due to HCT levels.

Fumin Li†1, John Zulkoski1, Douglas Fast1 & Steve Michael11Covance Laboratories, Inc., Bioanalytical Chemistry, 3301 Kinsman Boulevard, Madison, WI 53704, USA †Author for correspondence:E-mail: [email protected]

2321ISSN 1757-6180Bioanalysis (2011) 3(20), 2321–233310.4155/BIO.11.219 © 2011 Future Science Ltd

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Key Terms

Hematocrit: Also called packed cell volume, defined as the percentage of red blood cells that occupy blood volume.

Perforated dried blood spot: Microsampling technique where a fixed volume of whole blood is deposited on a perforated filter paper disc.

the process of sample punching can result in sample carryover, either from manual punchers or semi­automated punchers. Third, it is diffi­cult to obtain an accurate assessment of analyte recovery. There are two general ways for recov­ery evaluation. One is to estimate the total areas of the DBS [9]. Calculation is based on the area punched and the (averaged) areas of the spots. The recovery is a rough estimation and there are factors such as storage conditions [24] and hema-tocrit (HCT) [25] that can impact the distribution of analytes on papers, and thus the recovery. The other way is to cut and extract the entire blood spots. However, the recovery obtained does not truly mimic actual ana lysis, where only a small fraction of a DBS spot is analyzed. Why does the recovery need to be determined accurately? This is because it is very difficult to distinguish low recovery from analyte stability in DBS, both of which are reflected in low analyte accuracy. Lastly, there is room to further reduce blood sam­ple drawn per time point. A volume of 80–100 �l of blood is required for four DBS samples, which is still substantial for rodents.

We have developed a novel format of DBS sampling, called perforated dried blood spot (PDBS). PDBS discs, 6.35 mm in diameter and 0.83 mm thick, were prepared from regular filter papers (grade 220, Fisher) with a single­hole paper puncher. The proof­of­concept study was carried out on a PDBS LC–MS/MS assay development for the quantitation of lansoprazole in human whole­blood (K

3EDTA). An accurate amount

of blood sample (5–10 �l) was deposited on the center of the PDBS discs. The samples were dried at room temperature for 2 h and stored in sealed plastic bags with desiccant. Upon sample ana­lysis, PDBS samples were pushed into 96­well plates with single­use pipette tips, eliminating the need for mechanical punching and potential sample carryover. Extraction with a mixture of aqueous organic solvent was systematically inves­tigated, where acetonitrile:water (9:1, v:v) pro­vided the maximal lansoprazole recovery from PDBS samples. A full method validation was conducted under GLP regulations [26] with 5 �l lansoprazole whole­blood samples deposited on PDBS. Complete sample utilization was realized by extracting the whole PDBS discs. No sampling carryover was noted because there is no need for punching. Long­term storage stability for lanso­prazole in PDBS was established for 112 days at room temperature. Lansoprazole recovery from PDBS was readily attained with a high degree of confidence, which was consistent from low to

high concentrations. Recovery was demonstrated to be the key to achieve consistent results inde­pendent of HCT. In addition, good precision, accuracy and linearity were obtained. The results clearly demonstrated that the PDBS format offers additional advantages to DBS of complete sam­ple utilization, no requirement for punching, ease of recovery assessments, and even less blood sampling in preclinical and clinical studies.

Experimental�� Chemicals & reagents

Lansoprazole (purity 100%) was purchased from Sigma­Aldrich (MO, USA) and deuterated lansoprazole­d4 (purity 100%) was obtained from CDN Isotopes (Quebec, Canada). Formic acid (≥96%) and HPLC­grade acetonitrile were from Sigma­Aldrich. Human whole­blood in K

3EDTA anticoagulant was purchased from

Biochemed Pharmacologicals (VA, USA).

�� Preparation of blood samples with different HCT levelsFresh whole­blood was obtained from Biochemed, where the HCT was determined in­house (Advia, Siemens) to be 37%. Aliquots of the unaltered blood samples were centrifuged to separate plasma from red blood cells. Blood samples of different HCT levels (from 18.5 to 74.0%) were prepared by either removing or adding the appro­priate amount of plasma, and the resulting sam­ples were mixed gently. Lansoprazole was then prepared at 75 ng/ml with blood samples of vary­ing HCT values. Aliquots of 5 µl of the prepared samples were deposited to prepare PDBS samples.

�� Chromatographic conditionsHPLC was a Prominence HPLC system (Shimadzu, MD, USA) equipped with a binary pump. The chromatography was developed on an Aquasil C18 column (50 × 2.1 mm, 3 �m, Thermo). The HPLC was controlled via Shimadzu web control software. Column tem­perature was maintained at 35°C. Mobile phase A was composed of 0.2% formic acid in water, while mobile phase B consisted of 0.2% formic acid in acetonitrile. The following gradient was used: 0.01 min (10% B), 0.10 min (10% B), 1.00 min (60% B), 1.10 min (85% B), 2.00 min (85% B), 2.01 min (10% B) and 3.00 min (10% B, pump stop). The flow rate was kept at 0.6 ml/min.

�� Mass spectrometric conditionsAn API 5000 triple quadrupole mass spec­trometer (MDS­Sciex, Concord, Canada) with

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TurboIonSpray (TIS) interface was operated in positive ionization mode. The optimized instrument parameters for lansoprazole and lansoprazole­d4 were as follows: TIS tempera­ture: 600°C; TIS voltage: 3000 V; curtain gas: 20; nebulizing gas: 65; TIS gas: 65; collision gas: 10; declustering potential: 60 V; entrance potential: 10 V; collision energy: 16 eV; collision cell exit potential: 20 V. The following precursor to product ion transitions were used for the mul­tiple reactions monitoring of lansoprazole and lansoprazole­d4, at m/z 370.2→252.1 and m/z 374.2→252.2, respectively. The mass spectrom­eter was operated in multiple reactions monitor­ing mode with both quadrupoles maintained at unit resolution (0.7 mass units at half height). Analyst v.1.5.1 software (Applied Biosystems/MDS SCIEX Instruments) was used to acquire and process all other data. A weighted (1/concen­tration2) linear least­squares regression was used to generate the calibration curve.

�� Lansoprazole calibrators & QC samples preparationLansoprazole calibrators and QC samples were prepared in human whole­blood (K

3EDTA).

Eight levels of calibrators were prepared at 1.00, 2.00, 10.0, 50.0, 200, 500, 800 and 1000 ng/ml. Five levels of QC samples were prepared at 1.00 (LLOQ), 3.00 (LQC), 75.0 (MQC), 750 (HQC) and 3750 ng/ml (dilution QC or DQC). Unless otherwise mentioned, aliquots of 5 �l of lanso­prazole samples were spotted onto PDBS discs, which were allowed to dry at room temperature for at least 2 h. PDBS samples were stored in sealed plastic bags with desiccant. PDBS samples were pushed by single­use pipette tips to a 96­well plate (Axygen) upon sample ana lysis. Aliquots of 200 �l of lansoprazole­d4 (2 ng/ml) inter­nal standard (IS) solution in acetonitrile:water (9:1, v:v) were added to all PDBS calibrators and QC samples. For blank samples, 200 �l of acetonitrile:water (9:1, v:v) was added instead. The plate was vortexed for 10 min and centri­fuged at approximately 2560 × g at room tem­perature for 5 min. A volume of 100 �l of the supernatant was transferred to a TruTapered 96­well plate (Analytical Sales). Aliquots of 25 �l of reconstitution solvent, methanol:water (7:3, v:v), were added. The plate was vortex mixed for 1 min and ready for LC–MS/MS ana lysis.

Results & discussionFigure 1A shows a schematic diagram of one DBS card and a typical sampling strategy. A typical

spot has a diameter of 10 mm, or 78.5 mm2 in area. Upon whole­blood sample application, nor­mally 20 �l, the blood sample spreads across the spot. A small section of the spot, for example 3 mm, is punched out for sample ana lysis. The size corresponds to 7.06 mm2. Assuming that the 20 �l whole­blood sample occupies the entire 10 mm spot, only approximately 10% of the sam­ple was used for ana lysis. The majority (~90%) of the sample is wasted. We proposed a novel sam­pling strategy, called PDBS, for accurate micro­sampling, illustrated in Figure 1B–D. The filter paper is perforated with a fixed size (Figure 1B) that has a diameter of 6.35 mm. The purpose of perforation is that paper discs are mostly cut so that there is no need for mechanical punching during sample ana lysis. The discs, nevertheless, are slightly attached to facilitate sample storage, transportation and archiving. A cross is printed in the middle of the discs for visual aid so that blood samples can be reliably deposited in the center to prevent samples from spreading out the boundary. Upon sample collection, an accurate amount of samples, for example, 5 �l, is spotted (Figure 1C). Samples spread across the perforated discs and are dried at room temperature to form PDBS samples as depicted in Figure 1D.

For the proof­of­concept study, the filter papers were not actually perforated due to lack of in­house equipment that can perform per­foration. However, card perforation should be relatively straightforward with the right equip­ment, where mass production is achievable. Instead, a single­hole paper puncher was used

ID: 10 mm78.5 mm2

– DBS sampling is ∼10% of the total volume with a 3 mm punch

– 20 µl of whole blood was spotted Only ∼2 µl of samples available for LC–MS/MS analysis

– ~90% of waste

ID: 3 mm7.06 mm2

ID: 6.35 mm

The spot isperforated

Accuratelypipette 5 µlto the spot

Dry at RTfor 2 h

x

x

x

Bioanalysis © Future Science Group (2011)

Figure 1. Comparison of (A) a regular DBS and (B–D) a perforated DBS sampling scheme for LC–MS/MS bioana lysis.DBS: Dried blood spot; ID: Diameter; RT: Room temperature.

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to punch paper discs (6.35 mm diameter) from regular filter paper (Grade 222, 0.83 mm thick, Fisher Scientific). Figure 2A shows a piece of filter paper card with 20 discs being punched. The detached discs are then put back into the punched holes. Figure 2B shows of the filter paper with 20 punched discs assembled. The discs were held tightly. During the entire experiment period spanning several months there have been no cases where the PDBS discs were detached accidentally. The middle section of the paper card is for sample labeling purposes.

Blood samples in DBS are present in a solid format, where the analytes are solidified in filter papers in whole­blood medium. The first step of sample preparation is to extract analytes out of dried spots prior to further sample cleanup, such as liquid–liquid extraction or solid­phase extrac­tion to remove matrix components. Aqueous organic solvent mixtures are frequently used to elute the analytes from DBS [27]. Water has been employed to soak or soften dried spots to facilitate analyte elution prior to organic sol­vent extraction. Systematic investigations were undertaken to probe the effects of organic sol­vent (i.e., acetonitrile and methanol) concen­trations on the overall lansoprazole extraction efficiency from PDBS with or without water pre­treatment at 75 ng/ml. For water pretreatment experiments, the PDBS samples were pushed into an Axygen 96­well plate and 20 �l of water

was added. The plate was centrifuged briefly to ensure that the PDBS samples came into contact with water. Samples sat at room temperature for 5 min, allowing the PDBS samples to be fully soaked. Aliquots of 20 �l of IS (20 ng/ml) in ace­tonitrile lansoprazole­d4, were added. Aliquots of 160 �l of acetonitrile/water or methanol/water in the ratio of 0:1, 1:3, 1:1, 3:1 and 1:0 (v:v) were added, resulting in final acetonitrile or methanol concentrations of 10, 30, 50, 70 and 90%. For direct extractions with aqueous organic mixtures, 200 �l of aqueous organic solvents containing 10, 30, 50, 70 and 90% of acetonitrile or methanol with 2 ng/ml of lan­soprazole­d4 were added to the PDBS samples. The remaining sample preparation procedures are as described previously.

Figure 3 depicts lansoprazole peak areas or extraction efficiency from PDBS samples (n = 3) as a function of organic solvent compositions, direct extraction with aqueous methanol mix­ture, direct extraction with aqueous acetonitrile mixture, water pretreatment followed by aque­ous methanol mixture, and water pretreatment followed by aqueous acetonitrile mixture. The results demonstrated that there is virtually no difference in terms of lansoprazole responses with or without water pretreatment regardless of acetonitrile or methanol concentrations in the range of 10 to 90%. The extraction efficiency is generally higher from the aqueous aceto nitrile mixture than from the aqueous methanol mix­ture. Specifically, with water pretreatment, 23, 39, 100, 98 and 90% higher lansoprazole responses were observed from acetonitrile than from methanol at 10, 30, 50, 70 and 90%, respectively. For direct elution or without water pretreatment, 1, 39, 113, 75 and 103% higher lansoprazole responses were observed from ace­tonitrile than from methanol at 10, 30, 50, 70 and 90%, respectively. The results indicated that a mix of acetonitrile and water can better extract lansoprazole from PDBS samples than metha­nol and water mixture. While prewetting PDBS samples did not seem to impact lansoprazole extraction appreciably in current experiments, HCT should be considered and evaluated as will be discussed in the coming sections. It should be noted that blood used for the validation was from normal and healthy donors that have a finite range of hemaocrit values. Extractions of lansoprazole from blood samples of small HCT difference would likely not be distinguished with or without water pretreatment. In all, such a systematic assessment of extraction efficiency

Sample label

Sample label

Figure 2. (A) A card with 20 perforated dried blood spot discs punched and (B) 20 perforated dried blood spot discs were assembled. 

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in terms of organic concentrations with or with­out water pretreatment is straightforward in extraction optimization, and is recommended as part of bioanalytical method development. The procedures should work equally for other dried matrices such as dried plasma spot [22].

Proof­of­concept experiments were conducted to demonstrate the feasibility of bioanalytical assay development on PDBS. Representative lansoprazole chromatograms from a PDBS sample at LLOQ (1.00 ng/ml) and a PDBS matrix blank from human whole­blood can be found in SupplementAry Figure 1. With 2 �l injections, the LLOQ showed a S/N of 21, well above the requirement for reliable quantitation. Lansoprazole eluted at approximately 1.4 min and a small interference peak at approximately 1.6 min is baseline resolved from lansoprazole, facilitated by the use of 3 �m particles. There is no interference at the elution of lansoprazole as shown in the bottom trace. SupplementAry Figure 2 is a calibration curve from lansopra­zole PDBS samples, from 1.00 to 1000 ng/ml. The diamonds were obtained experimentally, whereas the line is the linear regression of the experimental data using 1/(concentration)2

regression. The calibration curve is linear over the range of 1.00 to 1000 ng/ml with an r2 of 0.9994. Excellent linearity suggests that lanso­prazole was quantitatively extracted. tABle 1 lists the statistics for four levels of PDBS lansoprazole QC samples with 5 �l of whole­blood samples spotted. Very good precision and accuracy were obtained for all four levels QCs. The intra­assay CV was between 3.29 and 6.79%, and intra­assay mean accuracies were between 94.3 and 103.1% (n = 6) of the theoretical concentra­tions. The results clearly demonstrated that the current LC–MS/MS bioanalytical method works well for lansoprazole quantitation in the PDBS format.

The PDBS disc (6.35 mm diameter and 0.83 mm thickness), shown in Figure 4A, has a volume of 26.3 �l, which acts like a container and can theoretically hold up to 26.3 �l of whole­blood samples. For practical purposes, the actual blood volume that can be supported by the disc would be less. Assuming that blood samples spread approximately 56% of the sur­face areas (i.e., ~75% of diameter) and pen­etrate 75% of the thickness, this volume corre­sponds to approximately 11 �l, as illustrated in Figure 4B. To evaluate how much blood samples can actually be spotted onto the PDBS discs with ease, aliquots of lansoprazole whole­blood

samples (75 ng/ml) in 5, 6, 7, 8, 9 and 10 �l (n = 4) were deposited onto the PDBS discs. The samples were extracted and analyzed. Figure 4C shows lansoprazole mass spectrom­etry response in terms of absolute peak areas as a function of the spotted volume from 5 to 10 �l. The squares were obtained from average lansoprazole peak areas, whereas the line is the linear regression of the experimental data. The curve is linear over the range of 5 to 10 �l with an r2 of 0.9979. Good linearity suggests that lansoprazole was quantitatively extracted from PDBS at various spotted volumes. It should be noted that the amount of matrix present in the extracts was different because different volumes of blood samples were spotted. However, simi­lar linearity can be obtained (r2 = 0.9984) if the ratios of lansoprazole to lansoprazole­d4 were used instead. The results supported that the current PDBS discs can hold a blood volume up to 10 �l. To enhance sampling ruggedness, only 5 �l of samples were spotted for all the

Organic solvent (%)

0 20 40 60 80 100

Lan

sop

razo

le p

eak

area

(cp

s)

0.0

3.0e+4

6.0e+4

9.0e+4

1.2e+5

1.5e+5

1.8e+5

2.1e+5

Acetonitrile pre-soak (n = 3)Methanol pre-soak (n = 3)Acetonitrile all (n = 3)Methanol all (n = 3)

Figure 3. Effect of acetonitrile and methanol concentrations on the extraction efficiency of lansoprazole from perforated dried blood spot samples with and without water pretreatment.

Table 1. Precision and accuracy of four levels of lansoprazole perforated dried blood spot QCs.

  Conc.  (ng/ml)

Replicate (n)

Mean conc.  (ng/ml)

RSD (%)

Accuracy (%)

LLOQ 1.00 6 0.943 4.84 94.3

LQC 3.00 6 2.98 6.79 99.3

MQC 75.0 6 73.1 3.29 97.4

HQC 750 6 773.6 5.33 103.1RSD: Relative standard deviation.

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coming experiments. The above results amply demonstrated that PDBS worked essentially the same as conventional DBS for the quantitation of lansoprazole in whole­blood. It would be interesting to see how conventional DBS per­formed compared with PDBS using the same filter papers. It is worth mentioning that the filter paper used in the study is approximately twice as thick as commercial cards. Eight lev­els of lansoprazole calibrators and five levels of QC samples were prepared as described previ­ously. A volume of 20 �l of samples was spot­ted onto the filter papers (grade 222, 0.83 mm thick, Fisher) used for PDBS. The samples were allowed to dry for 2 h at room temperature. Samples of 4.7 mm diameters were punched by a manual mechanical puncher (Tomtec, CT, USA) and placed in a 2 ml, 96­well plate (Axygen). Optimized PDBS extraction proce­dures were followed. For dilution samples, the extracts from DQC were diluted tenfold with extracts obtained from control samples contain­ing lansoprazole­d4 only. SupplementAry tABle 1 lists the statistics for five levels of lansoprazole DBS QC samples using a 4.7 mm punch. Good precision and accuracy were obtained for five levels of lansoprazole DBS QC samples. The intra­assay CV was between 2.6 and 10.5%, and intra­assay mean accuracies were between 94.4 and 102.2% (n = 6) of the theoretical con­centrations. Obviously, the DBS LC–MS/MS

bioanalytical method works well for lansopra­zole quantitation using normal filter papers in the DBS format. Shown in Figure 5 are lan­soprazole chromatograms from a DBS matrix blank and a DBS LLOQ sample (1 ng/ml). The matrix blank sample was the third one punched after a highest calibrator, 1000 ng/ml. To minimize carryover from the puncher, the first two punches of DBS matrix blank were discarded. The peak area of the matrix blank is approximately 18% of the response from LLOQ. The carryover was from the mechani­cal puncher instead of LC carryover because no chromatographic carryover was observed from the PDBS experiments using the same chro­matography. The results illustrated the general concern of sample carryover from DBS ana­lysis, where sample punching is required. The results on the other hand established the advan­tage of PDBS where no punching is needed for sample processing.

Method validation of the PDBS LC–MS/MS for lansoprazole quantitation in human whole-blood (K3EDTA)A full method validation in accordance with internationally accepted guidelines [26] was conducted for the quantitation of lansoprazole in PDBS format using LC–MS/MS. Three consecutive analytical batches were used to

r=3.175 mm0.83 mm

0.62 mm

r=2. 38 mm

r = 3.175 mm0.83 mm

0.62 mm

r = 2.38 mm

Spotted blood volume (µl)

4 5 6 7 8 9 10 11

Lan

sop

razo

le p

eak

area

(cp

s)

4.50e+4

6.00e+4

7.50e+4

9.00e+4

1.05e+5PDBS MQC (n = 4)

Figure 4. The loading capacity of a perforated dried blood spot disc (6.35-mm diameter, 0.83-mm thick). A blank perforated dried blood spot (PDBS) disc (A) and a predicated PDBS sample where 75% of surface areas and 75% of height are occupied by blood samples (B). Lansoprazole mass spectrometry responses (peak areas) as a function of the volume of lansoprazole blood samples (75 ng/ml; n = 4) spotted on PDBS discs in the range of 5 to 10 µl (C).PDBS: Perforated dried blood spots.

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assess the precision and accuracy. Each batch contained a single set of calibration standards and six replicates of four regular QC samples at 1.00 (LLOQ), 3.00 (LQC), 75.0 (MQC) and 750 ng/ml (HQC). In one of the batches, six replicates of the dilution QC samples at 3750 ng/ml were analyzed to assess the preci­sion and accuracy for samples over the curve. QC samples and other test samples were inter­spersed among calibration standards. Extracts of PDBS matrix blanks were analyzed in duplicate immediately following the two highest calibra­tion standards, at least one evaluation positioned early in the batch and at least one evaluation towards the end of the batch to determine carry­over of the LC–MS/MS system. Peak area ratios of lansoprazole to lansoprazole­d4 were calcu­lated. Calibration curves were generated using a weighted (1/x2) linear least­squares regression.

tABle 2 shows the accuracy of eight lanso­prazole PDBS calibrators from three validation batches. For LLOQ and ULOQ samples, the inter­assay CV was 2.9 and 5.2% and inter­assay mean accuracies were 100.6 and 98.1% of theoretical concentrations, respectively. The precision and accuracy data for four levels of QC samples from three validation batches (n = 18) are summarized in tABle 3. For QC samples, the inter­assay CV was between 3.2 and 6.0%, and inter­assay mean accuracies were between 96.2 and 105.5% of theoretical con­centrations. It is worth noting that one level of LQC at 3.00 ng/ml failed with high accuracy (~200%). This is likely due to a double spik­ing error, where two PDBS QCs were extracted. Overall, good precision and accuracy were obtained for lansoprazole calibrators and QC samples in the PDBS format, which are similar to those obtained from either liquid matrix or regular DBS assays.

One of the challenges associated with DBS or any other dried matrix is how to carry out dilu­tion experiments. This is primarily due to the solid nature of DBS/PDBS samples. The dilution for liquid samples is straightforward. For DBS/PDBS, dilution QC samples are typically pro­cessed simultaneously with control­0 samples in which blank matrix samples were extracted with IS. After extraction, the extracts of dilution QCs are diluted with control­0 extracts by a desirable dilution factor. For instance, a tenfold dilution is realized by combing 10 �l of extracts from dilution QC samples with 90 �l of extracts from control­0 samples. Accurate and precise liquid handling of small volume is crucial to achieve accurate and precise measurement of dilution

Table 2. Precision and accuracy of eight levels of lansoprazole perforated dried blood spot calibrators from three consecutive validation batches.

Analysis Theoretical concentrations (ng/ml) 

1.00 2.00 10.0 50.0 200 500 800 1000

Group 1 1.03 1.85 10.7 52.6 201 502 763 957

Group 2 0.97 2.11 10.1 50.5 188 494 792 1039

Group 3 1.02 1.91 10.7 52.6 204 491 771 946

n 3 3 3 3 3 3 3 3

Mean 1.01 1.96 10.5 51.9 198 496 775 981

SD 0.0290 0.132 0.348 1.23 8.54 5.60 14.8 50.7

RSD (%) 2.9 6.8 3.3 2.4 4.3 1.1 1.9 5.2

Accuracy (%) 100.6 97.8 104.8 103.8 98.9 99.2 96.9 98.1RSD: Relative standard deviation.

Retention time (min)

0.8 1.0 1.2 1.4 1.6 1.8 2.0

0.0

0.5

1.0

1.5

2.0

2.5

Area: 210 cps~18% of LLOQ

No

rm. s

ign

al2.0

Area: 1131 cps

DBS matrix blankLLOQ: 1 ng/ml

Figure 5. Representative lansoprazole chromatograms from (A) a dried blood spot matrix blank punched after a ULOQ sample and (B) a dried blood spot sample at LLOQ (1 ng/ml).DBS: Dried blood spot.

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QC samples. Liu and co­workers introduced an alternative DBS sample dilution, ‘IS­tracked dilution’, for LC–MS/MS assays using an IS to track the dilution step [28]. The IS for dilution QCs is prepared at an elevated concentration, which is X­times more concentrated than the one used for other samples, including calibra­tors, QCs and study samples that do not require dilution. X represents the desired dilution factor. This IS was added to the samples needing dilu­tion before sample processing. The processed samples were then diluted into the assay curve range before LC–MS/MS ana lysis with extracts from blank matrix. The approach offers a partic­ular advantage in that the physical dilution step

is not a volume­critical step because dilution is tracked by an IS. tABle 4 listed the precision and accuracy from six replicates of dilution QCs at 3750 ng/ml obtained from the traditional dilution and ‘IS tracked’ approach, respec­tively. Comparable precision and accuracy were obtained from either approach, 107.8 ± 5.6% (n = 6) from conventional dilution scheme and 106.2 ± 4.9% (n = 6) from IS­tracked strategy.

Another challenge of conventional DBS is that it is not straightforward to calculate abso­lute analyte recovery compared with liquid matrices. This is ascribed to the uncertainty of how much analyte is punched out from the DBS cards. Recovery is typically carried out where a

Table 4. Precision and accuracy of lansoprazole perforated dried blood spot DQCs using conventional dried blood spot dilution and internal standard tracked dilution strategy. 

Replicate DQC conc. (ng/ml) conventional strategy

DQC accuracy (%) conventional strategy

DQC conc. (ng/ml) IS-tracked strategy

DQC accuracy (%) IS-tracked strategy

1 4210 112.2 3640 97.0

2 4300 114.6 4070 108.6

3 3960 105.7 4080 108.8

4 4060 108.4 4200 112.1

5 4070 108.5 3890 103.7

6 3650 97.3 4020 107.2

Average 4041 107.8 3983 106.2

SD 226 – 196 –

RSD (%) 5.6 – 4.9 –DQC: Dilution concentration quality control sample; IS: Internal standard; RSD: Relative standard deviation.

Table 3. Precision and accuracy of four levels of lansoprazole perforated dried blood spot QCs from three consecutive validation batches.

  LLQC (1.00 ng/ml) LQC (3.00 ng/ml) MQC (75.0 ng/ml) HQC (750 ng/ml)

Day 1 (n = 6)

Mean 0.966 3.16† 78.9 798

RSD (%) 3.5 2.6 3.9 3.5

RE (%) -3.3 5.6 5.2 6.4

Day 2 (n = 6)

Mean 0.957 3.08 79.1 804

RSD (%) 9.6 5.2 3.8 1.4

RE (%) -4.3 2.6 5.4 7.3

Day 3 (n = 6)

Mean 0.962 3.13 79.3 767

RSD (%) 4.2 2.0 3.2 2.1

RE (%) -3.8 4.5 5.7 2.3

Interday (n = 18)

Mean 0.962 3.12† 79.1 790

RSD (%) 6 3.6 3.4 3.2

RE (%) -3.8 4.2 5.5 5.3†One level of LQC failed.RE: Relative error; RSD: Relative standard deviation.

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whole section of DBS card containing a known amount of analyte is punched. Extractions of the entire area are followed and recovery is calculated. Nevertheless, the practices do not mimic the real sample ana lysis procedures. In comparison, PDBS overcomes this disadvan­tage by extracting the entire PDBS discs with a known amount of analytes. As a result, it is easy to accurately assess recovery efficiency from PDBS. Recovery of lansoprazole using direct acetonitrile:water (9:1, v:v) extraction proce­dure was evaluated by comparing the mean peak areas of the QC sample (n = 6) at 3.00, 75.0 and 750 ng/ml extracted from PDBS to those of matrix recovery samples prepared by adding lansoprazole and lansoprazole­d4 to postextraction matrix blanks at the correspond­ing concentration (n = 3). The absolute lanso­prazole recoveries at 3.00, 75.0 and 750 ng/ml are 84, 83, and 86%, respectively. The results demonstrated that lansoprazole recoveries from PDBS were consistent from low to high concen­trations, crucial to achieve accurate and reliable quantitation. This was verified by good preci­sion and accuracy obtained for all calibrators and QC samples in validation.

Dried matrices such as DBS typically do not include IS, which do not truly compensate for the elution of analytes from dried spots. IS are introduced in the extraction solvent, which only compensates for the LC–MS/MS ana­lysis. The practice represents a rather significant drawback in bioana lysis compared with liquid matrices, where IS and samples can be mixed prior to extractions. The matrix factors were evaluated by comparing the mean peak area ratios of lansoprazole to lansoprazole­d4 from matrix recovery samples (n = 3) to those from reagent blank recovery samples (n = 3). Matrix recovery samples were prepared by adding lan­soprazole and lansoprazole­d4 to postextraction matrix blanks at the corresponding concentra­tions while reagent blank recovery samples were prepared by adding lansoprazole and lansopra­zole­d4 to postextraction reagent blanks at the

corresponding concentrations. The matrix fac­tors at 3.00, 75.0 and 750 ng/ml are 1.02, 1.04 and 1.01, respectively, indicating that there is minimal matrix effect of the current PDBS LC–MS/MS method. One possibility offered by PDBS is that a fixed amount of IS can be added to PDBS discs prior to sample spotting. Work is ongoing and will be communicated elsewhere.

For a typical bioanalytical method validation, it is critical to evaluate long-term stability (LTS) of an analyte in a particular matrix. The pro­cess of obtaining LTS for liquid matrices is well established and becomes a routine procedure. LTS in DBS may pose challenges. It is primarily due to the difficulty in distinguishing stability from extraction efficiency, both of which can result in low measured accuracy. This is compli­cated by the indirect approach to obtain recovery in DBS as outlined previously. As demonstrated, it is simple to attain absolute analyte recovery from PDBS. The ambiguity in recovery evalua­tion in DBS due to uncertainty in blood spread­ing and punching is eliminated in PDBS. Our approach with PDBS is to develop an extraction that provides maximal and consistent recovery from low to high concentrations during method development. As such, any loss observed from stored samples can be confidently linked to on­card analyte stability. tABle 5 lists the precision and accuracy of lansoprazole PDBS LTS samples at 3, 75 and 750 ng/ml after being stored at room temperature for 112 days. The results demon­strated that lansoprazole is stable in PDBS at room temperature for at least 112 days.

Impact of HCT on PDBSHCT or packed cell volume is defined as the percentage of red blood cells that occupy blood volume. Human HCT varies with age, gender and health conditions, with the majority falling within the range of 28 to 67% [25]. The viscos­ity of blood changes with HCT, that is, high HCT results in high viscosity. As a result, HCT could potentially influence the pharmacokinetic data obtained from DBS samples where a fixed

Table 5. Long-term stability of lansoprazole perforated dried blood spot samples stored at room temperature for 112 days.

  Concentration (ng/ml) Replicate (n)

Mean concentration (ng/ml)

RSD (%)

Accuracy (%)

LQC 3.00 6 2.99 5.6 99.8

MQC 75.0 6 72.5 3.6 96.7

HQC 750 6 737 2.2 98.3RSD: Relative standard deviation.

Key Term

Long-term stability: Duration during which analyte(s) are stable in a matrix under a specific condition.

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area/spot is used for bioana lysis. Assuming the same amount of blood is spotted, samples of higher HCT would produce smaller spot sizes compared with those of lower HCT. More ana­lyte is punched from higher HCT DBS samples when the punch diameter is fixed. Denniff et al. demonstrated that the size of DBS samples on cellulose paper­based substrates decreased pro­portionately and linearly with the increase in HCT level of human blood [25]. However, no obvious trend was observed regarding the effect of HCT level on the performance of quantitative bioanalytical assays, which depended on analyte and type of cellulose paper.

In comparison, one immediate benefit PDBS offers is that each PDBS sample contains 5 �l of blood samples (for instance) or a known amount of analyte regardless of HCT levels as far as all the blood is contained within the discs. Therefore, the uncertainty in the amount of analyte taken out for ana lysis, which could be impacted by HCT/viscosity of blood samples, is completely eliminated. Figure 6A shows the averaged lansoprazole responses (n = 6) obtained from PDBS samples prepared at 75 ng/ml using peak areas of lansoprazole and using peak area ratios of lansoprazole to lansoprazole­d4 as a function of HCT from 18.5 to 74.0%. It should be noted that the optimized extraction using a mixture of acetonitrile:water (9:1, v:v) was employed. All the data were normalized to samples at 37% HCT (unaltered). The results clearly showed that the responses of lansoprazole decreased almost linearly with the increase of HCT. For instance, lansoprazole’s response at 18.5% HCT is 103.4 ± 5.3% (n = 6), whereas the response at 74% HCT is 83.8 ± 7.0% (n = 6). The observed percentage difference between the lowest and highest HCT levels is 19.6% and is greater than the normally accepted 15%. Reduced responses may be ascribed to the stability of lansoprazole in PDBS, matrix effect or reduced recovery from samples of dif­ferent hemotocrit levels. Matrix stability shall not be an issue since up to 112 days of stabil­ity at room temperature was established. Blood samples of various HCT levels can be considered as having different matrices. Therefore, matrix effects could cause the drop in lansoprazole’s peak areas assuming matrix suppression is the leading cause. Nevertheless, this possibility can be ruled out because the peak area ratios of lansoprazole to lansoprazole­d4 exhibit a nearly identical trend. Furthermore, a close examination showed that the peak areas of the

stable­labeled IS, lansoprazole­d4, are virtually unchanged with HCT, indicating that there is no HCT­related matrix effect.

Recovery seems to be a reasonable explana­tion for the reduced lansoprazole responses observed for samples of high HCT. Higher HCT means less plasma (water) or more red blood cells. Samples of higher HCT are more viscous. It was hypothesized that high viscos­ity led to low recovery from PDBS samples. To increase the recovery of lansoprazole, the PDBS samples were first soaked in 75 �l of water (in a 96­well plate), which was sonicated in a water bath for 15 min. Aliquots of 225 �l acetonitrile containing 2.66 ng/ml of lansoprazole­d4 were added to all samples except blanks and double blanks. The plate was vortexed for 20 min at high speed. Aliquots of 150 �l of the super­natant were transferred to a clean plate for ana­lysis. Figure 6B shows the averaged lansoprazole responses (n = 6) obtained from PDBS samples prepared at 75 ng/ml using peak areas of lanso­prazole and using peak area ratios of lansoprazole to lansoprazole­d4 as a function of HCT from 18.5 to 74.0%. All the data were normalized to samples at 37% HCT (unaltered). Evidently, the responses of lansoprazole, either peak areas or peak area ratios over lansoprazole­d4, were not changed with HCT. The results supported the hypothesis that the low responses at elevated HCT values seen previously were due to low ana­lyte recovery from PDBS. The results were the first to positively relate recovery to HCT levels for DBS samples. A combination of pretreatment with water, sonication and prolonged vortex time helped release lansoprazole from PDBS samples of high HCT levels. The unique capabilities offered by PDBS in providing a known amount of analytes present in each sample and accu­rately assessing analyte recovery are crucial in solving the HCT issue. It should be noted that the recovery from PDBS or DBS is likely to be compound dependent. Therefore, it is critical to evaluate analyte recovery at different HCT levels. The optimized extraction method should be one that accomplishes reproducible recovery for samples of variable HCT levels expected for the study samples.

ConclusionA novel concept for accurate microsampling – PDBS – was developed. An accurate amount of blood samples, for example, 5�l, can be deposited onto the center of the PDBS discs (6.35 mm diameter and 0.83 mm thick). Upon

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sample ana lysis, the entire PDBS samples were analyzed. The proof­of­concept study was car­ried out on a PDBS LC–MS/MS assay devel­opment for the quantitation of lansoprazole in human whole­blood, which was successfully validated to GLP standards. The results dem­onstrated that PDBS offered multiple addi­tional distinctive advantages compared with conventional DBS. PDBS utilized 100% of blood samples compared with typically <25% in DBS, where only a fraction of sample in the center is punched for ana lysis. PDBS thus needs less blood drawn per time point compared with regular DBS, which makes the usage of toxico­logical animals instead of satellite animals more practical [29]. PDBS does not require punching because sample discs are perforated. Concerns of carryover from sample punching are eliminated. PDBS enables accurate assessment of sample

recovery. The ease in evaluating recovery is enormous, which allows for separation of recov­ery from analyte stability on the dried sample spots. Lastly, it was found that the responses of lansoprazole decreased almost linearly with the increase of HCT when the previous extraction procedures – not tested for samples of variable HCT levels – were used. Low recovery from PDBS samples of elevated HCT was responsible for reduced responses. A combination of water pretreatment, sonication and extended vortex­ing provided consistent recovery for lansopra­zole PDBS samples of different HCT, from 18.5 to 74.0%. In all, the results adequately demon­strated that the PDBS concept is practical, sim­ple and advantageous in LC–MS/MS bioana­lysis. The proof­of­concept of PDBS sampling in an in­house toxicology study is ongoing and will be published elsewhere. The PDBS concept

Hematocrit (%)

20 30 40 50 60 70 80

Per

cen

tag

e (%

)

80

90

100

110

120

Hematocrit (%)

20 30 40 50 60 70 80

Peak area ratio (n = 6, 75 ng/ml)Peak areas (n = 6, 75 ng/ml)

Peak area ratio (n = 6, 75 ng/ml)Peak areas (n = 6, 75 ng/ml)

Figure 6. The average lansoprazole responses obtained from perforated dried blood spot samples prepared at 75 ng/ml using absolute peak areas and peak area ratios of lansoprazole to lansoprazole d4 as function of hematocrit from  18.5 to 74.0%. (A) Optimized extraction conditions for method validation were used (see ‘Experimental’ section for details). (B) A combination of pretreatment with water, sonication and prolonged vortex was employed for sample extractions.

Executive summary

�� A novel concept for accurate microsampling – perforated dried blood spot (PDBS) – was developed. PDBS discs were prepared from regular filter paper, with a diameter of 6.35 mm and a thickness of 0.83 mm, which can easily hold 5–10 µl blood samples.

�� A proof-of-concept study was carried out on a PDBS LC–MS/MS assay development for the quantitation of lansoprazole in human whole blood (K

3EDTA) in the range of 1 to 1000 ng/ml with 5 µl blood samples. The method was successfully validated under GLP

guidelines, demonstrating good precision, accuracy, linearity and consistent recovery.

�� Compared with regular DBS using a fixed area for bioana lysis, PDBS provides additional unique advantages in 100% sample utilization, no need for punching, easy assessment of recovery and elimination of the impact of hematocrit (HCT).

�� The responses of lansoprazole decreased almost linearly with the increase of HCT when an extraction procedure not optimized for samples of variable HCT levels was used.

�� Low recovery from PDBS samples of elevated HCT levels was responsible for reduced responses.

�� A combination of water pretreatment, sonication and extended vortexing provided consistent recovery for lansoprazole PDBS samples of different HCT values, from 18.5 to 74.0%.

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can be readily adapted to other types of dried matrices as well. PDBS can also find applica­tions as a means for storage of a finite amount of samples that is, microsampling.

Future perspectiveThe last 3 years have witnessed an exponential increase of interest in applying DBS sampling in support of drug development in preclinical and clinical studies. The primary driver of DBS lies in reduced blood sampling (the 3 Rs) and logistical advantages such as room temperature sampling, shipping and storage. In terms of bio analysis, extensive research has been carried out on DBS assay development, mostly for small molecule drugs using LC–MS/MS platforms. Many technical hurdles had been overcome and in­depth understanding regarding DBS kinetics, in comparison with plasma data, was obtained. As a result, more and more companies are turn­ing their interests to actions in adapting DBS to their drug development portfolios. It is critically important to raise awareness of DBS to all staff involved in studies, from frontline technicians/scientists to people working in regulatory affairs. Based on our experience, staff training plays a central role in successful DBS implementations. Harmonization of best practices on DBS sam­pling and bioana lysis will be critical to obtain

high­quality data to support preclinical and clinical studies, which will ensure the long­term success of DBS microsampling and acceptance by regulatory agencies.

Supplementary dataSupplementary data accompanies this paper and can be found at www.Future-SCienCe.Com/Doi/Suppl/10.4155/Bio.11.219

Financial & competing interests disclosureThe  authors  have  no  relevant  affiliations  or  financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed  in  the manuscript. This  includes employment, consultancies, honoraria, stock ownership or options,  expert  t estimony,  grants  or  patents  received  or pendng, or royalties.

No writing assistance was utilized in the production of this manuscript. 

Ethical conduct of research The authors  state  that  they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addi-tion,  for  investi gations  involving  human  subjects, informed consent has been obtained from the participants involved.

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�� Extends the DBS concept to dried cerebrospinal fluid sampling.

24 Denniff P, Spooner N. Effect of storage conditions on the weight and appearance of dried blood spot samples on various cellulose­based substrates. Bioana lysis 2, 1817–1822 (2010).

25 Denniff P, Spooner N. The effect of hematocrit on assay bias when using DBS samples for the quantitative bioana lysis of drugs. Bioana lysis 2, 1385–1395 (2010).

�� Explores the impact of hematocrit levels on DBS bioana lysis.

26 Guidance for industry: bioanalytical method validation. US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM) (2001).

27 Liu G, Patrone L, Snapp HM et al. Evaluating and defining sample preparation procedures for DBS LC–MS/MS assays. Bioana lysis 2, 1405–1414 (2010).

28 Liu G, Snapp HM, Ji QC. Internal standard tracked dilution to overcome challenges in dried blood spots and robotic sample preparation for liquid chromatography/tandem mass spectrometry assays. Rapid Comm. Mass Spectrom. 25, 1250–1256 (2011).

�� Presents an alternative approach for DBS dilution using internal standards at elevated concentrations.

29 Scott W, Hunter R, Shen Z et al. Feasibility of applying dried blood spot (DBS) sampling in exploratory toxicity studies in the rat. Poster presentation at: SOT (2011).

Perforated dried blood spots: a novel format for accurate microsampling | ReseaRch aRticle

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