Research Article

For reprint orders, please contact [email protected]

Dried blood spot analysis for rat and dog studies: validation, hematocrit, toxicokinetics and incurred sample reanalysis Background: Execution of experiments to introduce dried blood spot (DBS) sampling for preclinical GLP studies and subsequent clinical studies. Results: Bridging data showed high concordance with DBS:plasma ratios of 0.9 in rats and 1.1 in dogs, demonstrating no preferential uptake or association with cellular components of the blood. The DBS methodology was fully validated incorporating additional experiments pertinent to DBS sampling, storage and analysis. Individual hematocrit (Hct) values in the test animals (rats and dogs) were within the validated Hct range. DBS concentration data and the resulting TK profiles were not impacted by an Hct bias. Incurred sample reanalysis showed high correlation in dogs (97%) and rats (100%) meeting acceptance criteria. Conclusion: Successfully validated and adopted DBS for preclinical GLP studies.

Background The periodic collection of blood is required for understanding the PK and TK properties during drug discovery and development. Historically the pharmaceutical industry has defaulted to using plasma samples, derived by spinning down blood, as the bioanalytical matrix for the quantification of the circulating drug and/or metabolite concentrations. The process involved in the collection of blood, processing it to generate plasma samples, as well as the volumes collected at each sampling time have undergone very little change over the past decades. Additionally, the bioanalytical assays used for the quantification of plasma samples often require the collection of relatively larger volumes of blood, which can be challenging when dealing with rodent species. There is a growing desire within the pharmaceutical industry to refine and improve this process – for both preclinical and clinical studies – and one of the recent advancements in this regard has been the adoption of microsampling techniques. Microsampling also benefits the adoption and compliance with the principles of the 3Rs (Reduction, Refinement and Replacement in animal usage) [1] . Different variations

10.4155/BIO.15.12  © 2015 Future Science Ltd

Enaksha Wickremsinhe Eli Lilly & Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA [email protected]

of microsampling has been reported including microsampling of blood stored as a liquid [2–4] , microsampling of plasma [5–7] , dried plasma spots [8] and dried blood spot (DBS) sampling [9,10] . Although the application of DBS sampling to support drug discovery was demonstrated in the early 2000s [9] , it was not until later in that decade that the technique gained broader implementation [10,11] . Overall, DBS seems to be the most practical and widely implemented microsampling technique, to date [12–14] . DBS involves the collection of a small volume of blood, typically 10 to 20 μl, which is applied to an absorbent matrix, allowed to dry and typically shipped and stored without the need for refrigeration or frozen storage. Although DBS has been selectively implemented across both preclinical and clinical studies, a majority of the applications have been for clinical use and supporting clinical trials [15] , most likely due to the significant advantages resulting from DBS sampling including the ease of implementation (globally, across multiple sites, remote sites that may lack proper instrumentation and infrastructure to generate plasma samples and its storage), use in neonatal and spe-

Bioanalysis (2015) 7(7), 869–883

part of

ISSN 1757-6180

869

Research Article  Wickremsinhe

Key terms Dried blood spot sampling: Technique for the collection, transport and storage of blood on cellulose-based absorbent papers. Blood-to-plasma (B:P) ratio: Concentration of the test compound in whole blood compared with plasma. DBS-plasma bridging studies: Collection of both blood (collected as DBS) and plasma for analysis from the same timepoint from an in vivo preclinical study. ISR: Repeat extraction and analysis of select samples performed in order to establish confidence in the reliability and reproducibility of a validated method.

cial populations [16,17] , and the significant savings in shipping costs [18] . However, the change of matrix from plasma to blood introduces an additional variable compared with what has become the gold standard ‘plasma’ and requires an understanding of the relationship of the partitioning/association of the drug to the red blood cells (RBC). This is typically accomplished by generating in vitro blood:plasma (B:P) data or by generating in vivo data  [3,19–21] . Understanding the blood-to-plasma (B:P) ratio as well as ruling out any time or concentration dependent differences in the partitioning/association of the drug with the RBCs is important in deciding if one should proceed with using DBS sampling. Additionally, once the B:P ratio is known, DBS data can be transformed to plasma equivalents and vice-versa [19] . One of the main challenges with DBS assays is related to the ‘hematocrit effect’ which results in an assay bias. Additionally, DBS assays are also impacted by assay sensitivity, since the volume of blood actually used for sample extraction and analysis would be equivalent to approximately 2 to 4 μl of blood (from a 3 mm diameter DBS punch), in contrast to using 50 to 100 μl of plasma for a standard analysis [13] . Although DBS sample collection process is relatively simple, compared with the generation of plasma, DBS sample analysis is more demanding since additional analytical challenges need to be overcome and extra validation experiments are needed [11,22,23] . The evolution and application of DBS and the challenges that need to be overcome have been discussed in detail in two recent reviews [12,13] . Although the implementation of DBS sampling and analysis to support drug development has been slow, mainly due to a combination of the lack of formal acceptance of the technique by regulatory agencies and the issue of the hematocrit (Hct) bias, it can be successfully adopted following the demonstration of concordance between the blood and plasma and the execution of carefully designed validation experiments [14] .

870

Bioanalysis (2015) 7(7)

This report describes the sequence of experiments that were executed during preclinical development of LY3023414 that included non-GLP DBS-plasma bridging studies employing fit-for-purpose methodologies to understand the relationship between blood and plasma as well as ex vivo blood:plasma ratios, the subsequent development and validation of a DBS LC– MS/MS methodology that meets the current regulatory guidelines [24] and its successful application for evaluating TK in rat and dog studies for LY3023414, an orally available, potent selective inhibitor of the class I PI3K isoforms and mTOR [25] . Evaluations of Hct as well as incurred sample reanalysis (ISR) are also demonstrated. DBS sampling and analysis were adopted in anticipation of compliance with the principles of the 3Rs (reduction in the number of TK animals) as well as the potential well-documented advantages in sample collection and shipping during the conduct of global clinical trials. Experimental section Chemicals & reagents

HPLC grade acetonitrile (ACN), HPLC grade methanol (MeOH) and formic acid (96%), were purchased from Sigma-Aldrich (MO, USA). ACS grade DMSO was purchased from Fisher (MA, USA). Type 1 water was obtained from an ELGA system (ELGA Labwater, Missisauga, Ontario, Canada). LY3023414 and its stable isotope-labelled internal standard (IS) (Supplementary Figure 1) were obtained from the Eli Lilly and Company molecule inventory (IN, USA). Fresh rat and dog blood was purchased from Biochemed (VA, USA) and stored refrigerated until used as described later (blood was shipped overnight and used within three days from receipt). Equipment

FTA DMPK-C cards (hereafter referred to as DBS cards) were purchased from GE Healthcare Life Sciences (PA, USA). Ninety-six deep-well Axygen plates were purchased from Corning Inc. (MA, USA). Semiautomatic DBS card puncher was purchased from Tomtec (CT, USA). Desiccant sachets were purchased from Sigma-Aldrich (MO, USA). All other items were standard laboratory equipment (balances, pipettors, vials, centrifuges, etc.). Stock solutions

Stock standard solutions for LY3023414 were prepared, in duplicate, at a final concentration of 400 μg/ml DMSO:MeOH (1:1, v:v) using the appropriate weight corrected for purity, moisture and salt correction, and compared. The calibration standards and the QC samples were prepared from separate stock standard

future science group

DBS analysis for rat & dog studies: validation, hematocrit, toxicokinetics & incurred sample reanalysis

solutions. A series of intermediate solutions were prepared at concentrations of 0.05, 0.1, 0.5, 2.5, 10, 25, 40 and 50 μg/ml in DMSO:MeOH (1:1, v:v) using positive displacement pipettes for the preparation of calibration standards. Additional intermediate solutions were also prepared at 0.5, 0.15, 2.5, 35 and 400 μg/ml for the preparation of QC samples. Stock standard solution for the IS was prepared at a final concentration of 100 μg/ml in DMSO:MeOH (1:1, v:v) using the appropriate weight corrected for purity, moisture and salt correction. This was diluted to a 500 ng/ml intermediate stock in ACN:MeOH:water (3:3:2, v:v:v), and finally to yield a 2 ng/ml working solution in ACN:MeOH:water (3:3:2, v:v:v). All solutions were stored in glass vials in a refrigerator at 2 to 8°C. Preparation of pooled calibration, QC & stability samples

All the calibration and QC samples were prepared by subsequently diluting the corresponding intermediate solutions by 20-fold in matrix (whole blood) to yield calibration standards at 2.5, 5, 25, 125, 500, 1250, 2000 and 2500 ng/ml and QC samples at 2.5, 7.5, 125, 1750 and 20,000 ng/ml. Dilutions were made into appropriate volumes of blank whole blood placed in to 2-ml polypropylene tubes from which an amount of whole blood equal to the volume of source solution to be added was removed and then the same volume of source solutions were added, using a positive displacement pipette and mixed by gently inverting several times (no vortexmix). Following which 20 μl of each was placed onto the center of the circular target area of the pre-labeled DBS cards. The spots were allowed to dry at room temperature for at least 4 h then stored at room temperature in plastic bags containing desiccant, until analyzed. DBS sample extraction

A 4.7-mm diameter disc was punched from each timepoint using a Tomtec semi-automatic puncher in to a 96 deep-well Axygen® plate. Three hundred microliters of ACN:MeOH:water (3:3:2, v:v:v) was added to the matrix blank and reagent blank. Three hundred microliters of the 2 ng/ml IS solution (in 3:3:2, v:v:v, ACN:MeOH:water) was added to all other samples, and the plate was vortex-mixed at medium speed for 25 min, then centrifuged at approximately 1640 × g for 5 min. One hundred microliters of the supernatant was transferred to a Axygen® 96-well collection plate following which 500 μl of MeOH:water (2:3, v:v) was added to each sample. The 96-well plate was covered with a dimpled sealing mat (Corning Inc., MA, USA), vortex-mixed at a low speed for approximately 1 min and analyzed. If needed, samples extracts were stored refrigerated prior to injection.

future science group

Research Article

LC system & chromatographic conditions

A Shimadzu HPLC system consisting of LC–20AD pumps, SIL-20AC autoinjector and CT-20AC column heater (Shimadzu Co, MD, USA) was used. An Xbridge® C18, 50 × 2.1 mm, 5 μm, HPLC column (Waters Corp., MA, USA) was used with a 0.5 μm prefilter (Sigma-Aldrich). Mobile phase solvent A consisted of 0.5% formic acid in HPLC–grade bottled water and solvent B consisted of 0.5% formic acid in ACN. The injector wash solution was 0.5% formic acid in ACN:water (2:8, v:v). Analytes were separated using a linear gradient starting and held at 10% solvent B for 0.3 min, increased to 15% at 0.6 min and held at 15% till 1.2 min. It was ramped to 90% solvent B at 1.8 min and held till 2.8 min and then reduced to 10% at 2.9 min. The HPLC column was held at 40°C and a flow rate of 0.8 ml/min was used with an injection volume of either 4 or 8 μl. The total cycle time was approximately 4 min. Mass spectrometric conditions

Mass spectrometric data were generated using an AB Sciex Triple Quadrupole 5500 mass spectrometer and acquired using Analyst ® Software, v 1.4 (Applied BioSystems, CA, USA). Full scan and selected reaction monitoring (SRM) acquisitions were performed at unit resolution using positive ion atmospheric pressure ionization at a source temperature of approximately 650°C and an IonSpray voltage of approximately 1500 V. Ultra High Pure (UHP) nitrogen was used as the nebulizer gas, curtain gas, collision gas as well as the turboionspray gas. Full scan and product ion spectra were acquired via direct infusion and the following SRM transitions were used for quantification; 407.2→335.2 for LY3023414 (dwell time 250 ms) and 411.2 →339.2 for the IS (dwell time 200 ms). A collision energy of 43 eV was used for both analytes. Calibration curve

Three batches of freshly prepared calibration curves ranging from 2.5 ng/ml (LLOQ) to 2500 ng/ml (ULOQ) were prepared and analyzed along with QC samples, in replicates of six at each concentration. The concentrations tested were 2.5 ng/ml (LLOQ), 7.5 ng/ml (LQC), 175 ng/ml (MQC) and 1750 ng/ml (HQC). In order to evaluate sample dilution analysis, six replicates of the dilution QC [DQC (20,000 ng/ml)] samples were extracted and diluted ten-fold (with blank blood DBS extract containing IS), to fall within the calibration range and extracted and analyzed. The internal standard responses were also evaluated from the calibration standards, LLOQ, LQC, MQC and HQC samples.

www.future-science.com

871

Research Article  Wickremsinhe Selectivity

Sample collection stability

Aliquots of blank dry blood spots from six different lots of rat and dog whole blood were extracted (without IS) and analyzed for endogenous interferences. The same lots of rat and dog blood were also spiked at the LLOQ (2.5 ng/ml) concentration, extracted (with IS) and analyzed. Additionally, blank matrix sample spiked with IS only and blank matrix sample spiked with LY3023414 at the 2500 ng/ml (ULOQ) without IS were extracted and analyzed in order to assess potential interferences that may affect either the analyte or the IS. Six replicates of LLOQ samples were also analyzed across three batches to assess assay sensitivity.

Whole blood samples pre-incubated to approximately 37°C were spiked at the LQC and HQC concentrations. The LQC and HQC whole blood pools were split into two portions (T0 and T4). Aliquots were spotted immediately post spike (T0), and after 4 h at room temperature conditions (T4). The samples were allowed to dry for at least 4 h, then extracted and analyzed. Stability was considered acceptable if the mean accuracy at each concentration was within the range of ±15.0% bias.

Matrix factor

Extracts of blank matrix from the same six lots were spiked post extraction at the LQC and HQC sample concentration with IS. In addition, three replicates of a pure solution containing LY3023414 and IS at the same concentration as the spiked extract were prepared and injected. The matrix factor was calculated as the ratio of the peak response in the presence of matrix ions to the mean peak response in the absence of matrix ions. Recovery

Recoveries were determined by comparing the mean peak area of extracted LQC, MQC and HQC samples with the mean peak area of recovery samples, prepared by adding LY3023414 and the IS to blank DBS extracts from rat and dog, at concentrations corresponding to the LQC, MQC and HQC samples.

Short-term matrix stability

Sets of six replicates each of LQC and HQC samples were stored at 40°C for 48 h, -20°C for 48 h and at room temperature for 24 h to account for extremes of temperature the samples may be subjected to en-route to the bioanalytical lab. The samples were extracted and analyzed and stability was considered acceptable if the mean accuracy at each concentration was within the range of ±15.0% bias. Long-term matrix stability

Six replicates of LQC, HQC and DQC samples were stored at room temperature in sealed plastic bags containing sachets of desiccant for a period of 3 to 6 months (sufficient to complete the analysis of the 1-month TK study samples). The samples were extracted and analyzed and stability was considered acceptable if the mean accuracy at each concentration was within the range of ±15.0% bias. Spotting volume assessment

Carryover

Extracts of blank matrix, in duplicate, were extracted and analyzed immediately following the two highest calibration standards. This would account for punch carry-over as well as auto-injector and LC system carryover. Stock solution stability

Solution stability was evaluated for the stock solutions of LY3023414 as well as the IS for 6 h at room temperature and in a refrigerator set to maintain 2 to 8°C. Stability was considered acceptable if the mean difference between the compared standard solutions was ≤5.0%. Reinjection reproducibility

An extracted calibration curve, and six replicates of LQC, MQC and HQC samples, were processed and stored at conditions similar to injector conditions over at least 4 days and reanalyzed. Stability was considered acceptable if the mean accuracy at each concentration was within the range of ±15.0% bias.

872

Bioanalysis (2015) 7(7)

Whole blood samples of the LQC and HQC concentrations were spotted in aliquots of 10, 20 and 30 μl on the DBS cards. This experiment was to evaluate the impact of the volume of blood placed on the DBS card. Six replicated of each QC at each aliquot volume were analyzed. Results were considered acceptable and ruled out any impact of the volume of blood spotted (within the range tested), if the mean concentration of the spiked samples had a percent RSD of ≤15.0% and mean accuracy within the range of ±15.0% bias. Hct assessment

Dog and rat blood adjusted to Hct 30, 45 and 60% were purchased from Biochemed (Winchester, VA, USA). LQC and HQC samples were prepared in whole blood with Hct levels of 30, 45 and 60%. Six replicated of each QC at each Hct level were spotted on the DBS cards and analyzed against a standard curve prepared with Hct 45% whole blood. Results were considered acceptable and ruled out any Hct bias (within the Hct range tested) if the mean concentration of the spiked

future science group

DBS analysis for rat & dog studies: validation, hematocrit, toxicokinetics & incurred sample reanalysis

samples had a percent RSD of ≤15.0% and mean accuracy within the range of ±15.0% bias. Dog TK study

Male and female Beagle dogs (Covance Research Products Inc., VA, USA) were administered 1, 3 or 6 mg/kg LY3023414 via oral gavage. LY3023414 was formulated in 1% hydroxyethylcellulose (w/v), 0.25% polysorbate 80 (w/v) and 0.05% Dow Corning® Antifoam 1510US (v/v) in reverse osmosis water and dosed once a day for 28 days. All dose preparations were made daily. The 1 and 3 mg/kg doses had three animals/sex/dose while the 6 mg/kg dose had five animals/sex. Blood samples (approximately 1 ml) were collected via a jugular vein into vacutainer tubes containing potassium (K 2) EDTA on Days 1, 14 and 28 of the dosing phase predose (Days 14 and 28 only) and approximately 0.5, 1, 2, 4, 8 and 24 h post dose. Four approximately 20-μl aliquots/time point were placed on the DBS card and allowed to dry for at least 4 h at room temperature. After drying, the DBS cards were stored in a sealed bag with desiccant at room temperature until analyzed. Rat TK study

Male and female Sprague-Dawley® rats (Charles River Laboratories, NC, USA) were administered 5, 15 or 30 mg/kg LY3023414 via oral gavage. LY3023414 was formulated in 1% hydroxyethylcellulose (w/v), 0.25% polysorbate 80 (w/v) and 0.05% Dow Corning® Antifoam 1510-US (v/v) in reverse osmosis water and dosed once a day for 28 days. All dose preparations were made daily. The 5 and 10 mg/kg doses had a total of ten animals/sex/dose while the 30 mg/kg dose had 15 animals/ sex. However, only three animals/sex/dose were used for blood sampling. Blood samples (approximately 80 μl) were collected via the lateral tail vein on Days 1, 14 and 28 of the dosing phase, predose (Days 14 and 28 only) and approximately 0.5, 1, 2, 4, 8 and 24 h post dose. Blood samples were collected from three animals/ group/sex. A needle (with no syringe) was inserted in to the tail vein and an EDTA coated capillary tube was placed against the hub of the needle to collect the DBS samples (total of four capillary tubes per timepoint). The needle would remain in place for all four collections for a single time point. Four spots of approximately 20 μl at each time point were placed on the DBS card and allowed to dry for at least 4 h at room temperature. After drying, the DBS cards were stored in a sealed bag with desiccant at room temperature until analyzed. The method validation, in vivo TK studies and the sample analyses described above were conducted at Covance, WI, USA. The bridging studies described below, which paved the way for using DBS, were conducted at Covance, IN, USA.

future science group

Research Article

Key term Method validation: Completion of experiments designed to enable reliable and reproducible quantitative measurement of analytes in a given biological matrix.

DBS-plasma bridging study to establish concordance in rats

Surgically modified (arterial catheter) male SpragueDawley ® rats (from Harlan Laboratories, IN, USA) were given a single oral dose by gavage at 10 and 30 mg/kg LY3023414 formulated in 1% hydroxyethylcellulose (w/v), 0.25% polysorbate 80 (w/v) and 0.05% Dow Corning® Antifoam 1510-US (v/v) in reverse osmosis water. Blood samples were taken at 0.5, 1, 2, 4, 8 and 24 h post dose. At each time point, approximately 250 μl of blood was collected via the arterial catheter using a needle and syringe. A heparin lock solution was maintained in the catheter between collections. The blood was immediately transferred to an EDTA tube, mixed and three 20 μl aliquots were spotted on to DBS cards. The remaining blood was immediately processed to generate plasma. The DBS cards were placed in zip-lock bags with desiccant sachets and shipped and stored without refrigeration. The plasma samples were shipped and stored frozen. DBS-plasma bridging study to establish concordance in dogs

Male Beagle dogs (from Charles Rivers, IN, USA) were given a single oral dose by gavage at 4.5 and 9 mg/kg each of LY3023414 formulated in 1% hydroxyethylcellulose (w/v), 0.25% polysorbate 80 (w/v) and 0.05% Dow Corning ® Antifoam 1510-US (v/v) in reverse osmosis water. Approximately 500 μl of blood samples were taken at 0.5, 1, 2, 4, 8 and 24 h post dose via the jugular vein using an EDTA vacutainer tube. Three 20 μl aliquots were spotted on to DBS cards and the remaining blood was processed to generate plasma. The DBS cards and plasma were handled as described above. DBS-plasma bridging study sample extraction & analysis

The DBS and plasma samples were extracted and analyzed using a fit-for-purpose methodology established for non-GLP studies. A single 3-mm disc was punched from each timepoint, extracted with 100 μl 1:1 MeOH:ACN (containing IS). Whole blood used for standard curve preparation was used within 3 days of refrigeration. Twenty-five microliters aliquots of plasma from each time point were transferred to a 96-well plate, protein precipitated with 100 μl of 1:1 MeOH:ACN (containing IS) and quantitated by LC–MS/MS.

www.future-science.com

873

Research Article  Wickremsinhe Results Establishing concordance between plasma & DBS

DBS and corresponding plasma concentration data from the rat and dog bridging studies are depicted in Figure 1. The fit-for-purpose assay used to support this bridging study had a range from 1 to 5000 ng/ml. The concentration data show a good concordance between the plasma and DBS. The slope of the regression line depicts the DBS:plasma ratios (blood-to-plasma ratio). These data show a DBS:plasma ratio of approximately 0.9 in rats and 1.1 in dogs, demonstrating no preferential uptake or association of LY with the cellular components of the blood (mainly the RBC). Additionally, there were no apparent time or concentration dependent bias in either rats or dogs since no trends or aberrant data points were observed over the entire sampling period (0.5–24 h) and across the entire assay range (Cmax to LLOQ). Statistical analysis of the bridging data was conducted and showed good concordance between the two matrices (plasma and DBS), using previously described mthodology [19] . These data were the basis for using DBS sampling and analysis to support the 28-day rat and dog TK studies and the validation of the analytical methodology described below. Accuracy & precision

The validation data showed precise and accurate quantification of LY3023414 from both rat and dog DBS samples within a range from 2.5 ng/ml (LLOQ) to A

B

5000

2500 ng/ml (ULOQ) and also the ability to quantify concentrations up to 20,000 ng/ml following a tenfold dilution. Calibration curve was constructed using a weighted (1/x 2) linear least-squares regression. The assay precision as measured by the RSD was ≤15.0% (≤20.0% at the LLOQ) and the accuracy as measured by the% bias was within the range of ±15.0% (within ±20.0% bias at the LLOQ) for both rat and dog. Overall assay accuracy and precision data for the rat are summarized in Table 1 (data in dog were similar and are not shown). Selectivity

There were no significant interferences or interference peaks detected in all six lots/individuals of control rat blood and dog blood samples tested (no signals detected that were > 20.0% of the LLOQ response or >5.0% of the IS response). Each of the six lots of rat and dog blood spiked at the LLOQ concentration of LY3023414 and with IS demonstrated accuracy within the range of ±20.0% bias, and the percent RSD ≤ 20.0%. Blank matrix samples, reagent blanks, blank matrix samples spiked with IS only (control zero) and blank matrix sample spiked with LY3023414 alone at 2500 ng/ml and without IS were analyzed in order to assess potential interferences that may affect either the analyte or the IS. In all cases, the LY3023414 and IS regions were free from significant interference (no signals detected that were >20.0% of the LLOQ response or >5.0% of the IS response). This demonstrates the selectivity of the analytical methodology and also

5000 Dog

4000

DBS concentration (ng/ml)

DBS concentration (ng/ml)

Rat

3000

2000

1000

y = 0.940 x - 16.58 R2 = 0.979

0

4000

3000

2000

1000

y = 1.100x + 19.47 R2 = 0.975

0 0

1000

2000

3000

4000

Plasma concentration (ng/ml)

5000

0

1000

2000

3000

4000

5000

Plasma concentration (ng/ml)

Figure 1. Dried blood spot and corresponding plasma concentrations following the administration of a single oral dose of LY3023414 to (A) rats and (B) dogs. DBS: Dried blood spot.

874

Bioanalysis (2015) 7(7)

future science group

DBS analysis for rat & dog studies: validation, hematocrit, toxicokinetics & incurred sample reanalysis

Research Article

Table 1. Precision (% RSD) and accuracy (% bias) data from the three-batch validation of the rat dried blood spot assay. Run

QC 2.50 ng/ml

QC 7.50 ng/ml

QC 125 ng/ml

QC 1750 ng/ml

Batch 1 (day 1)

2.22

7.46

126

1860

 

2.32

8.23

126

1710

 

2.98

7.56

129

1790

 

3.04

7.26

133

1770

 

2.79

7.00

123

1790

 

2.89

7.77

129

1730

Intrarun RSD (%)

12.9

5.6

2.7

3.0

Intrarun% bias

8.4

0.7

2.4

1.7

Batch 2 (day 2)

2.77

7.88

128

1790

 

2.83

7.61

134

1810

 

2.68

7.67

132

1770

 

2.64

7.95

133

1850

 

2.58

7.37

131

1800

 

2.76

7.85

127

1800

Intrarun RSD (%)

3.4

2.8

2.1

1.5

Intrarun% bias

8.4

2.9

4.8

2.9

Batch 3 (day 3)

2.63

8.12

136

1980

 

2.43

7.58

133

1880

 

2.32

7.35

138

1870

 

2.47

7.61

134

1880

 

2.67

7.53

132

1910

 

2.42

7.57

135

1900

Intrarun RSD (%)

5.4

3.4

1.6

2.1

Intrarun% bias

-0.4

1.7

8.0

8.6

Inter-run RSD (%)

8.9

4.0

3.0

3.8

Inter-run% bias

5.6

1.7

4.8

4.6

†

†

>20% bias.

confirms the ability for unbiased quantification of LY3023414 and its IS from DBS samples. Matrix factor

The ratio of the peak responses between extracts of blank matrix from the same six lots/individuals spiked post extraction at the LQC and HQC sample concentration with IS compared with pure solution containing LY3023414 and IS at the same concentration ranged between 0.98 and 1.07 (CV = 3.7%) across the six lots of rat blood and 0.97 to 1.10 (CV = 2.6%) in across the six lots of dog blood. Value greater than 1 indicates ionization enhancement, value less than 1 indicates ionization suppression. Overall, there were no significant impact of the matrix factor that could potentially impact the quantification of LY3023414 and its IS.

future science group

Recovery

The recovery of LY3023414 and its IS were consistent across the three concentrations tested. Recoveries averaged approximately 76% for LY3023414 and 90% for the IS in rat DBS samples and approximately 84% for LY3023414 and 90% for the IS in dog DBS samples (Table 2) . Carryover

There are two potential contributing sources for carryover; the HPLC system and the DBS Puncher. During method development, carryover was noted that approximated between 10 and 30% of the response of LLOQ DBS sample (after an ULOQ DBS sample). A combination of the following were implement during validation and sample analysis, in order to control and manage carryover; punching a blank DBS card immediately after all

www.future-science.com

875

Research Article  Wickremsinhe

Table 2. Recovery data for LY3023414 and the IS in rat and dog dried blood spots.  Treatment

LY3023414

 

IS

Extracted QC sample peak area

Recovery sample peak area

Extracted QC sample peak area

Recovery sample peak area

Mean

11,051.0

14,712.1

154,392.7

174,267.8

SD

351.4

408.4

3265.7

1854.1

7.5 ng/ml rat

RSD (%)

3.2

2.8

2.1

1.1

Recovery%

 

75.1

 

88.6

Mean

191,678.9

250,218.2

156,931.7

170,741.4

SD

3587.9

4243.1

3588.6

3431.9

125 ng/ml rat

RSD (%)

1.9

1.7

2.3

2.0

Recovery%

 

76.6

 

91.9

750 ng/ml rat Mean

2,538,107.6

3,399,681.5

146,893.0

166,469.9

SD

141,757.5

15,720.2

7223.5

1962.9

RSD (%)

5.6

0.5

4.9

1.2

Recovery%

 

Overall rat recovery (%)

74.7

 

88.2

75.5

 

89.6

7.5 ng/ml dog Mean

5535.0

6359.5

70,072.0

75,342.3

SD

267.1

209.8

880.7

360.8

RSD (%)

4.8

3.3

1.3

0.5

Recovery%

 

87.0

 

93.0

90,444.1

112,594.4

70,865.0

80,433.5

125 ng/ml dog Mean SD

1973.4

1198.8

957.8

741.6

RSD (%)

2.2

1.1

1.4

0.9

Recovery%

 

80.3

 

88.1

1,301,274.3

156,5471.0

72,937.3

81,290.5

750 ng/ml dog Mean SD

30,485.6

17,925.8

1195.8

990.5

RSD (%)

2.3

1.1

1.6

1.2

Recovery%

 

83.1

 

89.7

83.5

 

90.3

Overall dog recovery (%)

high concentration samples (since the DBS puncher was identified as a source of carryover), processing (punching) DBS cards from low to high concentration and selecting an HPLC system with low carryover for the sample analysis. Although the individual carryover attributed respectively to the DBS puncher and the HPLC system were not separately evaluated, the component of DBS carryover was most likely due to its solid bore design where the entire area of the punch came in contact with the DBS

876

Bioanalysis (2015) 7(7)

spot (as opposed to a having a hollow bore cylinder design where only the circumference of the tool comes in contact with the DBS spot). Therefore, during validation and sample analysis, a blank card was punched immediately after all high concentration samples. Stability

Data show that the rat DBS samples were stable for 189 days and dog DBS samples for 92 days (tested at 7.5,

future science group

DBS analysis for rat & dog studies: validation, hematocrit, toxicokinetics & incurred sample reanalysis

1750 and 20,000 ng/ml) when stored at room temperature in sealed plastic bags containing sachet of desiccant. The established room temperature was sufficient to cover the duration of this study as well as the analysis of all rat and dog study samples generated from future studies. Stability was also established over a period of 48 h at 40°C and 48 h at -20°C in an attempt to capture potential extremes of temperature the samples may get subjected to during the process of shipping and handling  [26] . A summary of all the stability experiments (e.g., stock solution and extract stability) are shown in Table 3. Reinjection reproducibility was established over 5 days for dogs and 6 days for rats. This would ensure sufficient stability to enable reinjection of a failed batch left on the autosampler over a long weekend.

Research Article

corresponding data are summarized in Table 5. The results show a negative bias when using Hct 30 blood and a positive bias when using Hct 60 blood, compared with Hct 45 blood, as expected and well-reviewed in the literature [30,31] . However, the bias was within the ±15% allowed for assay validations. The Hct values of all the rats (n = 100) and dogs (n = 32) used for the rat and dog toxicology/TK study are shown as Figure 2. The data show that all the Hct values in dogs ranged between 37 and 51 (overall mean and SDs of 43 ±3) and in rats, between 39 and 52 (overall mean and SDs of 45 ±3) and in agreement with published standard references  [32,33] . Therefore all the values were well within the validated Hct range described above. TK study sample analysis

Spotting volume

The impact of the actual volume of blood placed/ spotted on the DBS cards was evaluated using both rat and dog blood volumes of 10, 20 and 30 μl and summarized in Table 4. The results depict that there was no impact of the actual volume of blood spotted and therefore a standard 20 μl capillary or disposable pipette could be used for the placement of blood on to the DBS cards. For this study the DBS cards were spotted using a 20 μl capillary tubes and was executed by the animal facility technicians without any issues or errors. Analysis was feasible as long as the spot was larger than the area of the DBS punch (4.7 mm diameter). The data presented here are in excellent agreement with previous reports showing the absence of a blood volume effect, thus avoiding the need for using a calibrated measuring device to accurately measure the volume of blood placed on the DBS cards [10,27–29] . Hct

The impact of the blood Hct was evaluated using both rat and dog blood at Hct values of 30, 45 and 60. The

A total of 402 rat samples and 640 dog samples were analyzed. Each analytical batch consisted of at least one calibration curve, a matrix blank, a control zero (matrix blank containing IS), a reagent blank and duplicate QC samples at three concentrations within the calibration range. Hct 45 blood was used to prepare all matrix blank, calibration curve and QC samples. All the samples were quantified using the validated assay range. Dilution QC samples were included in batches where samples were diluted prior to analysis. No significant carryover issues were encountered during the analysis. The concentrations versus time profiles for LY3023414 across the three dose groups and both genders are shown in Figure 3 and the corresponding TK parameters are summarized in Table 6. Incurred sample reanalysis

The analysis of Incurred sample reanalysis (ISR) has been proposed as a means to improve the confidence in the reliability and reproducibility of a validated method during the analysis of study sample analysis [34] . In this study, 47 rat samples and 67 dog samples were selected

Table 3. Summary of stability experiments. Test

Conditions

Established stability

Stock standard solution of LY3023414

Room temperature

6h

Intermediate solution of LY3023414

Room temperature

6h

Stock standard solution of LY3023414

Refrigerated

48 days

Reinjection reproducibility (extract)

Refrigerated

6 days rat, 5 days dog

Sample collection – whole blood stability

Room temperature

5 h rat, 4 h dog

Short-term in matrix (DBS)

Approximately 40°C

48 h

Short-term in matrix (DBS)

Approximately -20°C

48 h

Short-term in matrix (DBS)

Room temperature

24 h

Long-term matrix stability (DBS)

Room temperature

189 days rat and 92 days dog

DBS: Dried blood spot.

future science group

www.future-science.com

877

Research Article  Wickremsinhe

Table 4. Influence of volume of blood used for spotting on assay performance. Aliquots of 10 μl dried blood spot and 30 μl dried blood spot compared with 20 μl dried blood spot using a 4.7 mm diameter punch.  Treatment

QC 7.50 mean ng/ml QC 7.50 % RSD (% bias)

QC 1750 mean ng/ml QC 1750 % RSD (% bias)

Rat 10 μl DBS

7.91 (5.5)

1.8

1830 (4.6)

1.6

Rat 20 μl DBS

8.06 (7.5)

5.3

1780 (1.7)

1.6

Rat 30 μl DBS

7.34 (-2.1)

2.4

1730 (-1.1)

2.7

Dog 10 μl DBS

7.51 (0.1)

3.7

1760 (0.6)

2.7

Dog 20 μl DBS

7.73 (3.1)

3.5

1810 (3.4)

2.5

Dog 30 μl DBS

7.73 (3.1)

3.8

1880 (7.4)

1.8

DBS: Dried blood spot.

(>10% of total number of samples from each species) and subjected to ISR. The samples were selected to represent all the animals used in the study and concentrations were selected to represent the maximum concentrations as well as samples from the elimination phase of the exposure profile. The data corresponding to the original analysis and the ISR analysis are plotted as Figure 4. The ISR acceptance criteria are defined as at least two-thirds (rounded up) of the repeat results and original results to be within 20% of each other [34] . Overall, the difference between the original and ISR analysis for all (100%) of the rat samples and 97% of the dog samples were within 20%, and overwhelmingly met the acceptance criteria for ISR. The ISR analysis was conducted using one of the unused additional spots (from a total of four spots), in contrast to thawing a frozen plasma sample and drawing an aliquot for ISR, consistent with methodology used in previously reported DBS ISR data [35] . Discussion To date, DBS has been used to support a wide diversity of studies including clinical and non-clinical PK studies to biomarkers during all phases of drug devel-

opment (discovery, preclinical and clinical studies) [36] . However, the introduction and implementation of DBS sampling and analysis has been received with mixed reviews, partially due to the need for generation of additional data to support such a change by the pharmaceutical industry as well as the lack of formal ‘acceptance’ of the methodology by the regulatory agencies [23] . Hence, individual companies have come up with their own best practices and guidelines with regard to adopting and implementing DBS sampling and analysis [37] . The rationale for implementing DBS sampling for this analyte was also based on similar guidelines as well as the guidance provided by Rowland and Emmons [38,39] . One of the key factors that have created concern among the bioanalytical community and the regulatory agencies is the ‘Hct effect’ [30,31] . This phenomenon is caused by the fact that blood with a low Hct will tend to disperse/spread easily on the DBS card and produce a ‘larger’ spot compared with blood that has a high Hct. Therefore, a potential bias is created as a result of sampling (punching out) a fixed area from the DBS card, depending on the Hct of the blood that is being sampled [40–43] . However, the extent of this bias could be well within the acceptance criteria of the

Table 5. Influence of blood hematocrit on assay performance. Hematocrit 30 dried blood spot and hematocrit 60 dried blood spot compared with hematocrit 45 dried blood spot using a 4.7 mm diameter punch.  Treatment

QC 7.50 mean ng/ml (% bias)

QC 7.50 % RSD

QC 1750 mean ng/ml (% bias)

QC 1750 % RSD

Rat Hct 30 DBS

6.72 (-10.4)

3.1

1580 (-9.7)

2.4

Rat Hct 45 DBS

7.75 (3.3)

6.3

1840 (5.1)

1.1

Rat Hct 60 DBS

8.13 (8.4)

6.4

1880 (7.4)

1.7

Dog Hct 30 DBS

7.14 (-4.8)

5.0

1660 (-5.4)

2.2

Dog Hct 45 DBS

7.67 (2.3)

4.2

1840 (5.1)

3.4

Dog Hct 60 DBS

7.74 (3.2)

2.2

1870 (6.9)

2.7

DBS: Dried blood spot; Hct: Hematocrit.

878

Bioanalysis (2015) 7(7)

future science group

DBS analysis for rat & dog studies: validation, hematocrit, toxicokinetics & incurred sample reanalysis

B 80

80

70

70

60

60 Hematocrit (%)

Hematocrit (%)

A

Research Article

50

50

40

40

30

30

20

20 Male

10

Female

Male 10

Rats

Female

Dogs

Figure 2s. Hematocrit values from the (A) 50 male and 50 female rats and (B) 16 male and 16 female dogs used in the toxicology/TK study. The values are from study day 1 and are arranged in ascending order for each gender. For color images please see online at: www.future-science.com/doi/full/10.4155/BIO.15.12.

10,000

Dried blood spot concentration (ng/ml)

1000

1 mg/kg male

1 mg/kg female

3 mg/kg male

3 mg/kg female

6 mg/kg male

6 mg/kg female

100

10

1

0.1 0

4

8

12

Time (h) Figure 3. Concentration versus time profiles following the administration of a single oral dose of 1 mg/kg, 3 mg/kg and 6 mg/kg of LY3023414 to male and female dogs.

future science group

www.future-science.com

879

Research Article  Wickremsinhe

Table 6. Mean ( ±SD) TK parameters in rats and dogs on day 1, representing a single oral dose of LY3023414. Parameter

Administered dose of LY3023414

Sex

M

F

Rat

5 mg/kg

M

F

M

15 mg/kg

F 30 mg/kg

AUC0–24 h (ng·hr/ml)

3948 ± 224

3554 ± 865

9525 ± 1864

12,036 ± 1494

21,209 ± 6209

14,700 ± 2593

Cmax (ng/ml)

342 ± 84

520 ± 267

1024 ± 228

1212 ± 317

2400 ± 685

1544 ± 558

Dog

1 mg/kg

3 mg/kg

6 mg/kg

AUC0–24h (ng·hr/ml)

631 ±220

324 ±99

2898 ±576

2122 ±478

6616 ±1107

5308 ±2173

Cmax (ng/ml)

211 ±83

115 ±27

907 ±204

611 ±173

1616 ±225

1494 ±452

validated assay and will depend on the expected Hct range in the actual study animals (or subjects). Typically, the variability in Hct within a given species of healthy laboratory animals is small [32] , and in these situations the expected Hct range could be easily validated using ‘normal’ healthy animal blood and tested against QC samples prepared using the extremes of the expected Hct range. If these QC samples fall within ±15% of its nominal value, the use of DBS sampling and analysis can be justified. Alternative approaches to countering the Hct bias would be to collect and spot an accurate and precise volume of blood and then punch out and analyze the entire spot. Several advancements have been recently reported incorporating novel approaches to mitigate the Hct effect including the use of novel absorbent blood collection matrices, disposable accurate blood collecting/spotting devices, direct on-line extraction of A

Conclusion This report describes a sequence of studies and validation experiments designed to introduce DBS sampling and analysis to support preclinical GLP studies. The conduct and outcome of the in vivo bridging studies, in both rats and dogs, provided the key data and scientific justification to proceed with DBS. The bridging study data were in agreement with the criteria outlined for concordance between the two matrices [19] , as well as the decision tree proposed by Rowland and Emmons [38,39] . Although the implementation of DBS sampling and analysis to support drug development has been slow, mainly due to a combination of the lack of formal acceptance of the technique by regulatory agencies and the issue of the Hct bias, it can be successfully adopted folB

ISR (concentration ng/ml)

ISR (concentration ng/ml)

Rat

entire spot, etc. [43–46] . However, the implementation of these have been slow and yet to be fully realized.

400

40

4

Dog

400

40

4 4

40 400 Original analysis (concentration ng/ml)

4

40 400 Original analysis (concentration ng/ml)

Figure 4. ISR data from the toxicology/TK studies. (A) rat and (B) dog. Solid line represents line of unity.

880

Bioanalysis (2015) 7(7)

future science group

DBS analysis for rat & dog studies: validation, hematocrit, toxicokinetics & incurred sample reanalysis

lowing the demonstration of concordance between the blood and plasma and execution of carefully designed validation experiments. The incorporation of DBS sampling during preclinical studies enables the generation of confidence in the methodology as well as the establishment of concordance between plasma and DBS. However, the overall value and cost savings will be more pronounced with the expansion of DBS to clinical development. The major benefit is the ability to collect PK samples from clinical sites and patients that did not have access or the infrastructure to generate and store/ship plasma samples, thus enabling the collection of PK data from large global trials. Additionally it also provides a convenient technique for collecting blood, which can be extremely beneficial in situations where blood draws can be collected within a home setting without the need for a visit to the clinic – especially for oncology trials and terminally ill patients. This program has continued to clinical development. Future perspective The advantages and challenges of implementing dried blood spot sampling and analyses for the quantification of new drug entities (and their metabolites) have been well documented [11–13] and the pharmaceutical industry have been cautiously adopting its use. Alongside, significant advances are being made across the pharmaceutical industry and the instrument manufacturers to counter the Hct effect as well as to improve the bioanalytical workflow (i.e., avoid punching, 96-well format ‘friendly’). DBS sampling and analyses is not a panacea and is not expected

Research Article

to replace the use of plasma analysis; however, it is expected that microsampling techniques (including improved formats of DBS) will gain wider acceptance and use. Overall, these approaches will enable the generation of better quality data along with the adherence to the 3Rs in preclinical studies and patient convenience and cost savings during the conduct of clinical studies. Acknowledgements The author would to thank H Huang, D Bedwell and K Ruterbories for their support during the early feasibility studies. The author would also like to thank L Lee and C Schmalz for the review of validation data and A Hagler for live-phase study design.

Financial & competing interests disclosure E Wickremsinhe is employed by Eli Lily and company where this work was carried out. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical conduct The author states that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary • Good concordance between blood (dried blood spot) and plasma data. • Successful adoption of dried blood spot sampling to support pivotal nonclinical TK studies. • Hematocrit effect was evaluated during method validation and effectively managed. • Excellent incurred sample reanalysis data.

References 1

Guhad F. Introduction to the 3Rs (Refinement, Reduction and Replacement). J. Am. Assoc. Lab. Anim. Sci. 44(2), 58–59 (2005).

2

Bateman KP, Castonguay G, Xu L et al. Reduction of animal usage by serial bleeding of mice for pharmacokinetic studies: application of robotic sample preparation and fast liquid chromatography-mass spectrometry. J. Chromatogr. B Biomed. Sci. Appl. 754(1), 245–251 (2001).

3

Jonsson O, Palma Villar R, Nilsson LB et al. Capillary microsampling of 25 microl blood for the determination of toxicokinetic parameters in regulatory studies in animals. Bioanalysis 4(6), 661–674 (2012).

future science group

4

Dillen L, Loomans T, Van De Perre G, Versweyveld D, Wuyts K, De Zwart L. Blood microsampling using capillaries for drug-exposure determination in early preclinical studies: a beneficial strategy to reduce blood sample volumes. Bioanalysis 6(3), 293–306 (2014).

5

Jonsson O, Villar RP, Nilsson LB, Eriksson M, Konigsson K. Validation of a bioanalytical method using capillary microsampling of 8 microl plasma samples: application to a toxicokinetic study in mice. Bioanalysis 4(16), 1989–1998 (2012).

6

Nilsson LB, Ahnoff M, Jonsson O. Capillary microsampling in the regulatory environment: validation and use of bioanalytical capillary microsampling methods. Bioanalysis 5(6), 731–738 (2013).

www.future-science.com

881

Research Article  Wickremsinhe 7

Bowen CL, Licea-Perez H, Karlinsey MZ et al. A novel approach to capillary plasma microsampling for quantitative bioanalysis. Bioanalysis 5(9), 1131–1135 (2013).

8

Barfield M, Wheller R. Use of dried plasma spots in the determination of pharmacokinetics in clinical studies: validation of a quantitative bioanalytical method. Anal. Chem. 83(1), 118–124 (2011).

23

Viswanathan CT. Perspectives on microsampling: DBS. Bioanalysis 4(12), 1417–1419 (2012).

24

Zimmer D. New US FDA draft guidance on bioanalytical method validation versus current FDA and EMA guidelines: chromatographic methods and ISR. Bioanalysis 6(1), 13–19 (2014).

25

Courtney KD, Corcoran RB, Engelman JA. The PI3K pathway as drug target in human cancer. J. Clin. Oncol. 28(6), 1075–1083 (2010).

26

Bowen CL, Dopson W, Kemp DC, Lewis M, Lad R, Overvold C. Investigations into the environmental conditions experienced during ambient sample transport: impact to dried blood spot sample shipments. Bioanalysis 3(14), 1625–1633 (2011).

27

Wickremsinhe ER, Abdul BG, Huang NH et al. Dried blood spot sampling: coupling bioanalytical feasibility, bloodplasma partitioning and transferability to in vivo preclinical studies. Bioanalysis 3(14), 1635–1646 (2011).

28

Li Y, Henion J, Abbott R, Wang P. Dried blood spots as a sampling technique for the quantitative determination of guanfacine in clinical studies. Bioanalysis 3(22), 2501–2514 (2011).

29

Damen CW, Rosing H, Schellens JH, Beijnen JH. Application of dried blood spots combined with highperformance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of vincristine and actinomycin-D. Anal. Bioanal. Chem. 394(4), 1171–1182 (2009).

30

O’mara M, Hudson-Curtis B, Olson K, Yueh Y, Dunn J, Spooner N. The effect of hematocrit and punch location on assay bias during quantitative bioanalysis of dried blood spot samples. Bioanalysis 3(20), 2335–2347 (2011).

31

De Kesel PM, Sadones N, Capiau S, Lambert WE, Stove CP. Hemato-critical issues in quantitative analysis of dried blood spots. challenges and solutions. Bioanalysis 5(16), 2023–2041 (2013).

32

Wolford ST, Schroer RA, Gohs FX et al. Reference range data base for serum chemistry and hematology values in laboratory animals. J. Toxicol. Environ. Health 18(2), 161–188 (1986).

33

Davies B, Morris T. Physiological parameters in laboratory animals and humans. Pharm. Res. 10(7), 1093–1095 (1993).

34

Rahavendran SV, Vekich S, Skor H et al. Discovery pharmacokinetic studies in mice using serial microsampling, dried blood spots and microbore LC–MS/MS. Bioanalysis 4(9), 1077–1095 (2012).

Fast DM, Kelley M, Viswanathan CT et al. Workshop report and follow-up – AAPS Workshop on current topics in GLP bioanalysis: assay reproducibility for incurred samples – implications of Crystal City recommendations. AAPS J. 11(2), 238–241 (2009).

35

21

Smith C, Sykes A, Robinson S, Thomas E. Evaluation of blood microsampling techniques and sampling sites for the analysis of drugs by HPLC–MS. Bioanalysis 3(2), 145–156 (2011).

Barfield M, Ahmad S, Busz M. GlaxoSmithKline’s experience of incurred sample reanalysis for dried blood spot samples. Bioanalysis 3(9), 1025–1030 (2011).

36

22

Jimmerson LC, Zheng JH, Bushman LR, Macbrayne CE, Anderson PL, Kiser JJ. Development and validation of a dried

Denniff P, Holliman C, Svensson L, Weng N, Patel S. Bioanalysis zone: DBS survey results. Bioanalysis 6(3), 287–291 (2014).

9

Beaudette P, Bateman KP. Discovery stage pharmacokinetics using dried blood spots. J. Chromatogr. B 809(1), 153–158 (2004).

10

Spooner N, Lad R, Barfield M. Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method. Anal. Chem. 81(4), 1557–1563 (2009).

11

Li W, Tse FL. Dried blood spot sampling in combination with LC–MS/MS for quantitative analysis of small molecules. Biomed. Chromatogr. 24(1), 49–65 (2010).

12

Demirev PA. Dried blood spots: analysis and applications. Anal. Chem. 85(2), 779–789 (2013).

13

Henion J, Oliveira RV, Chace DH. Microsample analyses via DBS: challenges and opportunities. Bioanalysis 5(20), 2547–2565 (2013).

14

Evans C, Arnold M, Bryan P et al. Implementing dried blood spot sampling for clinical pharmacokinetic determinations: considerations from the IQ Consortium Microsampling Working Group. AAPS J. (2014). (Epub ahead of print) 

15

Xu Y, Fang W, Zeng W, Leijen S, Woolf EJ. Evaluation of dried blood spot (DBS) technology versus plasma analysis for the determination of MK-1775 by HILIC-MS/MS in support of clinical studies. Anal. Bioanal. Chem. 404(10), 3037–3048 (2012).

16

Suyagh MF, Kole PL, Millership J, Collier P, Halliday H, Mcelnay JC. Development and validation of a dried blood spot-LC–APCI-MS assay for estimation of canrenone in paediatric samples. J. Chromatogr. B 878(9–10), 769–776 (2010).

17

Yakkundi S, Millership J, Collier P, Shields MD, Mcelnay J. Development and validation of a dried blood spot LC–MS/MS assay to quantify ranitidine in paediatric samples. J. Pharm. Biomed. 56(5), 1057–1063 (2011).

18

Amsterdam P, Waldrop C. The application of dried blood spot sampling in global clinical trials. Bioanalysis 2(11), 1783–1786 (2010).

19

Wickremsinhe ER, Huang NH, Abdul BG, Knotts K, Ruterbories KJ, Manro JR. Preclinical bridging studies: understanding dried blood spot and plasma exposure profiles. Bioanalysis 5(2), 159–170 (2013).

20

882

blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry. J. Chromatogr. B 944, 18–24 (2014).

Bioanalysis (2015) 7(7)

future science group

DBS analysis for rat & dog studies: validation, hematocrit, toxicokinetics & incurred sample reanalysis

Research Article

37

Xu Y, Woolf EJ, Agrawal NG, Kothare P, Pucci V, Bateman KP. Merck’s perspective on the implementation of dried blood spot technology in clinical drug development – why, when and how. Bioanalysis 5(3), 341–350 (2013).

42

De Vries R, Barfield M, Van De Merbel N et al. The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium. Bioanalysis 5(17), 2147–2160 (2013).

38

Emmons G, Rowland M. Pharmacokinetic considerations as to when to use dried blood spot sampling. Bioanalysis 2(11), 1791–1796 (2010).

43

39

Rowland M, Emmons GT. Use of dried blood spots in drug development: pharmacokinetic considerations. AAPS J. 12(3), 290–293 (2010).

Oliveira RV, Henion J, Wickremsinhe ER. Automated direct extraction and analysis of dried blood spots employing on-line SPE high-resolution accurate mass bioanalysis. Bioanalysis 6(15), 2027–2041 (2014).

44

Li F, Ploch S, Fast D, Michael S. Perforated dried blood spot accurate microsampling: the concept and its applications in toxicokinetic sample collection. J. Mass Spectrom. 47(5), 655–667 (2012).

45

Youhnovski N, Bergeron A, Furtado M, Garofolo F. Pre-cut dried blood spot (PCDBS): an alternative to dried blood spot (DBS) technique to overcome hematocrit impact. Rapid Commun. Mass Spectrom. 25(19), 2951–2958 (2011).

46

Denniff P, Spooner N. Volumetric absorptive microsampling: a dried sample collection technique for quantitative bioanalysis. Anal. Chem. 86(16), 8489–8495 (2014).

40

41

Vu DH, Koster RA, Alffenaar JW, Brouwers JR, Uges DR. Determination of moxifloxacin in dried blood spots using LC–MS/MS and the impact of the hematocrit and blood volume. J. Chromatogr. B (15–16), 1063–1070 (2011). Koster RA, Alffenaar JW, Greijdanus B, Uges DR. Fast LC–MS/MS analysis of tacrolimus, sirolimus, everolimus and cyclosporin A in dried blood spots and the influence of the hematocrit and immunosuppressant concentration on recovery. Talanta 115, 47–54 (2013).

future science group

www.future-science.com

883