The influence of the dried blood spot drying time on the recoveries of six immunosuppressants

One of the main advantages of DBS sampling is that it allows the patient to sample at home and send the DBS sample to the laboratory by mail [1-4]. This sampling is considered to be patient friendly because it is less invasive and saves patients transportation costs and time. DBS sampling also has a lower biohazard risk and requires a smaller amount of blood than venous sampling [2,4]. Solid organ transplant recipients are required to use a lifetime of immunosuppressant medications like tacrolimus (TaC), sirolimus (SiR), everolimus (EvE) and cyclosporin A (CyA) to prevent allograft rejection. Bioanalysis and Therapeutic Drug Monitoring of these drugs are necessary because efficacy and toxicity is associated with blood concentrations and/or pharmacokinetic parameters. Therefore, these patients could greatly benefit from immunosuppressant DBS analysis. Since the use of dried blood spot (DBS) analysis for therapeutic drug monitoring (TDM), more extensive validation procedures have been proposed in order to improve the quality of the analysis results. Variations of the hematocrit value, spot volume and DBS stability are among the parameters that should be investigated during method validation [1,5]. A perhaps unappreciated source of variability may be the drying time of a dried blood spot sample. After collection of the blood on the DBS card it should be dried dried at ambient temperature. It is already suggested by the European Bioanalysis Forum (EBF) that the required drying time may be influenced by the hematocrit (HT) and that this may affect the robustness and reproducibility of the assay [5]. Consequently it is recommended to investigate these parameters as part of the validation [5]. Although the DBS may appear dry after 3 hours, the extraction recoveries of the substances within the DBS JOURNAL OF APPLIED BIOANALYSIS, October 2015, p. 116-122. http://dx.doi.org/10.17145/jab.15.019 (ISSN 2405-710X) Vol. 1, No. 4


INtrODUctION
One of the main advantages of DBS sampling is that it allows the patient to sample at home and send the DBS sample to the laboratory by mail [1][2][3][4]. This sampling is considered to be patient friendly because it is less invasive and saves patients transportation costs and time.
DBS sampling also has a lower biohazard risk and requires a smaller amount of blood than venous sampling [2,4]. Solid organ transplant recipients are required to use a lifetime of immunosuppressant medications like tacrolimus (TaC), sirolimus (SiR), everolimus (EvE) and cyclosporin A (CyA) to prevent allograft rejection. Bioanalysis and Therapeutic Drug Monitoring of these drugs are necessary because efficacy and toxicity is associated with blood concentrations and/or pharmacokinetic parameters. Therefore, these patients could greatly benefit from immunosuppressant DBS analysis.
Since the use of dried blood spot (DBS) analysis for therapeutic drug monitoring (TDM), more extensive validation procedures have been proposed in order to improve the quality of the analysis results. Variations of the hematocrit value, spot volume and DBS stability are among the parameters that should be investigated during method validation [1,5].
A perhaps unappreciated source of variability may be the drying time of a dried blood spot sample. After collection of the blood on the DBS card it should be dried on air at room temperature. It is already suggested by the European Bioanalysis Forum (EBF) that the required drying time may be influenced by the hematocrit (HT) and that this may affect the robustness and reproducibility of the assay [5]. Consequently it is recommended to investigate these parameters as part of the validation [5]. Although the DBS may appear dry after 3 hours, the extraction recoveries of the substances within the DBS could still suffer from further drying effects after those 3 hours. This could especially be the case when substance binding to the DBS card matrix by the hydrogen bond acceptors of the substance with the hydrogen groups in the cellulose of the DBS cards is suspected [6][7][8]. Despite this recommendation, many publications describe a drying time of 2 to 3 hours without presenting data to support this period. The most recent review of DBS validation procedures also noted that none of 68 reports included in the review described the effect of aging on the recovery [9].
The objective of this study was to investigate the influence of the drying time of the DBS on the recoveries for the immunosuppressants TaC, SiR, EvE, CyA and their structural analogues ascomycin (AsC) and temsirolimus (TeM).

MAtErIALs AND MEtHODs chemicals and materials
TaC was purchased from USP (Rockville, MA, USA). EvE was purchased from Sigma-Aldrich Inc.
Combined stock solutions containing TaC, EvE, SiR, AsC and TeM were prepared at 2,500 ng/ mL in methanol and CyA at 50,000 ng/mL. Citrate whole blood was purchased from Sanquin (Amsterdam, The Netherlands). The whole blood was stored at 4°C and was used within two weeks after blood donation. To assure the quality of the blood, it was checked for hemolysis prior to use.
Whatman FTA DMPK-C cards (Kent, UK), which are not impregnated with chemicals, were used for the DBS analysis. A XN9000 hematology analyzer from Sysmex (Hyogo, Japan) was used for all hematocrit analyses. All experiments were performed on an Agilent 6460A (Santa Clara, Ca, USA) triple quadrupole LC-MS/MS system, with an Agilent 1290 series combined LC system. All technical parameters were used as described by Koster et al [6]. All precursor ions, product ions, optimum fragmentor voltages and collision energy values were tuned and optimized in the authors' laboratory and are shown in table 1. Agilent Masshunter software for quantitative analysis (version B.04.00) was used for quantification of the analysis results.

Sample preparation
The preparation of the different target hematocrit values was by centrifuging tubes of citrate whole blood with a known HT (measured by a Sysmex XN-9000 analyzer) for 5 minutes at 1,972g. The necessary volumes of plasma were omitted or added to achieve the target Dried blood spot: Drying time 157

3.7
HT values [10]. The prepared HT values were always measured with the Sysmex XN-9000 analyzer in order to confirm the correct HT preparation.
The sample preparation was performed according to a previously published method [6].

Influence of the DBS drying time on the recovery (full spot punch)
Since recoveries could be negatively affected by lower HT values, combined with high concentrations [6][7][8], blood with HT values of 0.10, 0.20, 0.30, 0.40 0.50 and 0.60 L/L were spiked at 100 ng/mL for TaC, SiR, EvE, AsC and TeM and at 2,000 ng/mL for CyA.
After the DBS preparation, the spots were dried for 3, 24 and 48 hours at ambient temperature, directly followed by processing and analysis (solutions A). For the extraction recovery, extracts of blank DBS were spiked at the tested concentrations (solutions B). The average peak area ratios of the substance with its internal standard were used to calculate the recovery. The calculation of the percentage recovery was as followed: recovery = A/Bx100.

rEsULts AND DIscUssION
The        It should be noticed that the observed effects are only related to the (full spot punch) recoveries and that the distribution of the blood on the DBS cards due to HT would create an additional error.
The influence of the DBS drying time may overlap with substance stability effects at a certain point in time. This could imply that instability of the substances in the liquid or dried blood interfered with the conclusion of this study. However, the stability of TaC, SiR, EvE and CyA was previously validated in whole blood for three days at ambient temperature and were all found stable [11]. And the stability of TaC, SiR, EvE and CyA was previously validated in DBS for at least 7 days at ambient temperature [6]. Both validations support the conclusion that the lowered recoveries were due to the observed drying time effects and not due to stability issues.
Published DBS analysis methods for immunosuppressants mentioned drying times of 3 hours [12], at least 3 hours [13][14][15] and overnight drying [16,17]. The lack of data supporting a 3 hour DBS drying time or more suggests that the effect of the drying time was underestimated.
When a DBS drying time of 3 hours is applied during the validation, this will provide misleading results for SiR, EvE, TeM and CyA with less extreme HT related recovery effects and relatively high recoveries. Therefore, discrepancies in drying times between prepared standards and patient DBS samples may introduce significant bias during analysis of patient samples.

cONcLUsIONs
It is clear that the DBS drying time is not yet acknowledged as a possible factor of influence in DBS analysis, since the most recent review did not include the DBS drying time in their recommended validation practices [9].
In conclusion, this study showed that the DBS drying time could have a significant influence on analyte recovery.

FUTURE PERSPECTIVE
All considered, a 3 hour DBS drying time is not recommended and a drying time of at least 24 hours is advised in order to minimize the risk on drying time related recovery effects for the compounds tested in this study. As a future perspective, DBS drying time effects should be investigated for a period of at least 48 hours during future method development and validation.