Dried Blood Spot Method Research Articles

Validation of a dried blood spot method for therapeutic drug monitoring of citalopram, mirtazapine and risperidone and its active metabolite 9-hydroxyrisperidone using HPLC–MS

Citalopram, mirtazapine and risperidone are frequently prescribed for psychiatric illnesses such as depression and psychosis or for aggressive behavior in elderly patients with dementia. The plasma concentrations vary greatly between patients, especially in elderly patients. Thus, therapeutic drug monitoring (TDM) increases the safety of antipsychotic treatment and a more rapid response to treatment may be achieved. To facilitate TDM, the objectives of this study were to develop and validate a reliable dried blood spot method to simultaneously quantify citalopram, mirtazapine and risperidone including its active metabolite 9-hydroxyrisperidone. The blood punches were extracted by methanol using an ultrasonic bath, purified by liquid–liquid extraction and analyzed by liquid chromatography/mass spectrometry (LC–MS). All acceptance criteria of the EMA and FDA guidelines for method validation were fulfilled. Linearity was shown over the range of 2.5–300μg/L for all substances. The analytes were stable for at least one month at all investigated storage conditions, including storing at room temperature exposed to light. Retrieving capillary blood by finger-pricking the assay was successfully applied in elderly patients. Venous serum samples were drawn simultaneously to compare capillary blood with serum concentrations. Given the validated results and the calculated capillary blood:serum ratio, the studied dried blood spot method offers an excellent application in TDM and can be applied in ambulatory care.

Dried blood spot validation of five immunosuppressants, without hematocrit correction, on two LC-MS/MS systems.

Hematocrit (Ht) effects remain a challenge in dried blood spot (DBS) sampling. The aim was to develop an immunosuppressant DBS assay on two LC-MS/MS systems covering a clinically relevant Ht range without Ht correction. The method was partially validated for tacrolimus, sirolimus, everolimus, cyclosporin A and fully validated for mycophenolic acid on an Agilent and Thermo LC-MS/MS system. Bias caused by Ht effects were within 15% for all immunosuppressants between Ht levels of 0.23 and 0.48 l/l. Clinical validation of DBS versus whole blood samples for tacrolimus and cyclosporin A showed no differences between the two matrices. A multiple immunosuppressant DBS method without Ht correction, has been validated, including a clinical validation for tacrolimus and cyclosporin A, making this procedure suitable for home sampling.

Rubella in Sub-Saharan Africa and sensorineural hearing loss: a case control study

BackgroundRubella infection can affect several organs and cause birth defects that are responsible for congenital rubella syndrome (CRS). Congenital hearing loss is the most common symptom of this syndrome, occurring in approximately 60% of CRS cases. Worldwide, over 100 000 babies are born with CRS every year. There is no specific treatment for rubella, but the disease is preventable by vaccination. Since 1969, the rubella vaccine has been implemented in many countries, but in Africa, only a few countries routinely immunize against rubella. The aim of this study was to estimate the rate of infection from the wild-type rubella virus in São Tomé and Príncipe by determining rubella seroprevalence with a DBS method. The goal of this study was to reinforce the need for implementation of the rubella vaccine in this country. As secondary objectives, the validation of a DBS method was first attempted and an association between seroprevalence and hearing loss was assessed.MethodsWe collected samples from individuals observed during humanitarian missions in São Tomé and Príncipe. All individuals underwent an audiometric evaluation, and a drop of blood was collected for the dried blood spot (DBS).We define two groups: the case group (individuals with unilateral or bilateral hearing loss (HL)) and the control group (individuals with two normal ears). Patients were excluded if they suffered from conductive HL, if they showed evidence of possible causes of HL, if they had developmental delay or if they refused to participate in the study.ResultsAmong the 315 subjects, we found 64.1% individuals with IgG for the rubella virus, 32.1% without immunity for the rubella virus and 3.8% who were borderline.In the control group, 62.6% were positive for the rubella IgG, whereas in the case group, 72% were positive. Analyzing both groups, with ages ranging from 2 to 14 years of age and from 15 to 35 years of age, we found a seroprevalence of 50.3% to rubella in the younger group and 82.1% in the older group, with a significant difference between cases and control group noted within the younger patients (p = 0.025).ConclusionsRubella is a disease that can be prevented. Rubella infections are still very common in São Tomé and Príncipe, and women of child-bearing age are still at risk for rubella infection during pregnancy, justifying the urgency of vaccination against rubella.A statistically significant association between the group of children under 14 years of age with HL and immunity for rubella was observed in this country, although this study did not allow us to establish a cause-effect relationship between rubella infection and SNHL.

A simple dried blood spot-method for in vivo measurement of ureagenesis by gas chromatography–mass spectrometry using stable isotopes

BackgroundClinical management of inherited or acquired hyperammonemia depends mainly on the plasma ammonia level which is not a reliable indicator of urea cycle function as its concentrations largely fluctuate. The gold standard to assess ureagenesis in vivo is the use of stable isotopes. MethodsHere we developed and validated a simplified in vivo method with [15N]ammonium chloride ([15N]H4Cl) as a tracer. Non-labeled and [15N]urea were quantified by GC–MS after extraction and silylation. ResultsDifferent matrices were evaluated for suitability of analysis. Ureagenesis was assessed in ornithine transcarbamylase (OTC)-deficient spfash mice with compromised urea cycle function during fasted and non-fasted feeding states, and after rAAV2/8-vector delivery expressing the murine OTC-cDNA in liver. Blood (5μL) was collected through tail vein puncture before and after [15N]H4Cl intraperitoneal injections over a two hour period. The tested matrices, blood, plasma and dried blood spots, can be used to quantify ureagenesis. Upon [15N]H4Cl challenge, urea production in spfash mice was reduced compared to wild-type and normalized following rAAV2/8-mediated gene therapeutic correction. The most significant difference in ureagenesis was at 30min after injection in untreated spfash mice under fasting conditions (19% of wild-type). Five consecutive injections over a period of five weeks had no effect on body weight or ureagenesis. ConclusionThis method is simple, robust and with no apparent risk, offering a sensitive, minimal-invasive, and fast measurement of ureagenesis capacity using dried blood spots. The stable isotope-based quantification of ureagenesis can be applied for the efficacy-testing of novel molecular therapies.

Clinical Application of the Dried Blood Spot Method in the Measurement of Blood Busulfan Concentration

The dried blood spot (DBS) method, which is a simple technique for blood sample processing involving the placement of a drop of whole blood onto filter paper, has been used recently in clinical pharmacology to determine blood concentrations of various drugs. This study examined the feasibility of the clinical application of the DBS method for individual busulfan dose adjustments. Pharmacokinetic (PK) parameters of blood samples for busulfan measurements determined using the DBS method were compared with those using plasma separation (the conventional method). Blood samples were collected from patients receiving i.v. busulfan as a conditioning regimen before allogeneic hematopoietic stem cell transplantation at Toranomon Hospital, Japan. Samples collected 2, 4, and 6 hours after the start of the first drip infusion were processed by DBS or the conventional method. The area under the blood concentration-time curve (AUC) and other PK parameters were calculated to compare the 2 methods. Divergence of <20% in each parameter was considered acceptable. The divergence range for each parameter was as follows: blood concentration at 2 hours after the start of drip infusion, .6 to 8.2%; at 4 hours, .3 to 10.0%; at 6 hours, .3 to 14.2%; and AUC0–∞, .0 to 10.3%. None of the PK parameters showed a divergence between the DBS method and the conventional method exceeding 20%, suggesting that both methods are well correlated. The clinical application of blood sample processing with the DBS method in the measurement of blood busulfan concentration may therefore be feasible, but further studies are needed to confirm these findings.

Prosfektif Metode Sampel Darah Kering dalam Bioanalisis

Biosampling of dried blood spot method became lately very interested in bioanalysis, some of the advantages given in this method compared to conventional venous canulla (venipuncture); less invasive making it more pleasant for the patient or subject, requires small volume, requires simple transportation process and storage did not need special equipment, suod stabilizes analit and reduces risk of infection. The method of dried blood spot is done by invasive light on the finger or heel prick and then spike on filter paper and dried at room temperature or under nitrogen flow with controlled humidity and stored in bag with desiccant packages, than it analyzed using validation bioanalyzing method. Dried blood spot method can be applied on bioanalysis test for pharmacokinetic, toksikokinetic , epidemiologic and Teurapeutic drug monitoring.

Estimation and Correction of the Blood Volume Variations of Dried Blood Spots Using a Postcolumn Infused-Internal Standard Strategy with LC-Electrospray Ionization-MS.

Dried blood spots (DBSs) have had a long history in disease screening in newborns but have gained attention in recent years in the medical care of adults because of the growing importance of personalized medicine. DBSs have several advantages, such as easy transportation, cost-effectiveness, and minimally invasive biological sampling. There are two strategies to process DBS samples. One method takes a fixed diameter of subsample, and another requires the extraction of the whole spot. The whole-spot extraction method is less affected by hematocrit-caused errors, but it requires calibration of the blood volume. We propose a novel strategy using a postcolumn infused-internal standard (PCI-IS) method with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for estimating and correcting blood volume variations on the DBS cards. By using PCI-IS to measure the extent of ion suppression in the first ion suppression zone in the chromatogram, the blood volume on the DBS cards can be calculated and further calibrated. We used reference blood samples with different volumes (5 to 25 μL) to construct a calibration curve between the blood volume and the extent of ion suppression. The calibration curve was used to estimate the blood volume on the DBS cards collected from 6 volunteers, with 5 designated volumes from each volunteer. The estimation accuracy of the PCI-IS method was between 74.5% and 120.3%. The validated PCI-IS method was used to estimate and calibrate blood volume variation and also to quantify the voriconazole concentration for 26 patients undergoing voriconazole therapy. A high correlation was found for the quantification results between the DBS samples and the conventionally used plasma samples (r = 0.97). The PCI-IS method was demonstrated to be a simple and accurate method for estimating and calibrating the blood volume variation on DBS cards, which greatly facilitates using the DBS method for therapeutic drug monitoring (TDM) for improving the efficacy and safety of drug therapy.

Dried blood spots analysis with mass spectrometry: Potentials and pitfalls in therapeutic drug monitoring

Therapeutic drug monitoring (TDM) relays in the availability of specialized laboratory assays, usually available in reference centers that are not accessible to all patients. In this context, there is a growing interest in the use of dried blood spot (DBS) sampling, usually obtained from finger pricks, which allows simple and cost-effective logistics in many settings, particularly in Developing Countries. The use of DBS assays to estimate plasma concentrations is highly dependent on the hematocrit of the blood, as well as the particular characteristics of the measured analyte. DBS assays require specific validation assays, most of them are related to hematocrit effects. In the present manuscript, the application of mass spectrometric assays for determination of drugs for TDM purposes in the last ten years is reviewed, as well as the particular validation assays for new DBS methods.

Stabilized dried blood spot collection

During the collection phase of the dried blood spot method, practitioners need to ensure that there is no smearing of the blood sample on the filter paper or else readings from it will be invalid. This can be difficult to accomplish in the field if there is relative motion between the site of blood discharge on the finger and the filter paper. In this article, a gyroscope stabilization method is introduced and demonstrated to provide consistent and improved dried blood spot collection within a circular guide region notwithstanding the presence of rocking.

Validation of an Optimized ELISA for Quantitative Assessment of Epstein-Barr Virus Antibodies from Dried Blood Spots

ABSTRACTOur objective was to validate a commercially available ELISA to measure antibody titers against Epstein-Barr virus (EBV) in dried blood spots (DBS) to replace a previously validated assay for DBS that is no longer available. We evaluated the precision, reliability, and stability of the assay for the measurement of EBV antibodies in matched plasma, fingerprick DBS, and venous blood DBS samples from 208 individuals. Effects of hematocrit and DBS sample matrix on EBV antibody determination were also investigated, and the cutoff for seropositivity in DBS was determined. A conversion equation was derived to enable comparison of results generated using this method with the former DBS method. There was a high correlation between plasma and DBS EBV antibody titers (R2 = 0.93) with very little bias (−0.07 based on Bland-Altman analysis). The assay showed good linearity and did not appear to be affected by the DBS matrix, and physiological hematocrit levels had no effect on assay performance. There was reasonable agreement between DBS EBV titer estimates obtained using this assay and the previously validated assay (R2 = 0.72). The commercially available ELISA assay for EBV antibody titers that we validated for use with DBS will facilitate continued investigation of EBV antibody titers in DBS.

Application of dried blood spots to determine vitamin D status in a large nutritional study with unsupervised sampling: the Food4Me project.

An efficient and robust method to measure vitamin D (25-hydroxy vitamin D3 (25(OH)D3) and 25-hydroxy vitamin D2 in dried blood spots (DBS) has been developed and applied in the pan-European multi-centre, internet-based, personalised nutrition intervention study Food4Me. The method includes calibration with blood containing endogenous 25(OH)D3, spotted as DBS and corrected for haematocrit content. The methodology was validated following international standards. The performance characteristics did not reach those of the current gold standard liquid chromatography-MS/MS in plasma for all parameters, but were found to be very suitable for status-level determination under field conditions. DBS sample quality was very high, and 3778 measurements of 25(OH)D3 were obtained from 1465 participants. The study centre and the season within the study centre were very good predictors of 25(OH)D3 levels (P<0·001 for each case). Seasonal effects were modelled by fitting a sine function with a minimum 25(OH)D3 level on 20 January and a maximum on 21 July. The seasonal amplitude varied from centre to centre. The largest difference between winter and summer levels was found in Germany and the smallest in Poland. The model was cross-validated to determine the consistency of the predictions and the performance of the DBS method. The Pearson's correlation between the measured values and the predicted values was r 0·65, and the sd of their differences was 21·2 nmol/l. This includes the analytical variation and the biological variation within subjects. Overall, DBS obtained by unsupervised sampling of the participants at home was a viable methodology for obtaining vitamin D status information in a large nutritional study.

A simple dried blood spot method for clinical pharmacological analyses of etoposide in cancer patients using liquid chromatography and fluorescence detection

BackgroundTherapeutic drug monitoring of etoposide is not part of the routine clinical practice, however, measuring etoposide plasma concentration may be useful to prevent chemotherapy related adverse drug reactions. This paper describes the development and validation of a dried blood spot (DBS) assay for the determination of etoposide in blood samples of lung cancer patients. MethodsThe whole blood spot was cut out of the DBS card followed by sonication assisted liquid drug extraction. Extraction solution was evaporated and re-dissolved. A high-performance-liquid-chromatography method with fluorimetric detection ( λex=230nm; λem=330nm) was used. ResultsMethod met the validation criteria in terms of selectivity, linearity (0.5–20.0μg/mL), accuracy (≥96.1%), precision (≤10.1%) and stability (long term 4weeks at room temperature and 40°C). Haematocrit did not influence DBS etoposide concentration. Good correlation between measured plasma and DBS concentrations was observed. The equation considering only haematocrit value was used for conversion of DBS to plasma concentration. ConclusionsDBS sampling method showed comparable results to plasma samples. Therefore, it can be concluded that the developed and validated DBS method, which is more patient-friendly and requires less sample handling, is a reliable alternative to conventional plasma methods for measuring etoposide concentration in clinical pharmacological analyses.

DBS and beyond.

DBS and beyond.

Dried blood spot analysis without dilution: Application to the LC-MS/MS determination of BMS-986001 in rat dried blood spot.

Sample dilution is one major challenge in dried blood spot (DBS) bioanalysis. To resolve this issue, we applied a no-dilution strategy for DBS analysis by using a calibration curve with very wide linear range. We developed an LC-MS/MS DBS assay with a linear range of 5 orders of magnitude (50-5000,000ng/mL) for BMS-986001, an HIV drug under development, by simultaneously monitoring two selective reaction monitoring transitions of different intensity. The assay was validated and successfully applied to the analysis of DBS samples collected in a toxicology study in rats dosed with BMS-986001. All samples were analyzed without any dilution. We also compared the concentration data generated from the DBS method and a validated plasma assay for the same study. The two sets of data agreed well with each other, demonstrating the validity of this strategy for DBS analysis. This approach provides an effective and convenient way to eliminate complicated dilution for DBS and other sample collection techniques.

Personalized monitoring of therapeutic salicylic acid in dried blood spots using a three-layer setup and desorption electrospray ionization mass spectrometry.

Desorption electrospray ionization (DESI) mass spectrometry is an emerging technology for direct therapeutic drug monitoring in dried blood spots (DBS). Current DBS methods require manual application of small molecules as internal standards for absolute drug quantification. With industrial standardization in mind, we superseded the manual addition of standard and built a three-layer setup for robust quantification of salicylic acid directly from DBS. We combined a dioctyl sodium sulfosuccinate weave facilitating sample spreading with a cellulose layer for addition of isotope-labeled salicylic acid as internal standard and a filter paper for analysis of the standard-containing sample by DESI-MS. Using this setup, we developed a quantification method for salicylic acid from whole blood with a validated linear curve range from 10 to 2000 mg/L, a relative standard deviation (RSD%) ≤14%, and determination coefficients of 0.997. The limit of detection (LOD) was 8 mg/L and the lower limit of quantification (LLOQ) was 10 mg/L. Recovery rates in method verification by LC-MS/MS were 97 to 101% for blinded samples. Most importantly, a study in healthy volunteers after administration of a single dose of Aspirin provides evidence to suggest that the three-layer setup may enable individual pharmacokinetic and endpoint testing following blood collection by finger pricking by patients at home. Taken together, our data suggests that DBS-based quantification of drugs by DESI-MS on pre-manufactured three-layer cartridges may be a promising approach for future near-patient therapeutic drug monitoring.

Limited sampling strategy for prolonged-release tacrolimus in renal transplant patients by use of the dried blood spot technique.

PurposeThe aim of this study was to develop a clinically applicable limited sampling strategy for ambulatory Caucasian kidney transplant patients to estimate area under the curve in a 24-h period (AUC0–24) of prolonged-release tacrolimus.MethodsTwenty six kidney recipients, at least 6 months after transplantation, receiving prolonged-release tacrolimus, were enrolled. In each patient, seven blood samples were collected during a period of 24 h by use of the validated dried blood spot method. Best subset selection multiple linear regression was performed to derive limited sampling strategy (LSS). The equations were constrained to include a maximum of three samples collected within 4 h after the intake to maintain clinical applicability. To assess the predictive performance of LSS, residuals for each patient were calculated based on models fitted to a dataset where that patient was omitted.ResultsThe prediction formula for the AUC0–24 using the time points 0, 2, and 4 h after ingestion (C0h-C2h-C4h) provided the highest correlation with the AUC0–24 (r2 = 0.95): AUC0–24 = 44.9 + 8.9 × C0h + 2.1 × C2h + 7.6 × C4h. Measures for bias and precision, i.e., median percentage prediction error (MPPE) and median absolute prediction error (MAPE), were 0.4 and 4.8 %, respectively. For the same patients, the correlation between C24h and AUC0–24 was worse (r2 = 0.77) while MPPE and MAPE were 6.2 and 7.2 %, respectively.ConclusionIn the outpatient department, a LSS using C0h-C2h-C4h can be used for reliable estimation of the AUC0–24 of prolonged-release tacrolimus.Electronic supplementary materialThe online version of this article (doi:10.1007/s00228-015-1863-6) contains supplementary material, which is available to authorized users.

Application of plasma clearance of iohexol in evaluating renal function in chinese children with chronic kidney disease.

To evaluate the accuracy of plasma clearance of iohexol (PCio) for glomerular filtration rate (GFR) measurement in Chinese children with chronic kidney disease (CKD) and assess the feasibility of single-blood-sample method or dried capillary blood spots in determining the PCio. Totally 45 CKD children were included,in whom the (99m) Technetium-diethylenetriaminepentaacetic acid ((99m)Tc-DTPA) plasma clearance and iohexol plasma clearance were simultaneously determined. Blood samples were obtained 2,4,and 5 hours after injection. In addition, we also evaluated the efficacy of single blood sample method and dried blood spots method in iohexol plasma clearance. Forty-five CKD children completed the iohexol plasma clearance and thirty-six children completed the (99m)Tc-DTPA plasma clearance at the same time among them. Thirteen children finished the iohexol dried blood spot clearance. The correlation coefficient between (99m)Tc-DTPA plasma clearance and iohexol plasma clearance was 0.941 and the bias was (6.53 ± 11.6) ml/ (min·1.73 m²), and the intraclass correlation coefficient (ICC) was high (ICC=0.947). The correlation between iohexol single-sample plasma clearance and double samples was also strong (r=0.958), with the bias being (4.26 ± 9.06)ml/(min·1.73 m²) and the ICC being 0.970. The iohexol clearance by dried blood spots showed a good correlation with the serum iohexol clearance (r=0.950), with the bias still being small [(0.48 ± 10.89)ml/(min·1.73 m²)]. Iohexol plasma clearance has satisfactory agreement with (99m)Tc-DTPA plasma clearance and can be used as an ideal method to measure GFR in CKD children. The single-sample method and dried blood spots method make iohexol plasma clearance more convenient and practical.

Collection and laboratory methods for dried blood spots for hemoglobin A1c and total and high-density lipoprotein cholesterol in population-based surveys

BackgroundThe Health Measures at Home Study was a study designed to evaluate the feasibility of incorporating dried blood spots (DBS) collection into the National Health Interview Survey and to compare the proficiencies between field interviewers and health technicians in obtaining DBS. MethodsDBS collection and venipuncture were attempted on 125 participants. The DBS were collected in the participant's home and venous blood was collected in the National Health and Nutrition Examination Survey (NHANES) mobile examination center. The DBS results were compared to venous results in the NHANES for the measurements of hemoglobin A1c (HbA1c) and total and high-density lipoprotein (HDL) cholesterol. ResultsField interviewers and health technicians were able to collect the DBS for greater than 95% of participants. For DBS, health technicians and field interviewers were highly correlated for HbA1c (r=0.92) and total cholesterol (r=0.89), but not for HDL cholesterol (r=0.72). The DBS results of interviewers and health technicians compared to the venous method for HbA1c (r=0.90), but did not compare well for HDL cholesterol (r=0.64–0.66) and total cholesterol (r=0.65–0.67). ConclusionDBS was comparable to venous HbA1c, but not for total and HDL cholesterol. Health technicians and field interviewers had similar performance for DBS methods, except HDL cholesterol.

Role of therapeutic drug monitoring in pulmonary infections: use and potential for expanded use of dried blood spot samples.

Respiratory tract infections are among the most common infections in men. We reviewed literature to document their pharmacological treatments, and the extent to which therapeutic drug monitoring (TDM) is needed during treatment. We subsequently examined potential use of dried blood spots as sample procedure for TDM. TDM was found to be an important component of clinical care for many (but not all) pulmonary infections. For gentamicin, linezolid, voriconazole and posaconazole dried blood spot methods and their use in TDM were already evident in literature. For glycopeptides, β-lactam antibiotics and fluoroquinolones it was determined that development of a dried blood spot (DBS) method could be useful. This review identifies specific antibiotics for which development of DBS methods could support the optimization of treatment of pulmonary infections.

Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis

In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[(2)H3] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 μmol/L (seven-point calibration curve), 116 to 7000 μmol/L (1-point calibration curve), and 1.00 to 400.0 μmol/L for creatinine-[(2)H3] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0% and a maximum bias of -5.9%. Creatinine in DBS was found stable at ambient temperature and 32 °C for 1 week and at -20 °C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement.