Validation of methods for determining pediatric midazolam using wet whole blood and volumetric absorptive microsampling.

Aim: Collection and quantitative analysis in dry blood using volumetric absorptive microsampling (VAMS™) potentially offers significant advantages over conventional wet whole blood analysis. This manuscript explores their use for pediatric sampling and explores additional considerations for the validation of the bioanalytical method. Results: HPLC-MS/MS methods for the determination of midazolam and its major metabolite 1-OH midazolam in both whole wet blood, and dry blood collected on VAMS were developed, validated, and used to support an observational clinical study to compare pharmacokinetic parameters in pediatric patients. Conclusion: Validation data met internationally accepted guideline criteria. A strong correlation was observed in calculated concentrations between wet and dry test samples, indicating that VAMS is a suitable technique for use in pediatric clinical studies.

Investigation of different approaches to incorporating internal standard in DBS quantitative bioanalytical workflows and their effect on nullifying hematocrit-based assay bias.

Hematocrit (HCT)-based assay bias (composed of area and recovery bias) is an important contributing factor to the barriers that currently hinder the development and acceptance of dried blood spots (DBS) as a widely used quantitative bioanalytical sampling technique for regulatory studies. This article describes the evaluation of a practical internal standard spray addition technique, used prior to LC-MS/MS analysis, which is demonstrated to nullify the effect of recovery bias. To our knowledge, this is the first time a potential solution to HCT-based recovery bias has been investigated in detail and reported in the literature. This new technique is coupled with accurate volume DBS sampling, whole-spot extraction, and automated direct elution techniques to demonstrate a workflow that both nullifies HCT-based assay bias and the additional manual extraction burden associated with DBS analysis.

Quantitative bioanalysis of paracetamol in rats using volumetric absorptive microsampling (VAMS).

Volumetric absorptive microsampling (VAMS) is a simple intuitive technique for collecting and quantitative analysis of dried blood samples. It enables the collection of an accurate blood volume (approximately 10µL) regardless of blood hematocrit. A bioanalytical method for the determination of paracetamol in dried blood supported on VAMS samplers has been validated and used to support a toxicokinetic (TK) study in rat. The calculated TK parameters were comparable to those obtained from blood-water (1:1, v/v) samples. VAMS is demonstrated to be a robust method that simplifies both the blood sample collection and bioanalytical laboratory procedures and generates high quality quantitative data. However, problems were encountered with controlling the bleed rate during sample collection, resulting in the VAMS tips being flooded and highlighting the need for bleeding methods to be compatible with microsampling techniques to avoid wasting blood. Alternative sample collection procedures are discussed that minimize these issues.

A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit.

AIMS: A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. MATERIALS & METHODS: An average blood volume of 10.6 µl was absorbed by the samplers across the different HCTs investigated (20-65%). RESULTS: No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. CONCLUSION: The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

Volumetric absorptive microsampling: a dried sample collection technique for quantitative bioanalysis.

Volumetric absorptive microsampling (VAMS) is a novel approach to obtaining a dried blood sample for quantitative bioanalysis that overcomes the area bias and homogeneity issues associated with conventional dried blood spot (DBS) sample when a subpunch is taken. The VAMS sampler absorbs a fixed volume of blood (∼10 µL) in 2-4 s with less than 5% volume variation across the hematocrit range of 20-70% with low tip-to-tip variability. There is no evidence of selective absorption by the tip of the plasma component over whole blood. Recommendations for best practice when collecting samples were developed based upon the results of tests examining a number of potential abuse scenarios.

Bioanalysis zone: DBS survey results.

Bioanalysis Zone carried out a survey to evaluate the use of and attitudes to DBS analysis among our readership in the bioanalytical community. DBS analysis has generated a huge amount of interest in recent years. We wanted to take a snapshot of the field and determine whether a consensus is emerging on the future of DBS. We asked you for your honest opinions and you delivered! You can view the full results at www.bioanalysis-zone.com. We have a selection of short opinion articles below, giving a range of viewpoints on the current and future use of DBS, alongside some of the survey results.

Assessment of the within- and between-lot variability of Whatman™ FTA(®) DMPK and 903(®) DBS papers and their suitability for the quantitative bioanalysis of small molecules.

BACKGROUND: To ensure that PK data generated from DBS samples are of the highest quality, it is important that the paper substrate is uniform and does not unduly contribute to variability. This study investigated any within and between lot variations for four cellulose paper types: Whatman™ FTA(®) DMPK-A, -B and -C, and 903(®) (GE Healthcare, Buckinghamshire, UK). The substrates were tested to demonstrate manufacturing reproducibility (thickness, weight, chemical coating concentration) and its effect on the size of the DBS produced, and the quantitative data derived from the bioanalysis of human DBS samples containing six compounds of varying physicochemical properties. RESULTS & DISCUSSION: Within and between lot variations in paper thickness, mass and chemical coating concentration were within acceptable manufacturing limits. No variation in the spot size or bioanalytical data was observed. CONCLUSION: Bioanalytical results obtained for DBS samples containing a number of analytes spanning a range of chemical space are not affected by the lot used or by the location within a lot.

Effect of ambient humidity on the rate at which blood spots dry and the size of the spot produced.

BACKGROUND: For shipping and storage, dried blood spot (DBS) samples must be sufficiently dry to protect the integrity of the sample. When the blood is spotted the humidity has the potential to affect the size of the spot created and the speed at which it dries. RESULTS: The area of DBS produced on three types of substrates were not affected by the humidity under which they were generated. DBS samples reached a steady moisture content 150 min after spotting and 90 min for humidities less than 60% relative humidity. All packaging materials examined provided some degree of protection from external extreme conditions. However, none of the packaging examined provided a total moisture barrier to extreme environmental conditions. CONCLUSION: Humidity was shown not to affect the spot area and DBS samples were ready for shipping and storage 2 h after spotting. The packing solutions examined all provided good protection from external high humidity conditions.

Method of applying internal standard to dried matrix spot samples for use in quantitative bioanalysis.

A novel technique is presented that addresses the issue of how to apply internal standard (IS) to dried matrix spot (DMS) samples that allows the IS to integrate with the sample prior to extraction. The TouchSpray, a piezo electric spray system, from The Technology Partnership (TTP), was used to apply methanol containing IS to dried blood spot (DBS) samples. It is demonstrated that this method of IS application has the potential to work in practice, for use in quantitative determination of circulating exposures of pharmaceuticals in toxicokinetic and pharmacokinetic studies. Three different methods of IS application were compared: addition of IS to control blood prior to DBS sample preparation (control 1), incorporation into extraction solvent (control 2), and the novel use of TouchSpray technology (test). It is demonstrated that there was no significant difference in accuracy and precision data using these three techniques obtained using both manual extraction and direct elution.

The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs.

BACKGROUND: As hematocrit levels are known to vary between individuals and with disease state, its effect on the physical characteristics of dried blood spot (DBS) samples and on the accurate quantification of analytes within these samples is examined. RESULTS: The area of DBS samples decreases with increasing hematocrit levels in a linear manner on the three cellulose paper substrates tested. Furthermore, a bias was observed in the concentrations of two analytes determined in DBS samples at different hematocrits, which in some cases exceeded acceptable values, particularly for hematocrits outside normal values. CONCLUSION: If it is expected that the hematocrit of study samples will vary from values considered normal, then its effect on the quantitative determination of an analyte in DBS samples should be investigated as part of the method development and validation. If an unacceptable effect is observed, then this will need to be addressed, by modification of the analytical method, or the inclusion of quality control samples at different hematocrit levels to show control of the assay.

Effect of storage conditions on the weight and appearance of dried blood spot samples on various cellulose-based substrates.

BACKGROUND: Before shipping and storage, dried blood spot (DBS) samples must be dried in order to protect the integrity of the spots. In this article, we examine the time required to dry blood spot samples and the effects of different environmental conditions on their integrity. RESULTS: Under ambient laboratory conditions, DBS samples on Whatman 903(®), FTA(®) and FTA(®) Elute substrates are dry within 90 min of spotting. An additional 5% of moisture is lost during subsequent storage with desiccant. When exposed to elevated conditions of temperature and relative humidity, the DBS samples absorb moisture. DBS samples on FTA lose this moisture on being returned to ambient conditions. DBS samples on 903 show no visible signs of deterioration when stored at elevated conditions. However, these conditions cause the DBS to diffuse through the FTA Elute substrate. CONCLUSION: Blood spots are dry within 90 min of spotting. However, the substrates examined behave differently when exposed to conditions of high relative humidity and temperature, in some cases resulting in the integrity of the substrate and DBS sample being compromised. It is recommended that these factors be investigated as part of method development and validation.