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.

Increasing the discrimination power of rapid evaporative ionisation mass spectrometry (REIMS) in analytical control tissue quality screening and cell line sample identification.

RATIONALE: Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has been evaluated as a tool to improve analytical efficiency and add capability in areas within Pharmaceutical Research and Development (Pharma R&D). This article reports the comparison of single MS, and tandem MS/MS REIMS (REIMS and REIMS/MS) methodologies to investigate which mode produces maximum discrimination power for screening applications. METHODS: Control tissue samples and cell line suspension samples were analysed using optimised REIMS and REIMS/MS to evaluate which technique produced optimal discrimination power for control tissue and cell line identification. The iKnife sampling tool and a prototype 'cell sampler' were utilised for tissue and cell analysis, respectively. The REIMS source was coupled to a hybrid Quadrupole-Time Of Flight (QTOF) mass spectrometer. Multivariate Analysis (MVA) was utilised to evaluate the resulting Mass Spectrometry (MS) data and discriminate between sample types. RESULTS: Proof of concept investigations demonstrating that REIMS/MS offered increased MVA discrimination for sample identification, compared with REIMS, is presented for the first time. Control tissue data showed discrimination by timepoint classification over 0-144 h storage after removal from the host. Timepoint discrimination was optimised using REIMS/MS with a collision energy that effectively maximised ion fragmentation. Similar optimisation was observed when REIMS/MS was applied to the identification of cell lines. CONCLUSIONS: The proof of concept results demonstrate that REIMS/MS can offer advantages over REIMS for control tissue quality screening, and cell line identification applications in Pharma R&D. Further work following this proof of concept investigation is being undertaken to implement the technology for these applications, utilising the optimised REIMS/MS methodology. REIMS/MS will also be used as an optimised tool for other applications.

Optimization of an automated IS addition system for use in high-throughput quantitative DBS analysis.

BACKGROUND: Automated DBS direct elution systems are available that incorporate IS spray modules which, unlike conventional IS addition via the extraction solvent, apply IS prior to DBS samples prior to extraction, allowing analyte and IS to be coextracted. RESULTS: IS spray system parameters were optimized to identify the conditions that produced the best analytical performance in quantitative bioanalytical assays, without interfering with the integrity of the DBS sample prior to extraction. CONCLUSION: LC-MS/MS method validations across four representative small molecule assays using the optimized IS spray conditions were demonstrated to produce analytical performance comparable to conventional methods of IS addition, demonstrating that the spray technique is a viable alternative.

DBS direct elution: optimizing performance in high-throughput quantitative LC-MS/MS analysis.

BACKGROUND: Automated DBS direct elution techniques eliminate the manual extraction burden of DBS bioanalysis, offer good quantitative performance, the ability to eliminate hematocrit-based assay bias, and, previous reports have demonstrated that significant increases in assay sensitivity compared with manual DBS extraction are possible. RESULTS: An investigation into elucidating parameters for optimized generic DBS direct elution for high sample throughput quantitative bioanalytical applications is presented for the first time. Generic direct elution conditions were identified that enabled LC-MS/MS assay sensitivity to be maximized while retaining acceptable chromatographic performance. CONCLUSION: Compared with generic conventional DBS manual extraction, assay sensitivity was demonstrated to be increased up to 33-fold across four representative small molecule compounds, using the recommended direct elution conditions.

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.

Multiplexed extraction and quantitative analysis of pharmaceuticals from DBS samples using digital microfluidics.

BACKGROUND: Dried blood spot (DBS) sampling is emerging as a valuable technique in a variety of fields, including clinical and preclinical testing of pharmaceuticals. Despite this popularity, current DBS sampling and analysis processes remain laborious and time consuming. Digital microfluidics, a microscale liquid-handling technique, characterized by the manipulation of discrete droplets on open electrode arrays, offers a potential solution to these problems. RESULTS: We report a new digital microfluidic method for multiplexed extraction and analysis of pharmaceuticals in DBS samples. In the new method, four DBS samples are extracted in microliter-sized droplets containing internal standard, and the extract is delivered to dedicated nanoelectrospray ionization emitters for direct analysis by tandem mass spectometry and selected reaction monitoring. CONCLUSION: The new method allows for an order of magnitude reduction in processing time and approximately three-times reduction in extraction solvent relative to conventional techniques, while maintaining acceptable analytical performance for most drugs tested.

Dried matrix spot direct analysis: evaluating the robustness of a direct elution technique for use in quantitative bioanalysis.

BACKGROUND: The surge in interest in switching from traditionally used wet plasma to dried matrix spot (DMS) sampling and analysis to support pharmaceutical drug development is due to the significant ethical, financial and data quality advantages on offer. Unfortunately these advantages do not extend to sample bioanalysis, as DMS extraction is more complex than the protein precipitation method typically used for wet plasma analysis. Direct elution techniques coupled to HPLC-MS/MS have been identified as a potential means to counter this additional complexity. RESULTS: The robustness and reproducibility of DMS HPLC-MS/MS data generated using a CAMAG DBS-MS 16 prototype automated direct elution instrument has been demonstrated to meet or exceed results obtained using a conventional manual extraction methodology. CONCLUSION: The data generated suggest that a simple and fast direct elution method of DMS samples that does not require additional sample or extract clean-up, offers sufficiently robust performance to be compatible with high-sample-throughput quantitative analysis. Further evaluation of the technique and the development of more advanced fully automated direct elution instrumentation is fully warranted.

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.

Quantitative analysis of therapeutic drugs in dried blood spot samples by paper spray mass spectrometry: an avenue to therapeutic drug monitoring.

A method is presented for the direct quantitative analysis of therapeutic drugs from dried blood spot samples by mass spectrometry. The method, paper spray mass spectrometry, generates gas phase ions directly from the blood card paper used to store dried blood samples without the need for complex sample preparation and separation; the entire time for preparation and analysis of blood samples is around 30 s. Limits of detection were investigated for a chemically diverse set of some 15 therapeutic drugs; hydrophobic and weakly basic drugs, such as sunitinib, citalopram, and verapamil, were found to be routinely detectable at approximately 1 ng/mL. Samples were prepared by addition of the drug to whole blood. Drug concentrations were measured quantitatively over several orders of magnitude, with accuracies within 10% of the expected value and relative standard deviation (RSD) of around 10% by prespotting an internal standard solution onto the paper prior to application of the blood sample. We have demonstrated that paper spray mass spectrometry can be used to quantitatively measure drug concentrations over the entire therapeutic range for a wide variety of drugs. The high quality analytical data obtained indicate that the technique may be a viable option for therapeutic drug monitoring.

Study to assess the effect of age of control human and animal blood on its suitability for use in quantitative bioanalytical DBS methods.

BACKGROUND: A study was performed to evaluate the suitability of stored EDTA-treated control whole blood for use in the preparation of calibration standards and quality control samples for quantitative bioanalytical methods employing dried blood spot (DBS) samples to support pharmaceutical exposure studies. RESULTS: It has been demonstrated that a storage time of 14 days for control human and animal blood is suitable for producing quantitative analytical results within internationally recognised acceptance criteria for two analytes. Furthermore, blood hemolysis and chill-thaw cycles have been evaluated and shown not to affect bioanalytical results notably. Aggressive mixing techniques can result in rat blood coagulation; however, this does not occur with other species tested and can be affected by the method of blood collection. CONCLUSION: Control whole blood handled and stored using the recommendations generated from this study will not notably affect quantitative bioanalytical results when used for the preparation of calibration standards and quality control samples for DBS assays. It was demonstrated that control human and animal blood can be stored for periods long enough to effectively eliminate wastage.

Direct quantitative bioanalysis of drugs in dried blood spot samples using a thin-layer chromatography mass spectrometer interface.

The CAMAG thin-layer chromatography mass spectrometer (TLC-MS) interface has been assessed as a tool for the direct quantitative bioanalysis of drugs from dried blood spot (DBS) samples, using an MS detector, with or without high-performance liquid chromatography (HPLC) separation. The approach gave acceptable sensitivity, linearity, accuracy, and precision data for bioanalytical validations with and without the inclusion of HPLC separation. In addition, the direct elution technique was shown to increase assay sensitivity for a range of analytes representing a wide "chemical space" for pharmaceutical-type molecules over that obtained by conventional manual extraction of samples (punching of DBS and elution with solvent prior to HPLC-MS analysis). Investigations were performed to optimize extraction time, minimize sample-to-sample carry-over, and compare chromatographic performance. On the basis of this preliminary assessment, it has been demonstrated that the TLC-MS interface has the potential to be an effective tool for the direct analysis of drugs in DBS samples at physiologically relevant concentrations, an approach that could provide significant time and cost savings and greatly simplify bioanalytical procedures compared to current manual practices. Further, the increased sensitivity compared to that of manual extraction may enable the analysis of analytes not currently amenable to DBS sampling due to limitations in assay sensitivity.