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A liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the determination of therapeutic levels of caffeine in dried blood spot (DBS) samples. Caffeine is used in the treatment of Apnoea of Prematurity (AoP) in newborn children. Calibration DBS samples were prepared by spotting 15 μL of whole blood spiked with the a...
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Citations
... Published assays quantifying analyte concentrations in DBS show a preference for preparing DBS standards and QCs by first spiking working solutions into whole blood. These whole blood standards and QCs are then spotted onto the DBS cards to create the DBS standards and QCs [21][22][23][24][25][26]. In the initial attempts in developing the method, DBS standards and QCs were made by spiking working solutions directly into whole blood to prepare whole blood standards and QCs, which were then spiked onto DBS cards to prepare the DBS standards and QCs. ...
Introduction
Adherence to medication is an important determinant of outcomes in chronic diseases like heart failure. Drug assays provide objective adherence biomarkers. Dried blood spots (DBS) are appealing samples for drug assays due to less demanding transportation and storage requirements.
Objectives
To analytically validate a LC-MS/MS method for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS and evaluate the feasibility of using the method as an adherence determining assay. To validate the assay further clinically by establishing correlation and agreement between plasma and DBS samples from a pharmacokinetic pilot study.
Methods
The method was validated over a concentration range of 1.00 – 200 ng/mL according to FDA guidelines. Adherence tracking ability of the assay was evaluated using a pharmacokinetic pilot study. Correlation and agreement were evaluated through Deming regression and Bland-Altman analysis, respectively.
Results
Accuracy, precision, selectivity, and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested. Stability of the analytes in the matrix and throughout sample processing was proven. The full range of concentrations of the pharmacokinetic pilot study could be quantified for enalaprilat, but not for carvedilol and perindoprilat. The difference between the observed and calculated plasma concentrations was less than 20% of their mean for >67% of samples for all analytes.
Conclusions
The assay is suitable as a screening tool for carvedilol and perindoprilat, while suitable as an adherence determining assay for enalaprilat. Equivalence between observed and predicted plasma concentrations proves DBS and plasma concentrations can be used interchangeably.
... 103 DBS screening is typically combined with LC-MS/MS analysis. 104,105 DBS analysis has the potential to facilitate caffeinelevel determination in neonates, [106][107][108] allowing optimal strategies for precision medication. Thus, the neonatal population would definitely benefit from studies that enable individualized treatment, because most drugs used in neonatal medicine are prescribed off-label. ...
Drug pharmacodynamics is monitored by directly measuring physiological indices of therapeutic response. For many drugs, however, methods for the direct measurement of a drug’s effect are either not available or not sufficiently sensitive. In these cases, the percentage of a drug, or its active moiety that actually reaches the systemic circulation and becomes available to its target, known as the drug bioavailability, has become the most accepted indicator of efficacy. This explains the importance of therapeutic drug monitoring (TDM), which is the analysis, assessment, and evaluation of the circulating concentrations of drugs in serum, plasma, or whole blood in order to ensure that the patient's drug concentrations are within the established therapeutic range for the respective indication. In this month’s special feature, Christoph Borchers and co‐workers review the advances, challenges, and limitations of the existing repertoire of TDM methods, and discuss future opportunities for TDM‐based precision medicine. Dr. Borchers is Professor of Oncology in Faculty of Medicine at McGill University (Montreal Canada). His research involves the improvement, development, and application of proteomics and metabolomics technologies, especially quantitative techniques for clinical diagnostics.
... 103 DBS screening is typically combined with LC-MS/MS analysis. 104,105 DBS analysis has the potential to facilitate caffeinelevel determination in neonates, [106][107][108] allowing optimal strategies for precision medication. Thus, the neonatal population would definitely benefit from studies that enable individualized treatment, because most drugs used in neonatal medicine are prescribed off-label. ...
Therapeutic Drug Monitoring (TDM) is typically referred to as the measurement of the concentration of drugs in patient blood. While in the past, TDM was restricted to drugs with a narrow therapeutic range in order to avoid drug toxicity, TDM has recently become a major tool for precision medicine being applied to many more drugs. Through compensating for inter‐individual differences in a drug’s pharmacokinetics, improved dosing of individual patients based on TDM ensures maximum drug effectiveness while minimizing side effects. This is especially relevant for individuals that present a particularly high inter‐variability in pharmacokinetics, such as newborns, or for critically/severely ill patients. In this article, we will review the applications for and limitations of TDM, discuss for which patients TDM is most beneficial and why, examine which techniques are being used for TDM, and demonstrate how mass spectrometry is increasingly becoming a reliable and convenient alternative for the TDM of different classes of drugs. We will also highlight the advances, challenges, and limitations of the existing repertoire of TDM methods, and discuss future opportunities for TDM‐based precision medicine.
... Dried blood spot (DBS) collection is a low blood volume technique that offers several advantages compared to traditional blood sampling techniques [10][11][12][13]. Furthermore, DBS have previously demonstrated utility for measuring systemic exposures and testing for inborn errors of metabolism in pediatric populations, including neonates [11,[14][15][16]. This article describes the development and validation of an ultra-high-performance liquid chromatography -tandem mass spectrometry (UHPLC-MS/MS) method for measuring budesonide concentrations in DBS to assess the systemic exposure of budesonide following intra-tracheal instillation in ELGAN. ...
Budesonide is a potential therapeutic option for the prevention of bronchopulmonary dysplasia in mechanically ventilated premature neonates. The dose and concentrations of budesonide that drive effective prophylaxis are unknown, due in part to the difficulty in obtaining serial blood samples from this fragile population. Of primary concern is the limited total blood volume available for collection for the purposes of a pharmacokinetic study. Dried blood spots (DBS), which require the collection of <200 μL whole blood to fill an entire card, are an attractive low-blood volume alternative to traditional venipuncture sampling. We describe a simple and sensitive method for determining budesonide concentrations in DBS using an ultra-high-performance liquid chromatography – tandem mass spectrometry assay. Budesonide was liberated from a single 6 mm punch using a basified methyl tert-butyl ether extraction procedure. The assay was determined to be accurate and precise in the dynamic range of 1 to 50 ng/mL. The validated assay was then successfully applied to DBS collected as part of a multi-center, dose-escalation study of budesonide administered in surfactant via intra-tracheal instillation to premature neonates between 23 and 28 weeks gestational age. These findings show that DBS are a useful technique for collecting pharmacokinetic samples in premature neonates and other pediatric populations.
... In addition, method development may be further complicated due to interferences originating from e.g. DBS filter paper or oral fluid collection devices 18,114 . Unfortunately, the above-mentioned additional variables are often not included in standard validation guidelines and matrix-specific guidelines are not always available. ...
Alternative sampling strategies such as dried blood sampling, liquid microsampling and the sampling of oral fluid, hair, meconium, interstitial fluid, sweat, exhaled breath condensate and sputum offer interesting opportunities for many applications in clinical routine. Here, we provide an overview of different applications, with special attention to the pivotal role of LC-MS/MS in facilitating analysis of the collected matrices. Covered clinical fields include newborn screening, endocrinology, therapeutic drug monitoring, phenotyping, toxicology, proteomics and metabolomics. Furthermore, specific advantages, challenges and limitations of each alternative sampling strategy are discussed, along with recent advances and future trends that may contribute to routine implementation of these sampling strategies. Given the development of many recent potentially valuable clinical applications, the possibility of home sampling and the opportunity to obtain information that is hard to procure using traditional sampling, a well-balanced role for alternative sampling strategies can be envisaged in patient healthcare in the (near) future.
... Using ICH guidelines, the LoD and LoQ of caffeine in water was found to be 0.013 μg/mL and 0.039 μg/mL, respectively while, following extraction from DBS, caffeine LoD and LoQ of 0.24 μg/mL and 0.73 μg/mL, respectively, were obtained. These findings are similar to those previously reported in a study investigating DBS therapeutic caffeine monitoring in neonates, where the LoQ was found to be 0.50 μg/mL [48]. For glucose measurements, the fluorescence intensity was measured using the fluorescence plate reader and glucose concentration calculated by comparison with a standard curve for glucose ranging from 0-1600 nmol/mL. ...
... For glucose measurements, the fluorescence intensity was measured using the fluorescence plate reader and glucose concentration calculated by comparison with a standard curve for glucose ranging from 0-1600 nmol/mL. The results obtained using this analytical method are in keeping with those reported in similar studies which analysed caffeine [48], theophylline [49] and glucose [50] using HPLC and liquid chromatography-mass spectrometry analysis. ...
We describe, for the first time the use of hydrogel-forming microneedle (MN) arrays for minimally-invasive extraction and quantification of drug substances and glucose from skin in vitro and in vivo. MN prepared from aqueous blends of hydrolysed poly(methyl-vinylether-co-maleic anhydride) (11.1% w/w) and poly(ethyleneglycol) 10,000 daltons (5.6% w/w) and crosslinked by esterification swelled upon skin insertion by uptake of fluid. Post-removal, theophylline and caffeine were extracted from MN and determined using HPLC, with glucose quantified using a proprietary kit. In vitro studies using excised neonatal porcine skin bathed on the underside by physiologically-relevant analyte concentrations showed rapid (5 min) analyte uptake. For example, mean concentrations of 0.16 μg/mL and 0.85 μg/mL, respectively, were detected for the lowest (5 μg/mL) and highest (35 μg/mL) Franz cell concentrations of theophylline after 5 min insertion. A mean concentration of 0.10 μg/mL was obtained by extraction of MN inserted for 5 min into skin bathed with 5 μg/mL caffeine, while the mean concentration obtained by extraction of MN inserted into skin bathed with 15 μg/mL caffeine was 0.33 μg/mL. The mean detected glucose concentration after 5 min insertion into skin bathed with 4 mmol/L was 19.46 nmol/L. The highest theophylline concentration detected following extraction from a hydrogel-forming MN inserted for 1 h into the skin of a rat dosed orally with 10 mg/kg was of 0.363 μg/mL, whilst a maximum concentration of 0.063 μg/mL was detected following extraction from a MN inserted for 1 h into the skin of a rat dosed with 5 mg/kg theophylline. In human volunteers, the highest mean concentration of caffeine detected using MN was 91.31 μg/mL over the period from 1 to 2 h post-consumption of 100 mg Proplus® tablets. The highest mean blood glucose level was 7.89 nmol/L detected 1 h following ingestion of 75 g of glucose, while the highest mean glucose concentration extracted from MN was 4.29 nmol/L, detected after 3 hours skin insertion in human volunteers. Whilst not directly correlated, concentrations extracted from MN were clearly indicative of trends in blood in both rats and human volunteers. This work strongly illustrates the potential of hydrogel-forming MN in minimally-invasive patient monitoring and diagnosis. Further studies are now ongoing to reduce clinical insertion times and develop mathematical algorithms enabling determination of blood levels directly from MN measurements.
... Using ICH guidelines, the LoD and LoQ of caffeine in water was found to be 0.013 μg/mL and 0.039 μg/mL, respectively while, following extraction from DBS, caffeine LoD and LoQ of 0.24 μg/mL and 0.73 μg/mL, respectively, were obtained. These findings are similar to those previously reported in a study investigating DBS therapeutic caffeine monitoring in neonates, where the LoQ was found to be 0.50 μg/mL [48]. For glucose measurements, the fluorescence intensity was measured using the fluorescence plate reader and glucose concentration calculated by comparison with a standard curve for glucose ranging from 0-1600 nmol/mL. ...
... For glucose measurements, the fluorescence intensity was measured using the fluorescence plate reader and glucose concentration calculated by comparison with a standard curve for glucose ranging from 0-1600 nmol/mL. The results obtained using this analytical method are in keeping with those reported in similar studies which analysed caffeine [48], theophylline [49] and glucose [50] using HPLC and liquid chromatography-mass spectrometry analysis. ...
The emerging field of microneedle-based minimally invasive patient monitoring and diagnosis is reviewed. Microneedle arrays consist of rows of micron-scale projections attached to a solid support. They have been widely investigated for transdermal drug and vaccine delivery applications since the late 1990s. However, researchers and clinicians have recently realized the great potential of microneedles for extraction of skin interstitial fluid and, less commonly, blood, for enhanced monitoring of patient health.
We reviewed the journal and patent literature, and summarized the findings and provided technical insights and critical analysis.
We describe the basic concepts in detail and extensively review the work performed to date.
It is our view that microneedles will have an important role to play in clinical management of patients and will ultimately improve therapeutic outcomes for people worldwide.
... Despite the better recovery obtained with the chemical additives and pretreated cards, the nature and the amount of these additives used should be carefully decided. Unexpected interferences has been observed from blood spot cards coated with different chemicals leading to ionization suppression in mass spectrometry ana lysis and chromatographic interferences such as shifts in retention times and peak shape distortion [63,103]. ...
Malaria is the leading parasitic disease in emerging countries. Therapeutic drug monitoring of antimalarial drugs is becoming increasingly important due to their spreading resistance. Measuring systemic antimalarial drug concentrations is also vital for safety and PK evaluations during clinical development. The dried blood spot (DBS) technique is a convenient alternative sample-collection method to venipuncture, especially in resource -limited areas where the clinical studies of antimalarials are usually carried out. Various bioanalytical methods for antimalarial drug estimation utilizing DBS sampling have been reported. This review discusses the applicability and relevance of DBS in quantitative assessment of antimalarial drugs, the advantages and drawbacks of DBS, and the difficulties encountered during its implementation.
Caffeine is an ergogenic substance that is consumed globally in many forms. The use of buccally absorbable formulations instead of gastrointestinal uptake has become increasingly popular over the years, especially when accelerated absorption with minimal gastrointestinal stress is desired. This study investigated the impact of five different formulations and administration routes of caffeine on the whole blood concentrations of caffeine, paraxanthine, and theobromine: caffeinated capsules, tablets, shots, pouches, and chewing gums. A uniform dose of caffeine (200 mg) was administered to 16 healthy recreational athletes (26.0 ± 2.1 years) using a randomized crossover design. Samples were taken in the form of dried blood spots at 16 different time points in a 2-hr timeframe after drug administration. The samples were analyzed using a validated liquid chromatography–tandem mass spectrometry method. The results for caffeine showed no significant differences in the overall bioavailability (area under the concentration–time curve), maximal concentration, and time to maximum concentration. However, when analyzing the bioavailability of caffeine in the first 5, 10, and 15 min, the liquid caffeine formulation was superior to other administered forms ( p < .05). This indicates that caffeine solubility has a major influence on its absorption rate. In sports, the rate of caffeine absorption must be considered, not only when ingesting anhydrous caffeine, but also when choosing buccal absorption. These findings imply that general guidelines for ergogenic caffeine use should consider the formulation used and, accordingly, the corresponding route of absorption.
Dried blood spot (DBS) has been used as an alternative matrix in drug testing. In forensic testing it offers enhanced stability of analytes and ease of storage that requires minimal space. This is compatible with long term archiving of large numbers of samples for future investigation. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify alprazolam, α-hydroxyalprazolam, and hydrocodone in a DBS sample that has been stored for 17 years. We achieved linear dynamic ranges (0.1-50 ng/mL) that capture wide ranges of concentration of the analytes below and above their reported reference ranges, and limits of detection (0.05 ng/mL) of 40-100X lower than the lower limit of the analyte's reference ranges. The method was validated according to FDA and CLSI guidelines and successfully confirmed and quantified alprazolam and α-hydroxyalprazolam in a forensic DBS sample.
