An evaluation of metabolite profiling of six drugs using dried blood spot

1. Dry blood spot (DBS) analysis has been extensively used for the quantitative analysis of drugs by mass spectrometry; however, utilization of DBS for qualitative metabolite profiling has been very limited.

2. In the present study, we investigated the use of DBS for metabolite profiling of genistein, carbamazepine, losartan, sunitinib, sildenafil and zoniporide representing a range of Phase I and Phase II biotransformations following oral and intravenous dosing to rats. Plasma and DBS were collected for PK and metabolite profiling. Analyte extraction recovery from DBS was optimized using the parent compound and metabolite standard. Rat DBS metabolite profiles from all six compounds were similar to plasma metabolite profiles, however Phase II metabolites appeared to be extracted less efficiently from DBS compared to plasma, and compounds that were unstable in blood showed different metabolite profiles.

3. In summary, this study showed that in addition to PK bioanalytical analysis, DBS samples may also be utilized for metabolite profiling and a comparison of plasma and DBS metabolite profiling can also provide partitioning/association of major circulating metabolites compared to the parent drug even in the absence of a metabolite standard.

     

The screening of forensic blood, urine, and tissue specimens for xenobiotics using ion-trap liquid chromatography-tandem mass spectrometry

During the investigation of aviation accidents, postmortem specimens from accident victims including blood, urine, and tissue are submitted to the Federal Aviation Administration's Civil Aerospace Medical Institute (CAMI) for toxicological analysis. The first, and perhaps most important, step in the analysis process is the initial screening of biological specimens for illicit, medically prescribed, and over-the-counter compounds that may be present and potentially be a cause and/or factor in the accident. Currently, our general unknown screening (GUS) procedure involves, in part, both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) with both diode-array detection (DAD) and fluorescence detection. Both GC and LC techniques have inherent limitations that prevent the detection of certain types of compounds. The decreased specificity and sensitivity of LC-DAD has been an impediment to the existing GUS procedure. Therefore, our laboratory set out to develop and validate an LC-MS-MS procedure that is superior to LC-DAD. The limits of detection of 359 forensically important xenobiotics have been established following solid-phase extraction from whole blood and analysis by LC-MS-MS. Although whole blood was used as the matrix during instrument validation, the method has been successfully applied to both forensic urine and tissue specimens as well.     

Fully automated dried blood spot sample handling and extraction for serological testing of SARS-CoV-2 antibodies

At the beginning of 2020, an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reached pandemic dimensions. Throughout the event, diagnostic tests function as an essential tool for understanding, mitigating, and implement strategies to curb and reduce infections. Here, we present a novel method for the fully automated dried blood spot (DBS) sample handling and extraction for serological testing of human IgG antibodies against SARS-CoV-2 using a commercial enzyme-linked immunosorbent assay (ELISA) testing kit. This proof-of-principle pilot study successfully demonstrates the recovery of antibodies in their intact form from DBS using automated, direct sample elution within 100 μl of extraction buffer. The use of minimally invasive DBS sampling provides an alternative to existing analytical procedures such as sampling by venipuncture or nasal swabs. Due to the ease of DBS collection, no third party need be involved, making at-home sampling possible (e.g., during quarantine).     

On the road of dried blood spot sampling for antidoping tests: Detection of GHRP-2 abuse

Dried blood spots (DBSs) sampling is gaining support by the antidoping community because of simplicity and cost-effective characteristics, especially in collection, transport, and storage. Nevertheless, DBS applicability demands specific studies for each of the analytes proposed for testing. Here, GHRP-2 has been selected as a representing member of the growth hormone-releasing peptides (GHRPs) family to provide further evidence of DBS suitability for GHRPs abuse detection in sport testing. An analytical procedure to extract GHRP-2 and its main metabolite (AA-3) from DBS and to detect them by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed. The method has been validated for the detection of GHRP-2. Specificity and identification capabilities have been assessed in agreement with antidoping guidelines. The low AA-3 levels found in DBS samples prevented its effective application for the determination of this metabolite. The limit of detection (LoD) for GHRP-2 has been established at 50 pg/ml. Long-term stability (>2 years) has been confirmed. The procedure has been successfully applied to actual DBS samples from an administration study with a single intravenous dose of GHRP-2 (100 μg) being detected up to 4 h after drug injection. GHRP-2 concentrations have been higher in venous blood DBS than in capillary blood DBS. Despite the observed differences, a similar detection window has been achieved independently of the type of blood used. In summary, this study provides specific evidence supporting DBS usefulness to detect GHRP-2, and potentially other GHRPs family members, for antidoping tests.     

Evaluation of fibronectin 1 in one dried blood spot and in urine after rhGH treatment

Since the appearance of recombinant human growth hormone (rhGH) in the 1980s, its expansion and acquisition through the black market has increased, so the detection of its abuse continues to be a challenge. New biomarkers that are more reliable and sensitive, allowing a larger detection window, are still needed. In this line, Fibronectin 1 (FN1) has been proposed as a potential genetic and protein biomarker of rhGH abuse in peripheral blood lymphocytes, serum, and plasma. However, logistic problems associated with current blood collection in sports drug testing point towards potential new alternative matrices that could be good candidates to be evaluated. Results obtained in this study showed high ELISA FN1 levels in one dried blood spot and in urine samples in ten healthy male volunteers treated with rhGH. Results showed that especially dried blood spots appear as a potential good matrix to detect rhGH abuse by means of FN1 biomarker. Copyright © 2016 John Wiley & Sons, Ltd.     

Application of automated serial blood sampling and dried blood spot technique with liquid chromatography-tandem mass spectrometry for pharmacokinetic studies in mice

The goal of this work was to obtain full pharmacokinetic profiles from individual mice with the use of an automated blood sampling system and dried blood spot (DBS) technique. AMG 517, a potent and selective vanilloid receptor (VR1) antagonist, was dosed to mice (n = 3) intravenously and blood samples were collected using the automated blood sampling system with the “no blood waste” method. The collected blood samples were a mixture of 25 μL blood and 50 μL of heparinized saline solution. Two 15 μL aliquots were manually spotted onto a DBS card and dried at room temperature for at least 2 h before being stored in zip bags with desiccant. The remaining samples (45 μL) were stored at −70 °C until analysis. Both the DBS and the whole blood samples (diluted with saline (1:2, v/v)) were extracted and analyzed by liquid chromatography-tandem mass spectrometry. The overall extraction recovery of the analyte from the dried blood spots was determined to be about 90%. The pharmacokinetic parameters calculated using the whole blood or the DBS concentration data were comparable, and were obtained from only 3 mice, whereas conventional sampling and analysis would have required up to 27 mice to achieve the same result. The analyte was shown to be stable in the diluted whole blood (blood:saline 1:2) at room temperature for at least 4 h and in the DBS for at least 34 days when stored at room temperature. These results indicated that the automated blood sampling system and DBS collection are promising techniques to obtain full pharmacokinetic profiles from individual mice and reduce the use of animals.     

A fast screening method for the detection of CERA in dried blood spots

Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia. However, misuse of CERA for doping in endurance sports has been reported. Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 μg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days post-injection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport.     

Automated online dried blood spot sample preparation and detection of anabolic steroid esters for sports drug testing

Dried blood spots (DBSs) provide a valuable complementary sample matrix for routine doping analysis, and the full automation of analysis for DBS samples is achievable to avoid extensive and repetitive manual laboratory work. In the current study, a fully automated online DBS preparation and detection method for the screening and quantification analysis of 13 anabolic steroid esters by means of the DBSA-TLX-HRMS system was developed and validated, based on the purpose for the determination of the abuse of anabolic steroid esters in athletes. Validation of the method yielded linear (R² > 0.99), precise, and accurate (RSD% and Re% < 20% at low, medium, and high concentration levels) results. The LOD of testosterone laurate was established at 0.5 ng/ml and at 0.2 ng/ml for other steroid esters. The extraction recovery of the target compounds from DBS ranged from 10.5% to 88.9%, and matrix effects were moderate. Furthermore, the developed and validated method was applied in the analysis of DBS samples collected after the oral administration of a single dose of 80 mg testosterone undecanoate demonstrating its applicability. Evaluation of analyte stability showed that testosterone undecanoate are more stable (8 weeks) in DBS samples of administration study when stored in frozen (−20°C) condition compared with cold storage (4°C). Collectively, these findings demonstrate the applicability of automated DBS analysis in doping control for detection of anabolic steroid esters.     

Comparison of urine analysis and dried blood spot analysis for the detection of ephedrine and methylephedrine in doping control

When the misuse of stimulants is determined in doping control tests conducted during the in‐competition period, athletes are asked to account for the violation of the rules. This study was designed to evaluate whether the urinary threshold values (10 µg/mL) for ephedrine and methylephedrine set by the World Anti‐Doping Agency (WADA) can be exceeded after the oral administration of each substance (25 mg). In addition, the study describes the validity of a liquid chromatography‐tandem mass spectrometric method using dried blood spot testing to detect ephedrine and methylephedrine by comparing it to a quantitative laboratory urine assay. After administration of ephedrine, the urinary concentration of ephedrine did not exceed the threshold at 4–10 h in two subjects, whereas the threshold was exceeded in both the subjects at 12 h after administration. For methylephedrine, the urinary concentrations of all the subjects failed to reach the threshold for up to 10 h after administration. The concentrations reached the threshold at 12–24 h after administration in some volunteers. In contrast, the blood concentrations of ephedrine and methylephedrine reached their maximum levels at 2–8 h after administration. The blood concentrations showed a low inter‐individual variability, and the results suggested that the urinary excretion of ephedrine and methylephedrine can be strongly affected by urine pH and/or urine volume. These facts suggest that urinary concentrations cannot reflect the psychoactive level of ephedrines in circulation. Thus, dried blood analysis might be suitable for the adequate detection of stimulants during in‐competition testing. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of the dried spot sampling technique for rat cerebrospinal fluid sample collection and analysis

Dried blood spotting (DBS) sample collection is gaining favor in the pharmaceutical industry due to benefits that include reduced animal usage and easier sample shipment and storage when compared to traditional plasma collection/analysis. The applicability of the DBS card to alternate, limited-volume, matrices has not been as fully characterized as their use with whole blood. In this paper we explored the application of the DBS sample collection technique to rat cerebrospinal fluid (CSF). A reverse phase HPLC-MS/MS method was developed and characterized for the quantitative bioanalysis of the α7 neutonal nicotinic acetylcholine receptor agonist PHA-00543613 in CSF using the dried spot sampling technique. The characterized assay and dried spot sampling technique was employed to analyze serially collected in vivo rat CSF samples after a single 4mg/kg dose of PHA-00543613 in CSF-cannulated rats. The DBS strategy enabled the collection of more timepoints and produced comparable exposure results to those obtained by the collection and analysis of liquid CSF samples but notably with eight less animals.     

Analysis of six anticonvulsant drugs using solid-phase extraction, deuterated internal standards, and gas chromatography-mass spectrometry

A rapid method for simultaneously determining the anticonvulsant drugs carbamazepine, ethosuximide, phenobarbitone, phenytoin, primidone, and valproic acid is described. Blank plus single-point calibration gives reliable quantitation from therapeutic to high fatal concentrations, except for ethosuximide, for which it gives semiquantitative results. Whole blood and liver tissue samples containing deuterated internal standards were extracted using Bond Elut Certify columns. Butyl derivatives were formed using n-iodobutane and TMAH under mild conditions and were extracted into ethyl acetate as a cleanup step. Recoveries were greater than 50%, except for valproic acid (42%). Sample preparation time was less than 2 h, and the GC run time was less than 20 min per injection. At least two ion pairs formed by electron impact ionization were monitored for each drug. Intraday CVs were less than 6.28% (4.20%) and interday CVs less than 14.1% (for midtherapeutic concentrations in blood [liver], except for ethosuximide). Linearity was observed from subtherapeutic to high fatal levels for all drugs. This method has been applied to forensic cases and has significantly reduced analytical time while improving case-work quality. Results of a case study involving anticonvulsant drugs are given.     

Development and clinical applications of the dried blood spot method for therapeutic drug monitoring of anti‐epileptic drugs

Anti‐epileptic drugs (AEDs) have various pharmacokinetic profiles, inter‐individual variabilities, high possibilities of drug‐drug interactions and narrow therapeutic indices. To provide optimal treatment for patients, therapeutic drug monitoring (TDM) is necessary. However, TDM requires sufficient quantities of blood samples to measure drug concentrations. Therefore, TDM could be a burden, particularly in paediatric cases. A good alternative that overcomes these disadvantages is the dried blood spot (DBS) method, which is simple, convenient to use and less invasive, requiring a lower quantity of blood than traditional blood sampling methods. However, the DBS method is affected by haematocrit (Hct) levels to varying extents depending on the drug properties. In addition, different papers with varying characteristics are available for use when applying the DBS method. Therefore, it has not yet been applied to TDM in clinical practice. To achieve this, several steps are required, including method development, method validation and clinical validation. Currently, the development status of the DBS method is different for each AED and unclear. Therefore, we assessed the development status of the following 19 AEDs in 26 studies: lamotrigine, valproic acid, levetiracetam, phenytoin, topiramate, carbamazepine, carbamazepine epoxide, gabapentin, phenobarbital, pregabalin, clobazam, clonazepam, ethosuximide, felbamate, monohydroxycarbamazepine, nitrazepam, rufinamide, vigabatrin and zonisamide. Among them, carbamazepine, lamotrigine, topiramate and valproic acid have been developed such that they are nearly available for TDM. In addition, Whatman 903 Protein Saver Cards and concentration analysis by liquid chromatography with triple quadrupole mass spectrometer were used most often.     

A single-step extraction for screening whole blood for basic drugs by capillary GC/NPD

Basic drugs were routinely extracted from whole blood under alkaline conditions into n-butyl acetate. An aliquot of the n-butyl acetate was injected into a gas chromatograph equipped with a nitrogen-phosphorous detector and a wide-bore cross-linked 50% phenylmethyl silicone capillary column. Absolute and relative retention times were recorded for more than 100 extracted drug standards. Recovery from whole blood was determined for some of the more frequently encountered drugs. This one-step extraction proved to be reliable for general screening and has been used routinely in forensic and clinical toxicological analyses.     

Validation of a simple and cheap gelatin particle agglutination test for human immunodeficiency virus using dried blood spot samples

OBJECTIVES: To validate a Gelatin Particle Agglutination (GPA) test for HIV using dried blood spots with a view to applying the test in large epidemiological studies. DESIGN: A method comparison study with standard enzyme linked immunosorbent assay (ELISA) using the Recombigen HIV-1/2 kit as the gold standard. SETTING: Blair Research Laboratory, Harare, Zimbabwe. SUBJECTS: Sera and dried blood spots samples were available from 379 women from Mbare, Harare and Mupfure, Shamva District, Zimbabwe who had participated in HIV studies conducted by Blair Research Laboratory. MAIN OUTCOME MEASURES: Results of the GPA and Recombigen HIV-1/2 ELISA using serum and dried blood spots. RESULTS: With the Recombigen HIV-1/2 ELISA as the gold standard, sensitivity and specificity of the GPA were 100% and 99.2% respectively using serum. With dried blood spots sensitivity and specificity of the GPA test were 100%. The cost of analysing one sample, based on cost of reagents and accessory materials only, was Z$300 for GPA compared to Z$1,200 for ELISA. Furthermore, hands-on time was significantly reduced with the GPA compared to ELISA. CONCLUSION: The GPA method is simple, less labour intensive and much cheaper, yet is equally sensitive when compared to standard ELISA. The high sensitivity with blood spots makes the test ideal for large-scale epidemiological studies in remote rural areas with no infrastructure for advanced diagnostic methods.