Dried Matrix Spots Research Articles

The Use of Dried Matrix Spots as an Alternative Sampling Technique for Monitoring Neglected Tropical Diseases.

Neglected tropical diseases (NTDs) are a group of illnesses which usually present with a chronic clinical picture. NTDs can lead to permanent disability and are often associated with social stigma. In many developing countries where NTDs are endemic, there are no diagnostic tools for the safe storage and transport of biological samples, and there are no specialist diagnostic centers where the samples could be processed. The transport of biological samples (blood, urine) collected in field conditions and brought to laboratories located in developed countries requires the maintenance of the cold chain during transportation. Ensuring temperature control during transport could be problematic or even impossible to achieve; it is also expensive. A helpful solution to this problem is to use the dried matrix spot (DMS) technique, which seems to be a reliable method for collecting biological samples to be used for screening purposes and conducting epidemiological surveillance of NTDs in developing countries. This article is an overview of how DMSs can be used in the diagnosis of most neglected tropical diseases.

Advancements in clinical approaches, analytical methods, and smart sampling for LC-MS-based protein determination from dried matrix spots.

Determination of proteins from dried matrix spots using MS is an expanding research area. Mainly, the collected dried matrix sample is whole blood from a finger or heal prick, resulting in dried blood spots. However as other matrices such as plasma, serum, urine, and tear fluid also can be collected in this way, the term dried matrix spot is used as an overarching term. In this review, the focus is on advancements in the field made from 2017 up to 2023. In the first part reviews concerning the subject are discussed. After this, advancements made for clinical purposes are highlighted. Both targeted protein analyses, with and without the use of affinity extractions, as well as untargeted, global proteomic approaches are discussed. In the last part, both methodological advancements are being reviewed as well as the possibility to integrate sample preparation steps during the sample handling. The focus, of this so-called smart sampling, is on the incorporation of cell separation, proteolysis, and antibody-based affinity capture.

A simple and rapid HPLC-MS/MS method for therapeutic drug monitoring of amikacin in dried matrix spots

Individualized treatment of amikacin under the guidance of therapeutic drug monitoring (TDM) is important to reduce the occurrence of toxicity and improve clinical efficacy. In the present study, we developed and validated a simple and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the concentration of amikacin in dried matrix spots (DMS) which the matrix is serum. DMS samples were obtained by spotting volumetric blood onto Whatman 903® cards. Samples were punched into 3 mm diameter discs and extracted with 0.2 % formic acid in water. The HILIC column (2.1 mm × 100 mm, 3.0 µm) under gradient elution was applied, and the analysis time was 3 min per injection. The mass spectrometry transitions were m/z 586.3 → 163.0 for amikacin and m/z 591.4 → 163.1 for D5-amikacin. Full validation was conducted for DMS method, and the method was applied for the amikacin TDM and compared with serum method. The linearity was ranged from 0.5 to 100 mg/L. Both within-run and between-run accuracy and precision of DMS ranged from 91.8 % to 109.6 % and 3.6 % to 14.2 %, respectively. The matrix effect was 100.5 %–106.5 % of DMS method. Amikacin remained stable in DMS for at least 6 days at room temperature, 16 days at 4 °C, 86 days at −20 °C and −70 °C. A good agreement between the DMS method and serum method has been shown in Bland-Altman plots and Passing-Bablok regression. All of the results demonstrated that the DMS methods can be a favorable replacement for amikacin TDM.

Back Cover: Smart proteolysis samplers for pre‐lab bottom‐up protein analysis – Performance of on‐paper digestion compared to conventional digestion

DOI: 10.1002/sscp.202100062 The cover picture shows filter paper which is converted into a sample preparation unit for high-end bottom-up protein analysis. The trypsin covalently bound to cellulose digests proteins enabling shotgun and targeted proteomics experiments from dried matrix spots. These paper-based smart proteolysis samplers allow instant sample preparation starting at the moment of sampling with potential continuation during postal transport. The smart proteolysis sampler shows good stability (no activity loss for at least 4 months when refrigerated) and the proteolytic activity of bound trypsin is up to 50–60% of trypsin acting in solution

Genetic individual identification from dried urine spots: A complementary tool to drug monitoring and anti‐doping testing

The collection of liquid biological matrices onto paper cards (dried matrix spots [DMS]) is becoming an alternative sampling strategy. The stability over time of molecules of interest for therapeutic, sport drug monitoring, and forensic toxicology on DMS has been recently investigated representing a reliable alternative to conventional analytical techniques. When a tampering of a urine sample in drug monitoring or doping control cases is suspected, it could be relevant to know whether genetic profiles useful for individual identification could be generated from urine samples spotted onto paper (dried urine spot [DUS]). To understand the influence of sex, storage conditions, and time on the quality and quantity of the DNA, five female and ten male urine samples were dispensed onto Whatman 903 paper and sampled after different storage conditions over time, from 1 to 12 weeks. Direct PCR was performed starting from 2‐mm punches collected from each spot amplifying a panel of markers useful for individual identification. The female DUS stored in different conditions produced genetic profiles fully matching the reference samples. The same result was obtained for the male DUS but using urine 30X concentrated by centrifugation instead of the original samples. Our data show that this approach is valid for genetic individual identification of urine samples spotted onto paper cards up to 12 weeks after deposition and could be easily incorporated in anti‐doping or drug screening protocols to help on the suspicion of evidence tampering or to solve questions on the reliability of samples collection.

Dried matrix spots: an evolving trend in the toxicological field.

Dried matrix spot (DMS) is a sampling technique, primarily used to analyze blood to diagnose metabolic diseases in newborns. As this technique has several advantages, DMS has started to be employed for other purposes using other biological matrices and increasingly in toxicology over the last decade. The aim of this work was to review the analytical methods using DMS which can be applied to drugs of abuse and which have been published since 2010. Three different databases were searched, using dried, spots, and drugs of abuse as the descriptors and using a snowball search. After applying the exclusion criteria, 39 papers remained. The most common publications were related to the use of blood, which corresponded to 77% of the papers, followed by urine and oral fluid, which corresponded to 13 and 10% of the papers, respectively. The selected studies covered different illicit drugs, sample sizes of 5 to 250 µL and spot sizes ranging from 3 to 18mm in diameter. This review also examined the extraction techniques and the methods employed to analyze various biological matrices and drugs of abuse, mostly by liquid-extraction and liquid chromatography-tandem mass spectrometry. The benefits of DMS include: a simple sample pretreatment, better stability than liquid matrices, a simple extraction procedure, lower costs, and environmental benefits. DMS appears to be a promising technique in the field of toxicology and provides new perspectives for use in forensic laboratories.

A Simple and Rapid Hplc-Ms/Ms Method for Therapeutic Drug Monitoring of Amikacin in Dried Matrix Spots

A Simple and Rapid Hplc-Ms/Ms Method for Therapeutic Drug Monitoring of Amikacin in Dried Matrix Spots

Dried matrix spots in forensic toxicology.

Dried matrix spots (DMS) has gained the attention of different professionals in different fields, including toxicology. Investigations have been carried out in order to assess the potential of using DMS for the analysis of illicit substances, the main interest of forensic toxicologists. This technique uses minimal volumes of samples and solvents, resulting in simple and rapid extraction procedures. Furthermore, it has proved to increase analyte stability, improving storage and transportation. However, DMS presents some limitations: the hematocrit influencing accuracy and inconsistencies regarding the means of spotting samples and adding internal standard on paper. Thus, we provide an overview of analytical methodologies with forensic applications focusing on drugs of abuse and discussing the main particularities, limitations and achievements.

Enantiomeric methadone quantitation on real post-mortem dried matrix spots samples: Comparison of liquid chromatography and supercritical fluid chromatography coupled to mass spectrometry

This study describes two bioanalytical methods for the quantitation of the two methadone enantiomers in dried matrix spots using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and high performance supercritical chromatography tandem mass spectrometry (HPSFC-MS/MS). Dried matrix spots were obtained by spotting 10 µL of each sample fluid on a Whatman paper. Methadone and its main metabolite, EDDP, were extracted with 100 µL methanol and subsequently injected into the LC-MS/MS and SFC-MS/MS systems. Enantiomeric separation was achieved with AGP-column for the LC conditions and with Chiralpak IH-3 in SFC. The two methods were fully validated and 93 post-mortem samples were analysed with both analytical methods. Results from validation parameters and results obtained for all post-mortem samples were compared with a significant spearman correlation of rs = 0.9978 for R-methadone and rs = 0.9981 for S-methadone. The LC method provided better results in terms of uncertainty, retention factor and resolution, whereas SFC provides better sensitivity, with lower LOD. Median R-/S-methadone ratio in peripheral blood was found equal to 1.60 (N = 32), varying from 0.79 to 4.23. The reported values were in good agreement with previously published results.Based on the results obtained here, SFC-MS/MS can be considered a reliable alternative to the widely used LC-MS/MS for the quantitation of methadone enantiomers in bioanalysis and should be evaluated for other bioanalytical methods. Both methods can be easily and quickly used in toxicological routine analysis for the methadone quantitation in human fluids matrices, even if considering that the polysaccharide coated column IH-3 used in SFC does not allow the enantiomeric EDDP separation.

Extraction of dried oral fluid spots (DOFS) for the identification of drugs of abuse using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Dried matrix spots (DMS) have been utilized as an alternative sample collection method for drug identification in different biological specimens in clinical and forensic toxicology scenarios. Oral fluid has demonstrated its potential to establish recent drug use in instances such as driving under the influence of drugs or workplace drug monitoring programs. Moreover, this matrix has become an excellent choice over invasive or prone to adulteration approaches, like blood or urine collection. The possibility of drying oral fluid on a paper substrate has open newer opportunities to facilitate sample distribution and analysis. In this study, a dried oral fluid spots (DOFS) method was used to investigate its performance for the analysis of a suite of drugs of toxicological interest. A direct liquid extraction from the paper substrate was assessed, and the detection and quantification were conducted using liquid chromatography-tandem mass spectrometry instrument. Amphetamine, methamphetamine, ketamine, benzoylecgonine, and mitragynine were selected as target drugs to evaluate the DOFS method. Figures of merit including selectivity, matrix effects, recovery (average of 82%, with the exception of mitragynine – 55%), process efficiency, LOQ (0.2 ng/mL), linearity carry over, intraday (3.2–8.4%) and inter-day precision (7.3–18.8%), accuracy (−1.0 to −12.2), and stability of DOFS samples (35 days) are presented. The DOFS method extraction capability was compared against a commercially available weak anion exchange (WAX-S) tips, and the results are presented herein. The method was also successfully applied to eight blind specimens to demonstrate proof of applicability and fit of purpose.

Assessment of paper tip angular position, carryover, matrix effects and dried blood spot storage effect on paper spray mass spectrometry

Experimental and validation parameters of paper spray mass spectrometry.

Comparison of dried matrix spots and fabric phase sorptive extraction methods for quantification of highly potent analgesic activity agent (2R,4aR,7R,8aR)-4,7-dimethyl-2-(thiophen-2-yl)octahydro-2H-chromen-4-ol in rat whole blood and plasma using LC–MS/MS

The methods for quantification of highly potent analgesic agent (2R,4aR,7R,8aR)-4,7-dimethyl-2-(thiophen-2-yl)octahydro-2H-chromen-4-ol in rat whole blood and plasma were developed and validated using dried matrix spots (DMS) or fabric phase sorptive extraction (FPSE) techniques in combination with LC–MS/MS. 2-Adamantylamine hydrochloride was used as an internal standard (IS). Chromatographic separation was carried out on a reversed-phase column (2.0×75 mm, 5 μm) using water containing 0.1% formic acid and methanol containing 0.1% formic acid as mobile phases in gradient mode at a flow rate of 200 μL/min. The mass spectrometric detection was performed using electrospray ionization (ESI) in positive ion mode. MRM transitions were m/z 284.5 → 137.2/157.4 for the analgesic agent and m/z 152.3 → 93.1/107.2 for IS. Calibration curves were linear within 20–5000 ng/mL in dried plasma spots (DPS) or dried blood spots (DBS) experiments. The linearity was obtained in the range of 20–5000 ng/mL and 50–5000 ng/mL for plasma-FPSE and blood-FPSE experiments, respectively. The intra- and inter-day accuracy and precision did not exceed acceptable limits. The mean extraction recovery (%) was 26 for DPS, 25 for DBS, 38 for plasma-FPSE, 31 for blood-FPSE.

Paper-based immunocapture for targeted protein analysis

A novel sampling concept for mass spectrometric bottom-up targeted protein analysis is here demonstrated with polymeric sampling spots integrated with instant immunocapture for analysis of dried matrix spots. The polymers 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) and pHEMA-Tosyl for covalent attachment of antibodies where investigated alongside with adsorption on non-treated filter paper. From performance characterization, the pHEMA-VDM had the best performance. The sampling spots demonstrated fast and easy sampling and preparation of human serum spiked with the biomarker human chorionic gonadotropin. The sampling spots enabled a detection limit of 1 ng/mL (26.4 pM) within a five point concentration curve from 1 ng/mL to 20 ng/mL (R2 = 0.97). The detection limit was demonstrated to be two times lower than previously demonstrated with standard DMPK-C sampling cards. A five point concentration curve from 100 ng/mL to 2000 ng/mL was also investigated (R2 = 0.998). Intra day precision was within 16% and 23% for concentration range 1 ng/mL to 20 ng/mL and 100 ng/mL to 2000 ng/mL, respectively. Inter day precision was within 20%. Accuracy was determined to 10% and 11% for 2.5 ng/mL and 20 ng/mL, respectively. The sampling spots were also demonstrated in a realistic setting where serum samples from two confirmed patients with testicular germ cell cancer were analyzed. These analyses confirmed an elevated hCG content in the sera of 418.5 ± 4.2 ng/mL and 21 ± 0.02 ng/mL hCG for patient one and two respectively.

New analytical approach to determine organophosphorus insecticides in blood by dried matrix spots sampling and GC-MS/MS

This work describes the optimization of a new method for the determination of five organophosphorus insecticides in whole blood. The analytes were extracted from the matrix (50μL) using the dried blood spot (DBS) approach and were analyzed by gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS). The studied compounds (diazinon, chlorpyrifos, parathion-ethyl, chlorfenvinphos and quinalphos) were chosen based on the statistics of intoxications in Portugal, and ethion was used as internal standard. The method was fully validated, taking into account international guidelines for bioanalytical method validation, such as those of the Food and Drug Administration (FDA), International Conference on Harmonization (ICH) and Scientific Working Group for Forensic Toxicology (SWGTOX). A linear range of 0.1-25μg/mL was obtained for all compounds, except for diazinon and quinalphos (0.05-25μg/mL and 0.25-25μg/mL, respectively), presenting determination coefficients above 0.99. Concerning precision, the coefficients of variation (CVs) were lower than 14% for all compounds. Those compounds were found to be stable in the samples. Although the values obtained for recovery were low (between 1 and 12%), the method proved to be sensitive, since detection limits between 0.05 and 0.1μg/mL were obtained. The novelty is the use of the DBS approach in the extraction of these compounds, and this is the first paper reporting it: DBS is a recent technique of bioanalysis in the field of toxicology, and in addition to its simplicity and sensitivity, it is applicable routinely in both clinical and forensic toxicology situations. Graphical abstract ᅟ.

Determination of the low-abundant protein biomarker hCG from dried matrix spots using immunocapture and nano liquid chromatography mass spectrometry

Using LC-MS/MS for determination of low-abundance protein biomarkers from dried blood spots is challenging due to the combination of low biomarker levels (low pM-level) and small sample volumes (typically <50 μL). In the present paper it is demonstrated that use of state-of-the-art nano liquid chromatography triple quadrupole mass spectrometry in combination with immunoaffinity sample clean-up enable determination of the low abundance biomarker human chorionic gonadotropin (hCG) from four different biological matrices (whole blood, serum, plasma and urine) at its upper reference level (low pM). Detection limits for hCG was determined for all matrices from both commercially available non-soluble DBS sampling material (DMPK-C) and the water-soluble material carboxymethyl cellulose (CMC). The detection limits (S/N = 3) were ranging from 5.0 IU/L (14.5 pM; whole blood) to 10.5 IU/L (30.5 pM; urine) for DMPK-C and from 2.1 IU/L (6.1 pM; urine) to 6.4 IU/L (18.6 pM; plasma) for CMC. A brief evaluation was performed for both sampling materials using serum as matrix resulting in sufficient linearity (r2 ≥ 0.93, range 20–1000 IU/mL (58–2900 pM) for DMPK-C and 10–1000 IU/mL (29–2900 pM) for CMC), repeatability (RSD% = 13–31%) and accuracy (95–106%). To demonstrate the applicability of the method to real samples, a serum sample from a patient previously diagnosed with cancer was also analyzed using both sampling materials. The concentration levels found using the two materials were similar (5280± 595 IU/L (15,312 ± 1726 pM, n = 3) in the DMPK-C spot and 5060 ± 430 IU/L (14,674 ± 1247 pM, n = 3) in the CMC spot). All in all this demonstrated that the tools for determination of low abundance biomarkers at upper reference level from dried matrix spots now is available through a combination of immunoaffinity enrichment and state-of-the-art LC-MS/MS.

Enantioseparation and determination of asenapine in biological fluid micromatrices by HPLC with diode array detection

Asenapine is a recent drug approved in the European Union for the treatment of bipolar disorder. An original approach has been developed for asenapine analysis in patients treated with the drug, including miniaturized microsampling procedures, separation and quantitation of drug enantiomers. An original enantioselective method based on high-performance liquid chromatography with diode array detection was developed and applied to the determination of asenapine enantiomer levels in innovative haematic samples: four micromatrices have been tested, two based on dried matrix spots (dried blood spots and dried plasma spots) and two based on volumetric absorptive microsampling (from blood and plasma). Chiral separation was achieved on a cellulose-tris(3,5 dimethylphenylcarbamate) column, with a mobile phase containing bicarbonate buffer and acetonitrile. The method was validated with satisfactory results of linearity and precision on all matrices that showed also a significant performance in terms of stability, feasibility and reliability, when compared to fluid plasma sampling, handling and processing. Among micromatrices, both volumetric absorptive microsampling types were superior to dried matrix spots in terms of data reproducibility and correspondence with plasma levels. The bioanalytical approach proposed herein provides for the first time a chiral high-performance liquid chromatographic method for the determination of asenapine enantiomers, coupled to a very effective microsampling strategy.

Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays.

Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.

Potential missing steps for a wide use of dried matrix spots in biomedical analysis.

Microsampling, mainly as DBS, has been significantly expanded in the biomedical and pharmaceutical communities in the last 10 years. In parallel, technology and methodology have evolved to overcome some of the issues associated with this sampling procedure. Despite the continuous developments and interest, only a few validated and routinely implemented clinical applications have arisen beyond the initial inborn screening. Based on the latest developments in this field, this perspective aims to discuss some of the missing steps (i.e., the habits to change, the Health Authorities acceptance and the shift for dried plasma generation), which may turn the current use of microsampling into an established and standard procedure in clinical and pharmaceutical analysis.

Enhanced stability of blood matrices using a dried sample spot assay to measure human butyrylcholinesterase activity and nerve agent adducts.

Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intraspot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intraspot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10 times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intraspot variability without the need to control for initial sample volume, and enhances analyte stability.

Quantitation of pregabalin in dried blood spots and dried plasma spots by validated LC–MS/MS methods

In this paper, novel LC–MS/MS methods for the determination of antiepileptic drug pregabalin in dried matrix spots (DMS) are presented. This attractive technique of sample collection in micro amount was utilized in the form of dried blood spots (DBS) and dried plasma spots (DPS). Following a pre-column derivatization procedure, using n-propyl chloroformate in the presence of n-propanol, and consecutive liquid–liquid extraction, derivatized pregabalin and its internal standard, 4-aminocyclohexanecarboxylic acid, were detected in positive ion mode by applying two SRM transitions per analyte. A YMC-Pack Octyl column (50mm×4.0mm, 3μm particle size) maintained at 30°C, was utilized with running mobile phase composed of acetonitrile: 0.15% formic acid (85:15, v/v). Flow rate was 550μL/min and total run time 2min. Established methods were fully validated over the concentration range of 0.200–20.0μg/mL for DBS and 0.400–40.0μg/mL for DPS, respectively, while specificity, accuracy, precision, recovery, matrix-effect, stability, dilution integrity and spot homogeneity were found within acceptance criteria. Validated methods were applied for the determination of pregabalin levels in dried blood and plasma samples obtained from patients with epilepsy, after per os administration of commercial capsules. Comparison of drug level in blood and plasma, as well as correction steps undertaken in order to overcome hematocrit issue, when analyzing DBS, are also given.