Microsampling Device Research Articles

Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device.

Therapeutic drug monitoring (TDM) for immunosuppressive (ISP) drugs is an important component of organ and tissue transplantation and chemotherapy management. Whole blood is the specimen type for the quantitative analysis of cyclosporine A, everolimus, sirolimus, and tacrolimus. Some alternatives to venous whole blood samples have the potential to reduce blood volume requirements and simplify sample collection and transport. The feasibility of ISP drug (cyclosporine A, everolimus, sirolimus, and tacrolimus) monitoring via microsampling device (MitraTM, Neoteryx) was assessed by comparing venous samples collected and extracted using microsampling device to conventional extraction procedure. Analysis was performed by LC-MS/MS. All analytes were found to be linear across the measurement range of 22.7-937.0 ng/mL (18.9-779.1 nmol/L) for cyclosporine A, 2.3-44.2 ng/mL (2.4-46.1 nmol/L) for everolimus, 2.2-47.2 ng/mL (2.4-51.6 nmol/L) for sirolimus, and 2.2-41.3 ng/mL (2.7-51.4 nmol/L) for tacrolimus. Imprecision was evaluated at concentrations within the therapeutic range and was found to be 10.1% and 5.8% for cyclosporine A, 10.0% and 10.0% for everolimus, 15.0% and 11.9% for sirolimus, and 6.8% and 8.5% for tacrolimus. Method comparison (n = 30 for each analyte, using Deming regression) indicated slopes of 1.08, 1.02, 0.90, and 1.15 and intercepts of -12.8 ng/mL (-10.7 nmol/L), 0.8 ng/mL (0.8 nmol/L), 1.5 ng/mL (1.7 nmol/L), and -0.3 ng/mL (-0.3 nmol/L) for cyclosporine A, everolimus, sirolimus, and tacrolimus, respectively. This feasibility study demonstrates that precision and bias of ≤15% can be achieved for microsampling-based ISP monitoring.

Challenges and opportunities in blood flow through porous substrate: A design and interface perspective of dried blood spot

Blood microsampling is desired in clinical, pharmaceutical and biomedical fields to overcome the challenges of conventional whole blood sampling. One of the popular methods for blood microsampling is the dried blood spot (DBS) kit and the collected sample is subsequently used for bioanalysis. The current practice of DBS is simple to use, cheap and very well standardized from sample collection to analysis. However, DBS suffers from several well documented challenges related to blood spot formation such as varying hematocrit volume, thin layer chromatography effect and subsequent bio-analysis resulting in a variable and ocassionally high failure rate. A major source of these problems is our limited understanding of blood flow in porous media under different ambient and material conditions. Therefore, it is highly desirable to understand the parameters that affect blood flow in a porous medium to enable a more robust design of DBS and generally blood microsampling kits. In this review, we discuss some existing blood microsampling techniques while focusing on the challenges associated with blood flow dynamics. We also review existing studies on the potential factors that affect the permeation (imbibition or wicking) and spreading of blood in a thin, porous substrate as means to understand and overcome the challenges in designing new DBS kits and blood microsampling devices. Thereafter, we have discussed recent advances in the design of passive flow-based devices to overcome these challenges of current blood microsampling by DBS. Finally, we present a few applications of DBS in clinical and non-clinical studies. This review can benefit researchers working at the interface of complex fluid flow, surface chemistry, and material and device design for biomedical and biological applications.

Fast and simple method using DLLME and FAAS for the determination of trace cadmium in honey

A novel method for the determination of trace cadmium in honey is presented. It consists of a preconcentration step based on dispersive liquid-liquid microextraction (DLLME) and measurement by flame AAS. Samples are dissolved in water. A mixture of carbon tetrachloride (extraction solvent) and acetonitrile (dispersion solvent) containing ammoniun pyrrolidine dithiocarbamate (APDC) as complexing agent is injected rapidly into the dissolved sample by means of a syringe pump. After centrifugation, an aliquot of the organic phase is injected in the nebuliser of an atomic absorption spectrometer by means of a lab-made microsampling device. Response (integrated absorbance) was linear with concentrations up to 12 μg L−1. Precision (sr(%)) at the 5 μg L−1 level was < 3.1%. Detection and quantification limits were 1.6 and 5.5 μg per 100 g of honey respectively. Six commercial samples were spiked at the 10 μg per 100 g level and analysed by the proposed method and by electrothermal atomic absorption spectrometry. The results did not differ at the 5% significance level, with recoveries for the DLLME/FAAS method ranging from 82 to 125%. The proposed method requires simple sample preparation and does not use expensive equipment, thus it could be used as screening method for the content of cadmium in honey.

112 Skin micro-sampling device, MicrobiopsyTM is more sensitive to detect known melanoma and non-melanoma skin cancer biomarkers on volunteers

Basal cell carcinoma (BCC) often appears on the face and neck and recurrence rates are significant. Our patented skin Micobiopsy is a minimally invasive device that does not require local anaesthetic nor sutures. The aims of this study were to evaluate 1) feasibility of microbiopsy sampling in suspected BCC lesions; 2) evaluate sample quality for Real-Time PCR biomarker analysis; and 3) compare BCC mRNA biomarker expression levels to conventional shave biopsy. We collected 1,170 microbiopsy samples from 106 patients with suspected BCC lesions. We took 290 microbiopsies from the head and neck and 880 from the rest of the body. Five microbiopsies were taken from lesions in situ and from peri-lesion skin 10mm from the lesion border. Afterwards the lesions were removed by shave biopsy. Out of 117 skin lesions, 77 were BCC and 36 were other non-melanoma skin cancer. The lesion half was micro-dissected to obtain separate lesion from peri-lesion samples. RNA was isolated from each sample. TaqMan assays were performed using published BCC markers (PTCH1, GLI1 and BAP1), SCC markers (CFH, COX7C, RAB31 and MAP4K4) and melanoma markers (CMIP, TYR and LINC00518). The study volunteer recruitment was efficient largely due to the minimally invasive nature of the device. There were no adverse events during the study. Dermatopathologists found no confounding artefacts when diagnosing the microbiopsied lesions. The Real-Time PCR results showed sufficient RNA for gene expression analysis based on GAPDH, B-actin and RPLPO. Comparison between microbiopsy and shave biopsy sampling revealed increased differential gene expression results with microbiopsy sampling. These data support the hypothesis that superficial, targeted sampling could improve patient compliance, reduce scaring and increase diagnostic testing sensitivity/specificity for BCC by providing a purer tumor sample.

SAT-011 Quantification of Testosterone, Androstenedione and 17-Hydroxyprogesterone Collected Using Mitra®Micro Sampling Devices

Introduction Measurement of total plasma testosterone (T), androstenedione (A4) and 17-hydroxyprogesterone (17OHP) typically requires a venous sample. A highly sensitive and specific assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after extraction from a Mitra® volumetric absorptive microsampling (VAMs) device. VAMs devices are small absorptive polymer tips which absorb a fixed amount of blood (1); they can be dried after collection of finger prick blood and stored prior to analysis. Differences in binding in whole blood compared to serum require the quantification of haematocrit using a published protocol (2), thus allowing an estimated serum result to be generated. Method Pre-mixed calibrator/sample (10 µL) was combined with deionised water and mixed internal standard, after vortexing MTBE was added and mixed by inversion for 1 minute. The supernatant was transferred to a clean 2 mL deep-well plate, dried down and re-constituted with 100 µL 50:50 methanol/water. Mitra® tips were prepared from anonymised whole blood samples in the laboratory and compared to the corresponding plasma using the same method. Results Mean recovery was 86% for A4, 102% for T and 97% for 17OHP, the lower limit of quantification was 1 nmol/L for A4, 1 nmol/L for testosterone and 4 nmol/L for 17OHP. Ion suppression was within acceptable criteria for all the analytes measured. For the samples prepared in the laboratory, comparisons for T, A4 and 17OHP the R-squared values were 0.95, 0.93 and 0.95 respectively with a minimal bias. Summary We have developed a robust assay for LC-MS/MS measurement of VAMs for T, A4, and 17OHP in a routine clinical laboratory. It is simple, reproducible and easy to perform and may have implications for monitoring of T therapy in hypogonadal males or screening/monitoring of patients with congenital adrenal hyperplasia. In-house comparisons of T, A4 and 17OHP compared well when haematocrit was used to adjust for the space occupying effect of red cells.

Simultaneous determination of vancomycin and creatinine in plasma applied to volumetric absorptive microsampling devices using liquid chromatography-tandem mass spectrometry

Vancomycin (VCM) is a glycopeptide antibiotic, widely used against methicillin-resistant Staphylococcus aureus infections, and its clearance is highly correlated to creatinine (CRE) clearance. Usually, VCM and CRE levels are measured in serum or plasma specimens obtained from venous blood, after phlebotomy. As the majority of hospitals in Developing Countries do not have on-site access to VCM measurements, the availability of data on patient’s VCM levels would involve the transport of liquid samples to specialized laboratories. An alternative to overcome the logistic difficulties of liquid biological specimens is the use of dried microsamples, such as plasma applied to volumetric absorptive microsampling (VAMS) devices. The aim of this study was to develop and validate a LC–MS/MS assay for the simultaneous determination of VCM and CRE in plasma applied to VAMS devices. VCM and CRE levels were determined in clinical relevant ranges with acceptable precision and accuracy and both compounds were stable in VAMS for up to two weeks at 45 °C. Concentrations measured in VAMS differed from those measured in plasma and the use of estimation approaches was necessary to estimate plasma concentrations. VCM levels in plasma can be estimated by multiplying VAMS measurements by the correction factor of 0.934. The unbiased estimation of CRE plasma levels from VAMS required the use of the equation CREplasma = 0.9808 * CREVAMS + 1.6621. VAMS can be used as an alternative for short-term storage and transportation of plasma at ambient and high temperatures, allowing a more widespread access to VCM therapeutic drug monitoring in limited-resources settings.

Evaluation of the Mitra microsampling device for use with key urinary metabolites in patients with Alkaptonuria.

Alkaptonuria is a disorder of tyrosine metabolism where elevated circulating homogentisic acid causes progressive dysfunction of collagenous tissues. Logistical, financial and patient experience concerns with collection and transport of specimens to central laboratories makes evaluation of microsampling attractive. Volumetric absorptive microsampling devices were evaluated for accuracy, precision and drying time. Elution methods were evaluated for several urinary metabolites of interest and stability assessed under multiple conditions. Comparison was performed between dried and liquid specimens via LC-MS/MS. Volumetric absorptive microsampling was found to be highly accurate and precise. Elution methods showed good recovery and reproducibility. Dried and liquid samples compared favorably. Analyte stability was variable, presenting barriers to implementation into routine use in this context.

Investigation of dried blood sampling with liquid chromatography tandem mass spectrometry to confirm human exposure to nerve agents

A method was developed to detect and quantify organophosphate nerve agent (OPNA) metabolites in dried blood samples. Dried blood spots (DBS) and microsampling devices are alternatives to traditional blood draws, allowing for safe handling, extended stability, reduced shipping costs, and potential self-sampling. DBS and microsamplers were evaluated for precision, accuracy, sensitivity, matrix effects, and extraction recovery following collection of whole blood containing five OPNA metabolites. The metabolites of VX, Sarin (GB), Soman (GD), Cyclosarin (GF), and Russian VX (VR) were quantitated from 5.0 to 500 ng mL−1 with precision of ≤16% and accuracy between 93 and 108% for QC samples with controlled volumes. For unknown spot volumes, OPNA metabolite concentrations were normalized to total blood protein to improve interpretation of nerve agent exposures. This study provides data to support the use of DBS and microsamplers to collect critical exposure samples quickly, safely, and efficiently following large-scale chemical exposure events.

Volumetric absorptive MicroSampling vs. other blood sampling materials in LC–MS-based protein analysis – preliminary investigations

The aim was to evaluate the performance of different microsampling materials in LC–MS-based protein analysis. The evaluated materials were the Volumetric Absorptive MicroSampling (VAMS) device, the pure cellulose sampling material (DMPK-C) and the water-soluble material (carboxymethyl cellulose, CMC), with the main emphasis on VAMS. Six proteins with different physicochemical properties were used as model proteins. A quick and generic sample preparation consisting of extraction/dissolution of the blood spot, tryptic digestion and subsequent matrix precipitation was applied prior to analysis. The recovery from VAMS, compared to DMPK-C, was dependent on the protein analyte: Lower recovery compared to DMPK-C was seen for β-lactoglobulin and myoglobin (74% and 80%, respectively) while higher recovery compared to DMPK-C was seen for cytochrome c and albumin (149% and 197%, respectively). The recovery from CMC was comparable to the recovery from DMPK-C for all proteins except for cytochrome c (76%). Hematocrit bias was evaluated for DMPK-C and VAMS, and the blood hematocrit influenced the protein analysis from both the materials. A preliminary evaluation was performed for VAMS: The correlation coefficient (R2 ≥ 0.983), the accuracy (71–101%) and the precision (RSD ≤ 20%) were determined for blood spiked with the six model proteins.

Microsampling Collection Methods for Measurement of C-peptide in Whole Blood.

Microsampling techniques are alternative methods to venous sampling for obtaining blood for measurement of circulating biomarkers, offering the convenience of reduced sample volume and elimination of the need for phlebotomists. Dried blood spot (DBS) microsampling methods have been used for many years while more recently a volumetric absorptive microsampling device (VAMS™) has been introduced. In diabetes mellitus, circulating C-peptide is commonly used as an indicator of endogenous insulin secretion and clinical measurement can aid in diagnosis as well as informing on therapy. This pilot study investigated the effectiveness of microsampling collection of capillary blood for measurement of C-peptide. Capillary blood was collected into capillary tubes and centrifuged for plasma samples. Simultaneous samples were also collected using both microsampling methods (DBS and VAMS). Blood from both microsamplers was extracted prior to assaying for C-peptide alongside the corresponding plasma samples, using specific immunoassays and results obtained from microsampling compared to the reference plasma concentrations. Stability was determined by collecting duplicate DBS and VAMS and assaying both in a single assay after storing one at -20°C immediately and one at room temperature for 48 hours post-collection. Good agreement was observed between C-peptide concentrations in plasma and equivalent DBS and VAMS samples ( R2 = .929 and .9231, DBS and VAMS, respectively), with mean differences of 75.7 and 8.4 pmol/L observed for DBS and VAMS. Small decreases in C-peptide of 11.6% and 0.1% were observed after 48 hours storage for DBS and VAMS, respectively. C-peptide collected using DBS and VAMS showed good agreement with reference plasma concentrations, suggesting both would be an effective microsampling method for collection and measurement of C-peptide.

Evaluation of a novel micro-sampling device, Mitra™, in comparison to dried blood spots, for analysis of praziquantel in Schistosoma haematobium-infected children in rural Côte d’Ivoire

Pharmacokinetic (PK) studies with paediatric populations are increasing in importance for drug development. However, conventional PK sampling methods are characterised by invasiveness and low patient adherence, unsuitable for use with sensitive population, such as children. Mitra™ is a novel volumetric absorptive micro-sampling device, which offers an alternative to the dried blood spotting (DBS) technique, a current popular sampling technique within PK studies. We tested Mitra™ for the first time in the framework of a randomised controlled trial in rural Côte d’Ivoire. Thirty-five school-aged children, infected with Schistosoma haematobium, were sampled with both DBS and Mitra™, at 10 time points after treatment with praziquantel (PZQ). An extraction method for PZQ from Mitra™ was developed, optimised and validated. Analytes, namely R- and S-praziquantel (R-/SPZQ) and the main human metabolite, R-trans-4-OH-praziquantel, were measured using liquid chromatography-tandem mass spectrometry and the results were compared with Bland-Altman analysis to determine agreement between matrices. PK parameters, such as maximal plasma concentration and area under the concentration-time curve, were estimated using non-compartmental analysis.While we observed strong positive correlation (R2 > 0.98) and agreement between both matrices within the calibration line and quality control samples, Mitra™ revealed higher concentrations of all the analytes in the majority of patients’ samples compared to DBS sampling, namely 63% samples for RPZQ, 49% for SPZQ and 78% for the metabolite were overestimated. While T1/2 and Tmax were in agreement between both matrices, area under the curve and maximal blood concentration were up to 2× higher for Mitra™ samples, with P < 0.005 for all parameters except Cmax of SPZQ, which was not significantly different between the two matrices. The reasons for the higher PZQ concentrations, more pronounced in incurred Mitra™ samples compared to spiked samples, are yet to be fully explored. Mitra™ appears superior to DBS in terms of simplicity and practicality however labelling issues and the high price of Mitra™ are difficult to overlook.

Novel volumetric adsorptive microsampling technique for determination of perfluorinated compounds in blood

Microsampling is an attractive option for significantly reducing the volume of blood taken for chemical analysis allowing for blood samples taken as a ‘finger-prick’ with a lancet. A novel, volumetric adsorptive microsampling (VAMS™) device, which reproducibly collects a small volume of 10 μL whole blood in a hematocrit-independent manner, is evaluated in a human biomonitoring setting, and has been utilized for analysis of several perfluoroalkyl acids (PFAA). The results show that the VAMS technique is applicable for PFAA analysis, method has good linearity, repeatability, accuracy and is sufficiently sensitive for samples from general populations. The stability of PFAAs with VAMS devices is shown to be acceptable, which supports the sampling and transportation strategy of several study designs. Furthermore, as well as allowing for a quick and efficient extraction and analysis flow path, the VAMS microsampler is an easy to use device in a real-world sample collection scenario.

P648 Clinical feasibility of dried blood sampling for infliximab in IBD patients

Therapeutic drug monitoring (TDM) is important to optimise outcome of infliximab (IFX) treatment in inflammatory bowel disease (IBD). Dried blood spot (DBS) sampling using capillary blood obtained via a finger prick could facilitate TDM, since patients can administer this finger prick themselves at any time and away from the hospital. We investigated the predictive performance and the feasibility of DBS for measuring IFX concentrations in IBD patients. We studied 40 adult IBD patients receiving IFX therapy according to standard guidelines. From each patient blood was obtained simultaneously via venepuncture and DBS via finger prick by a trained employee at 3 different time points (trough, peak, 3–5 weeks after infusion). One week before IFX infusion (time point 4), patients performed DBS at home and the sample was directly sent to Sanquin laboratories, Amsterdam, The Netherlands. The corresponding serum concentration for this time point was estimated using Bayesian pharmacokinetic analysis using the three samples obtained by venepuncture. Capillary blood was obtained by a microsampling device developed by Neoteryx™. Haematocrit (Hct) values of each individual patient were used to convert DBS eluate results to values which can be compared with (venous) serum concentrations. Spearman’s correlation coefficient was used to assess correlation and bias was calculated. Forty IBD patients were included with median [interquartile range] age: 41 [32–50], albumin: 43 mg/l [41–45], and CRP: 1.3 mg/l [0.4–4.4]. IFX concentrations obtained from the DBS method correlated strongly with serum results from the same patient for IFX trough- and mid-interval concentrations (Spearman correlation coefficient >0.88) and moderately for IFX peak concentrations (Spearman correlation coefficient = 0.69). IFX serum concentrations from the DBS performed at home showed strong correlation with the concentrations obtained by Bayesian analysis (Spearman correlation coefficient = 0.71). No structural bias was shown (Table 1). Concentration range and relative bias. Concentration range and relative bias. DBS via finger prick can be used for the assessment of serum IFX concentrations. More importantly, we showed the feasibility of using DBS via finger prick at home. This method greatly facilitates the use of TDM in the treatment of IBD patients using IFX.

High-Precision In Situ 87Sr/86Sr Analyses through Microsampling on Solid Samples: Applications to Earth and Life Sciences.

An analytical protocol for high-precision, in situ microscale isotopic investigations is presented here, which combines the use of a high-performing mechanical microsampling device and high-precision TIMS measurements on micro-Sr samples, allowing for excellent results both in accuracy and precision. The present paper is a detailed methodological description of the whole analytical procedure from sampling to elemental purification and Sr-isotope measurements. The method offers the potential to attain isotope data at the microscale on a wide range of solid materials with the use of minimally invasive sampling. In addition, we present three significant case studies for geological and life sciences, as examples of the various applications of microscale 87Sr/86Sr isotope ratios, concerning (i) the pre-eruptive mechanisms triggering recent eruptions at Nisyros volcano (Greece), (ii) the dynamics involved with the initial magma ascent during Eyjafjallajökull volcano's (Iceland) 2010 eruption, which are usually related to the precursory signals of the eruption, and (iii) the environmental context of a MIS 3 cave bear, Ursus spelaeus. The studied cases show the robustness of the methods, which can be also be applied in other areas, such as cultural heritage, archaeology, petrology, and forensic sciences.

Sponge Spray-Reaching New Dimensions of Direct Sampling and Analysis by MS.

Sample preparation for the analysis of clinical samples with the mass spectrometer (MS) can be extensive and expensive. Simplifying and speeding up the process would be very beneficial. This paper reports sponge spray-a novel sampling and direct MS analysis approach-attempting exactly that. It enables direct analysis without any sample preparation from dried blood, plasma, and urine. The tip of a volumetric absorptive microsampling device is used to collect an exact amount of sample and from that same tip an electrospray can be directed into a mass spectrometer. We demonstrate here that, although with significant matrix effects, quantitation of penicillin G, a common antimicrobial, is possible in plasma and in urine, with essentially no sample preparation.

Comparison of nanofluidic and ultra-high performance liquid chromatography-tandem mass spectrometry for high sensitive pharmacokinetic studies of estrogens starting from whole blood microsampling

Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the use of miniaturized analytical techniques may provide undeniable advantages in terms of sensitivity, sample and solvent consumption compared to the reference UHPLC–MS/MS methods In this study, we present the development of a nanofluidic-LC–MS/MS method to analyze two model analytes of therapeutic interest, namely estradiol (E2) and estetrol (E4) after microsampling with volumetric absorptive microsampling (VAMS) devices, an innovative sampling technique to collect small volumes of whole blood. The nanofluidic LC–MS/MS method was developed using an experimental design to find the optimal conditions to analyze both E2 and E4 with the highest sensitivity. Subsequently, the optimized method was validated according to ICH guidelines and compared to a previously developed UHPLC–MS/MS method. A limit of quantitation of 50pg/ml was reached with the LC-chip method, which is 50 times better than UHPLC–MS/MS. Both methods were then critically evaluated from the analytical and operational points of view. Finally, the quantitation of estrogens after whole blood microsampling was compared with the results obtained with the corresponding plasma samples.

Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition

Volumetric absorptive microsampling device (VAMS) was evaluated for bioanalysis of tacrolimus, which was used as a model drug with high blood cell partition. Aliquots of blood (ca. 10μL) with different hematocrits and fortified with tacrolimus were wicked up by VAMS and tacrolimus was extracted with a methanol-water mixture (1:1, v/v) via sonication. After chromatography on an AQUITY UPLC HSS T3 column (100×2.1 i.d., mm, 1.8μm), tacrolimus and the internal standard ascomycin, were detected in the positive ion mode with electrospray ionization by monitoring of transitions m/z 826.6→616.4 and m/z 814.6→604.0, respectively. An assay method to quantify tacrolimus from 1 to 250ng/mL in whole blood was qualified by ensuring that linearity, selectivity, intra- and inter-batch reproducibility, and stability were within the acceptance criteria. Consistent and high extraction recovery of tacrolimus was ensured from blood with low- (20%), mid- (45%), and high-hematocrit (65%) levels with minimal matrix effects. Apparent instability at ambient temperature or 4°C possibly due to reduced recovery suggests that tacrolimus in VAMS should be stored at −25°C until assay. Potential reduced recovery over time from VAMS should be taken into consideration in method optimization.

Application of volumetric absorptive microsampling for robust, high-throughput mass spectrometric quantification of circulating protein biomarkers

Volumetric absorptive micro sampling (VAMS™) allows accurate sampling of 10µL of blood from a minimally invasive finger prick and could enable remote personalized health monitoring. Moreover, VAMS overcomes effects from hematocrit and sample heterogeneity associated with dried blood spots (DBS). We describe the first application of VAMS with the Mitra® microsampling device for the quantification of protein biomarkers using an automated, high-throughput sample preparation method coupled with mass spectrometric (MS) detection.The analytical performance of the developed workflow was evaluated for 10 peptides from six clinically relevant proteins: apolipoproteins A-I, B, C-I, C-III, E, and human serum albumin (HSA). Extraction recovery from blood with three different levels of hematocrit varied between 100% and 111% for all proteins. Within-day and total assay reproducibility (i.e., 5 replicates on 5days) ranged between 3.2–10.4% and 3.4–12.6%, respectively. In addition, after 22weeks of storage of the Mitra microsampling devices at −80°C, all peptide responses were within ±15% deviation from the initial response. Application to data-independent acquisition (DIA) MS further demonstrated the potential for broad applicability and the general robustness of the automated workflow by reproducible detection of 1661 peptides from 423 proteins (average 15.7%CV (n=3) in peptide abundance), correlating to peptide abundances in corresponding plasma (R=0.8383).In conclusion, we have developed an automated workflow for efficient extraction, digestion, and MS analysis of a variety of proteins in a fixed small volume of dried blood (i.e., 10µL). This robust and high-throughput workflow will create manifold opportunities for the application of remote, personalized disease biomarker monitoring.

Whole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry

Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low volumes of biological material are available and low concentrations (ng/ml) are often expected. In this context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect 10 or 20μl of whole blood from tested subjects. In this study, we present the development and validation of ultra-high performance liquid chromatography coupled to tandem mass spectrometry method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a common consideration when using negative ionization mode, we demonstrated in this work that an excellent sensitivity could be reached by carefully optimizing the nature and concentration of the mobile phase additive. After the optimization of every experimental parameter, the stability, selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In addition, the excellent performances of the method were confirmed by two independent proof-of-concept pharmacokinetic studies of E4 after VAMS collection in a murine model.

Adhesive blood microsampling systems for steroid measurement via LC–MS/MS in the rat

IntroductionLiquid Chromatography Tandem Mass Spectrometry (LC–MS/MS) allows for the direct analysis of multiple hormones in a single probe with minimal sample volume. Rodent-based animal studies strongly rely on microsampling, such as the dry blood spot (DBS) method. However, DBS suffers the drawback of hematocrit-dependence (non-volumetric). Hence, novel volumetric microsampling techniques were introduced recently, allowing sampling of fixed accurate volumes. We compared these methods for steroid analysis in the rat to improve inter-system comparability. ExperimentalWe analyzed steroid levels in blood using the absorptive microsampling devices Whatman® 903 Protein Saver Cards, Noviplex™ Plasma Prep Cards and the Mitra™ Microsampling device and compared the obtained results to the respective EDTA plasma levels. Quantitative steroid analysis was performed via LC–MS/MS. For the determination of the plasma volume factor for each steroid, their levels in pooled blood samples from each human adults and rats (18weeks) were compared and the transferability of these factors was evaluated in a new set of juvenile (21days) and adult (18weeks) rats. Hematocrit was determined concomitantly. ResultsUsing these approaches, we were unable to apply one single volume factor for each steroid. Instead, plasma volume factors had to be adjusted for the recovery rate of each steroid and device individually. The tested microsampling systems did not allow the use of one single volume factor for adult and juvenile rats based on an unexpectedly strong hematocrit-dependency and other steroid specific (pre-analytic) factors. DiscussionOur study provides correction factors for LC–MS/MS steroid analysis of volumetric and non-volumetric microsampling systems in comparison to plasma. It argues for thorough analysis of chromatographic effects before the use of novel volumetric systems for steroid analysis.