Volumetric Absorptive Microsampling Research Articles

Patiromer Does Not Alter Tacrolimus Pharmacokinetics in Kidney Transplant Recipients When Administered Three Hours Post–Tacrolimus

Background. Hyperkalemia is common in kidney transplant (KTx) recipients. Patiromer, a potassium-binding polymer used to treat acute and chronic hyperkalemia, has the potential to bind charged particles in the gastrointestinal tract and thereby potentially affect the absorption of coadministered drugs. The immunosuppressive drug tacrolimus (Tac) has a narrow therapeutic window, is susceptible to drug-drug interactions (DDIs), and a potential gastrointestinal interaction with patiromer could elevate the risk of allograft rejection. We aimed to investigate the potential DDI between patiromer and Tac pharmacokinetics in KTx with hyperkalemia by sampling capillary blood using volumetric absorptive microsampling (VAMS). Methods. Thirteen KTx recipients on Tac twice daily (BID) with plasma potassium levels of >4.6 mmol/L were included. Two 12 h Tac pharmacokinetic investigations were performed with and without 8.4 mg patiromer/d for 1 wk. Oral Tac dose remained unchanged and patiromer was administered 3 h after Tac dose. Tac sampling was self-conducted using VAMS after mastering the technique. Results. Ten patients provided 2 evaluable pharmacokinetic profiles. The Tac area under the curve (AUC)0–12 ratio (AUCTac+patiromer/AUCTac) was 0.99 (90% confidence interval [CI], 0.86-1.14), and the Cmax ratio was 1.01 (90% CI, 0.86-1.19). Tac C0 and C12 fulfilled the bioequivalence criteria with a ratio of 0.98 (90% CI, 0.90-1.07) and 0.93 (90% CI, 0.83-1.04), respectively. Conclusions. When administered 3 h after the Tac morning dose, patiromer has no clinically relevant impact on Tac pharmacokinetics. We demonstrate that VAMS is a well-suited sampling method to simplify the execution of DDI studies.

Personalization of pharmacotherapy with sirolimus based on volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients-from LC-MS/MS method validation to clinical application.

The benefits of pharmacotherapy with sirolimus (SIR) in pediatric transplant recipients are well established. Traditionally, whole blood samples have been used to measure SIR concentrations. Volumetric Absorptive Microsampling (VAMS) is an alternative sampling strategy suitable for Therapeutic Drug Monitoring (TDM). In this study, we developed and validated two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determining SIR concentrations in whole blood (WB) and capillary whole blood samples collected using a VAMS-Mitra™ device. We used protein precipitation during WB sample preparation and dispersive liquid-liquid microextraction (DLLME) with methyl tert-butyl ether for VAMS sample preparation to optimise the analyte extraction process. The described validation protocols were cross-validated, confirming the equivalence of the whole-blood and VAMS-based methods. Furthermore, the developed methods were evaluated in two three-level rounds of an external proficiency-testing scheme. The analytical methods were successfully validated within the calibration range of SIR (0.5-60 ng/ml). The validation parameters met the European Medicines Agency (EMA) and the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDM&CT) acceptance criteria. No hematocrit (tested in the range of 24.3-64.1%), matrix, or carry-over effects were observed. Cross-validation confirmed the interchangeability between VAMS-LC-MS/MS and WB-LC-MS/MS methods. The developed methods were successfully implemented for SIR determination in 140 clinical samples (70 each of WB and VAMS) from pediatric renal transplant recipients, demonstrating their practicality and reliability. The VAMS-based method has been rigorously tested and is clinically equivalent to the reference WB-LC-MS/MS method. Additionally, clinical validation confirmed the utility of the presented methods for TDM of the SIR in the pediatric population after renal transplantation.

A-317 Development of a patient-focused volumetric absorptive microsampling strategy to assess urinary amino acid excretion

Abstract Background Biochemical determinations of urinary amino acid excretion patterns can diagnose specific inborn errors of metabolism (IEM). Clinical management of these genetic metabolic patients may require frequent urine amino acid tests. A simplified process for transporting home collected urine specimens would benefit the IEM community. Volumetric absorptive microsampling (VAMS) is an emergent alternative sampling technique for the collection of dried biological specimens. We describe the development and preliminary validation results of a VAMS strategy for the analysis of creatinine and amino acids (N=22) from microsampled dried urine. Methods Urine creatinine and amino acids were respectively measured in a clinical laboratory setting using established assays on a CobasPro (Roche) and a MassTrak ultra-performance liquid chromatography (UPLC) amino acid analysis (AAA) solution (Waters). Two 30 µL VAMS tips from a Mitra device (Neoteryx) were used to sample each random urine specimen. An ultrasonication-based solvent extraction procedure was optimized to extract the amino acids and creatinine from the VAMS tips. Solvent extracts were subsequently dried by centrifugal evaporation. Extract from one tip was re-dissolved in water and tested for creatinine using an enzymatic method. The other extract was re-dissolved in 0.02 N sodium hydroxide and tested according to the MassTrak AAA procedure, which deploys pre-column AccQTag derivatization with reversed-phase UPLC on a C18 column (2.1 × 150 mm; 1.7 µm) and UV detection at 260 nm. Accuracy, within-batch precision and short-term stability of the optimized VAMS protocol were determined. Results Extraction solvent with a 60:40 (v/v) water:methanol mixture provided the highest average amino acid recovery. This extraction solvent produced higher average amino acid recoveries at pH 11 (89±13%), than pH 3 (82±10%) or pH 7 (72±11%). Creatinine measured from the VAMS tips (N=44) had a consistent negative bias of 20% relative to neat urine (R=0.9905, slope=0.802, intercept=0.02). The creatinine corrected urine amino acid results from the Mitra device were corrected for this observed concentration bias. The accuracy of the amino acid results derived from the VAMS tip vs neat urine was 104 ± 14%. The average within-batch precision (CV%) of the amino acids was 9.8% (range=6.7% (cystine) to 17.6% (arginine)) and 6.2% for creatinine. The average 1 week storage stability at ambient temperature was 91% (range=73% (ornithine) to 107% (glycine)) for the amino acids and 107% for creatinine. The devised VAMS strategy was applied to the analysis of microsampled dried urine from a patient with cystinuria (OMIM no. 220100). Marked elevations in arginine, cystine, lysine and ornithine were respectively observed and are consistent with the biochemical diagnosis of the patient. Conclusions Creatinine corrected amino acid results from the devised VAMS strategy exhibit good precision and accuracy when compared to tradition analyses of neat urine. The potential use of microsampled dried urine for the diagnosis and monitoring of selected IEM has been demonstrated. The analytical and clinical performance of this VAMS protocol must be further validated, including the use of microsampled dried urine from home specimen collections.

B-156 Extraction strategy for therapeutic drug monitoring of sirolimus and tacrolimus using volumetric absorptive microsampling

Abstract Background Following organ transplantation, patients are placed on immunosuppressant drugs (ISDs) to minimize the risk of rejection. Sirolimus and tacrolimus are two of the most widely used ISDs, however, due to their narrow therapeutic indices, patients require frequent therapeutic drug monitoring (TDM). Recurrent venipuncture can become uncomfortable and disruptive, especially in the pediatric population. Therefore, we describe an extraction method using dried whole blood from volumetric absorptive microsampling (VAMS) devices for the detection of sirolimus and tacrolimus by turbulent flow liquid chromatography tandem mass spectrometry (TFC-MS/MS). The use of VAMS in the clinical laboratory will provide ISD patients with a convenient alternative to traditional blood draws. Methods MassTrak immunosuppressant calibrators based on a whole blood matrix (Waters) were applied to 30 μL VAMS Mitra devices (Neoteryx) and dried overnight. To extract the whole blood off of VAMS tip, we tested various methanol:water solvent ratios, of which 30 μL was added to the device. Tips were extracted using ultrasonication (30 min), vortexing (5 min), and centrifugation. Once extracted from the tip, 25 mM zinc sulfate was added to whole blood extract at a 1:1 (v/v) ratio. An acetonitrile and methanol extraction reagent was then added to the whole blood/zinc sulfate solution at a 2:1 (v/v) ratio. After brief vortexing, samples were centrifuged at 16,060 × g (10 min) and tested using an established TFC-MS/MS method which deploys a TLX-2 system for sample preparation coupled to a Q-Trap tandem mass spectrometer (AB Sciex 5500). Results A 80:20 (v/v) water:methanol extraction solvent yielded the greatest extraction efficacy for both sirolimus and tacrolimus from the VAMS tips compared to other tested ratios. We established a 5-point calibration curve to correlate the measured values of ISDs with known MassTrak calibration standard concentrations. The resulting average calibration curve from 3 independent experiments was highly linear for both sirolimus (R2= 0.992; slope= 1.1; intercept= 0.94) and tacrolimus (R2= 0.996; slope= 1.1; intercept = 0.8819). Our VAMS whole blood extraction strategy was correlated to the established neat whole blood protocol, which utilized all of the sample preparation steps outlined above except the initial extraction from the VAMS tip. The average extraction efficacy was 89.1% for sirolimus with an average coefficient of variation (CV%) of 12.1% across 3 independent experiments. For tacrolimus, we demonstrated an average extraction efficacy of 100.1% with an average CV% of 8.4%, again across 3 independent experiments. Conclusions We demonstrate the feasibility of using a VAMS approach to TDM of sirolimus and tacrolimus. Our results demonstrate favorable recoveries for both of the tested drugs, evidenced by high recoveries of the external calibration standards. Additionally, our method exhibited high extraction efficacy and low variability. Further validation studies, including the utilization of clinical samples is necessary to confirm our preliminary findings.

A VAMS‐based LC–MS/MS method for precise cenobamate quantification in epilepsy (patients)

AbstractObjectiveCenobamate (CNB), a recently approved antiseizure medication by the European Medicines Agency (EMA), serves as an adjunctive therapy for focal‐onset seizures in adult patients unresponsive to at least two other treatments. Administered in polytherapy, CNB can potentially interact with co‐administered drugs in epilepsy patients, necessitating dose adjustments and the need for effective therapeutic drug monitoring (TDM).MethodsIn this study, we introduce a novel LC–MS/MS method for precise CNB quantification using Volumetric Absorptive Microsampling (VAMS), following validation according to ICH guidelines M10. VAMS samples are efficiently extracted with 200 μL of methanol, with chromatographic separation achieved using an Acquity UPLC HSS PFP column. The method's efficacy was confirmed through its application to real samples from adult CNB‐treated patients.ResultsOur results demonstrate that the method exhibits linearity within the range of 0.05–30 mg/L, with intra‐ and inter‐run precision ranging from 1% to 8% and accuracy from 1% to 10% based on 30 μL of sample. Furthermore, CNB stability in VAMS is confirmed for up to 15 days at 25°C and −20°C. Importantly, no significant difference was observed between CNB concentrations in VAMS samples and those in plasma obtained from venous blood.SignificanceThis VAMS‐based LC–MS/MS method presents a robust alternative for TDM in CNB‐treated patients. Future investigations should explore CNB concentrations in capillary blood and assess their correlation with plasma levels to further enhance its clinical utility.Plain Language SummaryCenobamate is an antiepileptic drug and used for treatment of focal‐onset seizures in adult patients (≥18 age). TDM can prevent drug interactions and minimize drug toxicity. The aim of this work is to evaluate volumetric absorptive microsampling (VAMS) from capillary blood as an alternative strategy for TDM in patients treated with the newly antiepileptic drug. Our method is suitable for TDM, and this study suggests that VAMS allows monitoring of cenobamate concentration and can offer valuable support for personalized therapy in refractory epilepsy.

Factors affecting peripheral neuropathy induced by nanoparticle albumin-bound paclitaxel in patients with pancreatic cancer.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major toxicity limiting the use of nab-paclitaxel (Nab-P) in treating patients with pancreatic cancer. The aim of this study was to identify the factors affecting CIPN using patient-reported outcome measures and the minimally invasive volumetric absorptive microsampling (VAMS) technique. The maximum concentrations of paclitaxel (Cmax) were measured from 81 VAMS samples collected from 44 participants with pancreatic cancer. The association between CIPN development and demographic, clinical and pharmacokinetic factors was determined using univariable and multivariable logistic regression. The association between CIPN severity and the factors was evaluated using Spearman's rank correlation. The impact of Cmax and the number of treatment cycles on the severity was assessed using multivariable linear regression. The development of CIPN was significantly associated with cumulative dose (odds ratio 1.005, 95% confidence interval [CI] 1.003-1.007), treatment cycles (3.47, 2.25-5.85), alkaline phosphate (0.992, 0.985-0.998) and age (1.092, 1.020-1.179), with an area under the receiver operating characteristic curve of 0.89 (95% CI 0.83-0.95). The severity of CIPN significantly worsened with increasing cumulative dose (coefficient 0.58, 95% CI 0.44-0.69), treatment cycles (0.57, 0.44-0.68) and age (0.18, 0.00-0.35). The severity of CIPN was predictable from treatment cycles (P = .0002) and Cmax (P = .01). The higher the cumulative dose of Nab-P, treatment cycles and age, the more frequently and severely do the patients experience CIPN. In predicting the severity of CIPN using Cmax, minimally invasive VAMS is a feasible alternative to venous blood sampling.

Assessment of Dried Serum Spots (DSS) and Volumetric-Absorptive Microsampling (VAMS) Techniques in Therapeutic Drug Monitoring of (Val)Ganciclovir-Comparative Study in Analytical and Clinical Practice.

Ganciclovir (GCV) and its prodrug valganciclovir (VGCV) are antiviral medications primarily used to treat infections caused by cytomegalovirus (CMV), particularly in immunocompromised individuals such as solid organ transplant (SOT) recipients. Therapy with GCV is associated with significant side effects, including bone marrow suppression. Therefore, therapeutic drug monitoring (TDM) is mandatory for an appropriate balance between subtherapeutic and toxic drug levels. This study aimed to develop and validate three novel methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for GCV determination in serum (reference methodology), dried serum spots (DSS), and VAMS-Mitra™ devices. The methods were optimized and validated in the 0.1-25 mg/L calibration range. The obtained results fulfilled the EMA acceptance criteria for bioanalytical method validation. Assessment of DSS and VAMS techniques extended GCV stability to serum for up to a minimum of 49 days (at room temperature, with desiccant). Developed methods were effectively evaluated using 80 clinical serum samples from pediatric renal transplant recipients. Obtained samples were used for DSS, and dried serum VAMS samples were manually generated in the laboratory. The results of GCV determination using serum-, DSS- and VAMS-LC-MS/MS methods were compared using regression analysis and bias evaluation. The conducted statistical analysis confirmed the interchangeability between developed assays. The DSS and VAMS samples are more accessible and stable during storage, transport and shipment than classic serum samples.

Near-infrared-based hematocrit determination of dried blood samples collected by volumetric absorptive microsampling: An in-depth evaluation

BackgroundVolumetric absorptive microsampling (VAMS) has gained great interest in the past decade because of its capability to collect a fixed volume of blood independent of the hematocrit (Hct). This alternative microsampling technique tackles the well-known Hct-related area bias, a major issue in conventional partial-punch dried blood spot (DBS) analysis. However, as microsampling typically involves the collection of blood, whereas the ‘gold standard’ matrix used in clinical practice is plasma or serum, a major concern remains: how do we interpret or convert these dried blood microsample-based results? ResultsA method to determine the Hct of a VAMS sample based on non-contact NIR spectroscopy was developed and extensively validated. Using authentic patient samples (n = 102) and a dynamic measurement procedure (turning the VAMS samples in between measurements), accurate (maximum bias of –0.022 L/L) and precise (maximum total imprecision of 10.6 %) Hct determinations were obtained. Sample storage (up till one month at room temperature or lower), operator and lot of VAMS devices did not impact the resulting Hct, confirming the method’s robustness. Significance and noveltyThis is the first non-contact method allowing Hct determination of VAMS samples. Importantly, there is no ‘consumption’ of part of the microsample, which would impact subsequent analysis of target compounds. As one of the issues hampering wide implementation of microsampling is related to interpretation of (dried) blood-based results and conversion into plasma results, a simple and easy way to determine the Hct of a microsample, as presented here, may help to overcome this hurdle.

Clinical validation of two volumetric absorptive microsampling devices to support home-based therapeutic drug monitoring of immunosuppression.

Dried blood volumetric absorptive microsamples (VAMS) may facilitate home-based sampling to enhance therapeutic drug monitoring after transplantation. This study aimed to clinically validate a liquid chromatography-tandem mass spectrometry assay using 2 VAMS devices with different sampling locations (Tasso-M20 for the upper arm and Mitra for the finger). Patient preferences were also evaluated. Clinical validation was performed for tacrolimus and mycophenolic acid by comparison of paired VAMS and venipuncture samples using Passing-Bablok regression and Bland-Altman analysis. Conversion of mycophenolic acid VAMS to serum concentrations was evaluated using haematocrit-dependent formulas and fixed correction factors defined a priori. Patients' perspectives, including useability, acceptability and feasibility, were also investigated using established questionnaires. Paired samples (n =50) were collected from 25 kidney transplant recipients. Differences for tacrolimus whole-blood concentration were within ±20% for 86 and 88% of samples from the upper arm and fingerstick, respectively. Using correction factors of 1.3 for the upper-arm and 1.47 for finger-prick samples, 84 and 76% of the paired samples, respectively, were within ±20% for mycophenolic acid serum concentration. Patient experience surveys demonstrated limited pain and acceptable useability of the upper-arm device. Tacrolimus and mycophenolic acid can be measured using 2 common VAMS devices with similar analytical performance. Patients are supportive of home-based monitoring with a preference for the Tasso-M20 device.

Development of LC-MS/MS method for quantification of Lurasidone using volumetric absorptive microsampling (VAMS); a comparative study between dried blood and plasma samples

The ecological impact of biological, chemical, and analytical research practices, including toxic reagents and biohazardous waste, has led to the development of alternative sampling and extraction techniques for bioanalysis. Microsampling (sample volume < 50 µL) aligns with the 3Rs principle, allowing multiple sampling points from the same animal at different time points and improving animal welfare. A bioanalytical method was developed to investigate factors related to bioanalytical challenges and the implementation of microsampling techniques. An LC-MS/MS method for Volumetric Absorptive Microsampling (VAMS), 20 µL, was developed for quantifying Lurasidone using a liquid–liquid extraction technique. The method uses a C18, Phenomenex column for chromatographic separation and a mobile phase composition of Methanol, Acetonitrile, and Water with 0.1 % HFBA. The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and achieved acceptable precision and accuracy. The recovery for analyte from VAMS was approximately 40% at four different concentrations and is consistent (%CV < 15), with no significant differences among HCT levels. The matrix factor ranged between 85.00 and 115.00 %, showing no substantial issues with reduced or enhanced signal. The stability data showed no significant degradation of LUR in VAMS samples when stored at room temperature for 15 days. The newly established method for Lurasidone confirmed the use of VAMS sampling method and its analysis on LC-MS/MS. Further, the data obtained from microsampling techniques was compared with conventional (plasma) technique, as proof-of-concept, and it confirms the agreement between the two methods. The study supports the advantages of microsampling in protecting the environment and animals while maintaining scientific judgement.

A novel approach to therapeutic drug monitoring of Ciclosporin in pediatric renal transplant recipients using volumetric absorptive microsampling (VAMS) – Teaching old dog new tricks

Background and aimsCiclosporin (CSA) is an immunosuppressive agent that requires therapeutic drug monitoring (TDM). High partitioning in erythrocytes indicates that whole blood (WB) is a suitable matrix for CSA determination. Alternative sampling strategies, such as volumetric absorptive microsampling (VAMS), are novel possibilities for blood collection during TDM for various analytes, including immunosuppressants. This technique is attractive for vulnerable pediatric patients, including home-based self-sampling, remote therapy, and adherence control. Materials and methodsThis study aimed to develop and validate a new method for CSA determination based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of WB and VAMS samples. Additionally, these methods were applied for CSA determination in clinical samples from pediatric transplant recipients. A strong point of this study is the assessment of an external proficiency testing scheme. ResultsBoth methods were successfully validated within the 1–2000 ng/mL calibration range, with LOD 0.5 and 1 ng/mL for WB and VAMS methods, respectively. All the validation parameters fulfilled the international acceptance criteria for bioanalytical methods. Cross-validation confirmed the interchangeability of the LC-MS/MS method developed in this study. ConclusionThis study developed and validated novel methods for CSA determination in whole blood and VAMS using LC-MS/MS. Clinical validation and proficiency testing confirmed their utility in routine clinical practice.

Self-Sampling by Adolescents at Home: Assessment of the Feasibility to Successfully Collect Blood Microsamples by Inexperienced Individuals.

Blood microsampling has increasingly attracted interest in the past decades as a more patient-centric sampling approach, offering the possibility to collect a minimal volume of blood following a finger or arm prick at home. In addition to conventional dried blood spots (DBS), many different devices allowing self-sampling of blood have become available. Obviously, the success of home-sampling can only be assured when (inexperienced) users collect samples of good quality. Therefore, the feasibility of six different microsampling devices to collect capillary blood by inexperienced adolescents at home was evaluated. Participants (n = 95) were randomly assigned to collect blood (dried or liquid) at different time points using four of six different self-sampling devices (i.e., DBS, Mitra volumetric absorptive microsampling (VAMS), Capitainer B, Tasso M20, Minicollect tube and Tasso+ serum separator tube (SST)). The quality of the samples was visually inspected and analytically determined. Moreover, the participants' satisfaction was assessed via questionnaires. Although a majority succeeded based on the visual inspection, the success rate differed largely between the different devices. In general, the lowest success rate was obtained for the Minicollect tubes, although there is an opportunity and need for improvement for the other self-sampling devices as well. Hence, this also emphasizes the importance to assess the quality of samples collected by the target population prior to study initiation. In addition, visual classification by a trained individual was confirmed based on assessment of the analytical variability between replicates. Finally, self-sampling at home was overall (very) positively received by the participants.

Determination of warfarin in volumetric absorptive microsampling by liquid chromatography-tandem mass spectrometry

ObjectiveThis study aims to develop and validate bioanalytical method for quantifying warfarin in VAMS samples using liquid chromatography tandem mass spectrometry (LC-MS/MS), directly implementing the method to patients receiving warfarin therapy. MethodsThe UPLC-MS/MS method was developed and optimized, with quercetin as the internal standard. Sample preparation was carried out using protein precipitation with methanol-acetonitrile (1:3 v/v). ResultsChromatographic separation was achieved using Acquity® UPLC BEH C18 column with 0.1 % formic acid-acetonitrile-methanol (30:69:1 v/v) as mobile phase, in isocratic elution. Multiple Reaction Monitoring (MRM) detection was done using m/z values of 307.10 → 161.06 for warfarin and 301.03 → 150.98 for quercetin as internal standard, using Electrospray Ionization (ESI) negative ion source. The clinical application of the bioanalytical method was carried out on 25 patients receiving warfarin therapy at Universitas Indonesia Hospital and warfarin levels were well within the calibration range from 6.05 to 431.39 ng/mL. ConclusionA novel method has been developed to analyze warfarin in VAMS samples. This method has been fully validated according to guideline from FDA 2022 and is linear in the range of 5–500 ng/mL and the value of r ≥ 0.9977, and successfully applied for the analysis of warfarin in VAMS samples of clinical patients.

Surface-Enhanced Raman Spectroscopy Combined with Multivariate Analysis for Fingerprinting Clinically Similar Fibromyalgia and Long COVID Syndromes.

Fibromyalgia (FM) is a chronic central sensitivity syndrome characterized by augmented pain processing at diffuse body sites and presents as a multimorbid clinical condition. Long COVID (LC) is a heterogenous clinical syndrome that affects 10-20% of individuals following COVID-19 infection. FM and LC share similarities with regard to the pain and other clinical symptoms experienced, thereby posing a challenge for accurate diagnosis. This research explores the feasibility of using surface-enhanced Raman spectroscopy (SERS) combined with soft independent modelling of class analogies (SIMCAs) to develop classification models differentiating LC and FM. Venous blood samples were collected using two supports, dried bloodspot cards (DBS, n = 48 FM and n = 46 LC) and volumetric absorptive micro-sampling tips (VAMS, n = 39 FM and n = 39 LC). A semi-permeable membrane (10 kDa) was used to extract low molecular fraction (LMF) from the blood samples, and Raman spectra were acquired using SERS with gold nanoparticles (AuNPs). Soft independent modelling of class analogy (SIMCA) models developed with spectral data of blood samples collected in VAMS tips showed superior performance with a validation performance of 100% accuracy, sensitivity, and specificity, achieving an excellent classification accuracy of 0.86 area under the curve (AUC). Amide groups, aromatic and acidic amino acids were responsible for the discrimination patterns among FM and LC syndromes, emphasizing the findings from our previous studies. Overall, our results demonstrate the ability of AuNP SERS to identify unique metabolites that can be potentially used as spectral biomarkers to differentiate FM and LC.

Revolutionizing biomarker analysis one tip at a time—Coupling antibody to Volumetric Absorptive Microsamplers for sensitive LC-MS analysis

The constant pursuit of efficient and cost-effective methods for analyzing human samples drives ongoing innovation in biomedical research. Introduced in 2014, Volumetric Absorptive Microsampling (VAMS) technology ensures fixed blood volume absorption and enhanced sample uniformity. Our study aims to enhance VAMS functionality by integrating sample preparation onto the device primarily used for sample collection. This involves the adsorption of antibodies onto VAMS tips, enabling instant sample clean-up at the time of sample collection. Using human chorionic gonadotropin (hCG) as a model analyte, we evaluated the qualitative and quantitative performance of Affinity-VAMS. First, we showed that adsorbing and covalently binding monoclonal antibodies to VAMS tips results in capturing of the target analyte in comparison to unmodified VAMS. Due to the ease of preparation, we moved forward using antibody adsorption to VAMS tip and optimized the procedure. Optimization of the procedure involved fine-tuning the antibody coupling step, washing process, and determining the optimal amount of antibody required, leading to a streamlined process with significant time savings up to two days. Recovery experiments demonstrate successful capture of the target analyte by the Affinity-VAMS, while matrix effects and stability assessments indicate no negative effects from serum matrix or storage conditions. Finally, quantitative analysis shows promising performance of the Affinity-VAMS in detecting different concentrations of the target analyte in the concentration range between 7.5 - 25 ng·mL−1. The calculated correlation factor was R2 of 0.9988 and limit of detection 2.5 ng·mL−1. The precision lays within the ICH guidelines for bioanalytical method validation. While this report is focused on the proof of concept of the Affinity-VAMS, our findings demonstrate the potential for the usage of modified VAMS in remote sampling and integrated sample processing, enhancing biomarker analysis in clinical and research settings.

Pharmacokinetic profile and incurred sample stability of hydroxychloroquine in Volumetric Absorptive Microsampling (VAMS) using high-performance liquid chromatography-photodiode array

Background: Hydroxychloroquine (HCQ) is a quinoline compound derived from chloroquine used to treat SLE. An in vitro stability evaluation was conducted to develop and validate the hydroxychloroquine analysis method. However, an assessment of the incurred sample stability is needed to ensure drug effectiveness, as in vitro evaluation doesn't reflect drug metabolism processes in the body. Objective: This research aims to obtain a pharmacokinetic profile of hydroxychloroquine using Volumetric Absorptive Microsampling (VAMS) as a safe sampling technique to use during the COVID-19 pandemic and evaluate the incurred sample stability of hydroxychloroquine samples. Methods: The chromatographic conditions used were column C18 (Waters, XBridge; 250 × 4.6 mm; 5μm); mobile phase acetonitrile-1% diethylamine (65:35); flow rate of 0.8 mL/min; column temperature 45°C; PDA detector analysis wavelength of 332 nm; and chloroquine as internal standard. Results: The pharmacokinetic profile of hydroxychloroquine in VAMS samples gave results that the Cmax ranged from 322.61-505.32 ng/mL with an average of 425.33 ± 65.90 ng/mL; tmax was 4 hours; mean t1/2 was 23.32 ± 9.65 hours; mean AUC0-72 was 5103.63 ± 1419.66 ng.h/mL; mean AUC0-∞ was 5763.97 ± 2155.26 ng.h/mL, and the AUC ratio was above 80%. Conclusion: The incurred sample stability of hydroxychloroquine in VAMS met the 2011 EMEA Bioanalytical Guideline requirements up to day 30.

Development of an LC-HRMS/MS Method for Quantifying Steroids and Thyroid Hormones in Capillary Blood: A Potential Tool for Assessing Relative Energy Deficiency in Sport (RED-S).

Relative energy deficiency in sport (RED-S) is a condition that arises from persistent low energy availability (LEA), which affects the hypothalamic-pituitary axis and results in alterations of several hormones in both male and female athletes. As frequent blood hormone status determinations using venipuncture are rare in sports practice, microsampling offers promising possibilities for preventing and assessing RED-S. Therefore, this study aimed at developing a liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) method for quantifying relevant steroids and thyroid hormones in 30 μL of capillary blood obtained using Mitra® devices with volumetric absorptive microsampling technology (VAMS®). The results of the study showed that all validation criteria were met, including a storage stability of more than 28 days in a frozen state (-18 °C) and 14 days at room temperature (20 °C). The validated assay provided precise (<12%) and accurate (<13%) results for all the target analytes. Furthermore, as a proof of concept, autonomously collected VAMS® samples from 50 female and male, healthy, active adults were analyzed. The sensitivity of all analytes was adequate to quantify the decreased hormone concentrations in the RED-S state, as all authentic samples could be measured accordingly. These findings suggest that self-collected VAMS® samples offer a practical opportunity for regular hormone measurements in athletes and can be used for early RED-S assessment and progress monitoring during RED-S recovery.

Targeted and untargeted metabolomics and lipidomics in dried blood microsampling: Recent applications and perspectives.

Blood microsampling (BµS) offers an alternative to conventional methods that use plasma or serum for profiling human health, being minimally invasive and cost effective, especially beneficial for vulnerable populations. We present a non-systematic review that offers a synopsis of the analytical methods, applications and perspectives related to dry blood microsampling in targeted and untargeted metabolomics and lipidomics research in the years 2022 and 2023. BµS shows potential in neonatal and paediatric studies, therapeutic drug monitoring, metabolite screening, biomarker research, sports supervision, clinical disorders studies and forensic toxicology. Notably, dried blood spots and volumetric absorptive microsampling options have been more extensively studied than other volumetric technologies. Therefore, we suggest that a further investigation and application of the volumetric technologies will contribute to the use of BµS as an alternative to conventional methods. Conversely, we support the idea that harmonisation of the analytical methods when using BµS would have a positive impact on its implementation.

THE APPLICATION OF BIOANALYTICAL METHOD OF TAMOXIFEN AND ITS ACTIVE METABOLITES FOR THERAPEUTIC DRUG MONITORING IN BREAST CANCER PATIENTS: A REVIEW

Breast cancer is the most common cancer around the world and in Indonesia. The most widely used agent for breast cancer treatment is tamoxifen, with a fixed dose of 20 mg per day. Tamoxifen is metabolized by cytochrome P450 3A4 (CYP3A4) and 2D6 (CYP2D6) to endoxifen and 4-hydroxytamoxifen, which have 30-to 100-fold more potent antiestrogenic activity than tamoxifen. High variations of CYP3A4 and CYP2D6 genes can lead to interpatient variability in its metabolites concentration. The dose can be increased to 40 or 60 mg per day based on individual needs. Therapeutic drug monitoring (TDM) is required to measure the concentration of tamoxifen and its metabolites to decide the individualized dose. The measurement of drug levels should use a sensitive, selective, accurate, precise, and reliable bioanalytical method. Various bioanalytical methods have been developed in several matrices: urine, scalp hair, serum, plasma, dried blood spot (DBS), and volumetric absorptive microsampling (VAMS) samples, with different sample preparations, and frequently using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The bioanalytical method of tamoxifen and its metabolites in the DBS sample was more suitable in the TDM application due to the low invasive sampling technique, more stable sample, and rapid sample preparation. Therefore, it is more time-and cost-efficient than the other methods.

Microsampling Techniques Suitable for Therapeutic Drug Monitoring of Antipsychotics.

Therapeutic drug monitoring (TDM) of antipsychotics for dose titration or detection of noncompliance is not uncommon in daily practice. Normally, TDM implies measuring a drug concentration in venous blood samples. This technique is invasive and requires trained assistants and patients normally need to go to an outpatient clinic. Over the past decades, sensitivity of analytical equipment has improved leading to a growing interest in microsampling techniques. These techniques are minimally invasive, require a small volume (<100 μL), usually result in stable samples, and can be collected by the patient or a caregiver at home. Before a microsampling technique can be used in daily routine, proper method development and a clinical validation study should be performed. For this review, the databases of PubMed and Embase were systematically searched. Currently available microsampling techniques for antipsychotics in blood, serum, or plasma are summarized. Subsequently, it has also been assessed whether these techniques are sufficiently validated for TDM monitoring in daily practice. Several microsampling techniques are available today, for example, dried blood spot sampling, dried plasma extraction cards, and volumetric absorptive microsampling. Eighteen studies were identified in which a microsampling technique for 1 or a few antipsychotics was chemically analytically and clinically validated. However, the majority of these studies have relevant shortcomings that mean its usefulness for different antipsychotics is not yet well established. Microsampling for TDM can be recommended for patients using clozapine. For TDM of other antipsychotics, it is a very promising development.