<h4 style="text-align:justify;">Background</h4>TDM of immunosuppressants is extremely important topic, especially when it comes to sample collection at home coupled with simple and fast sample preparation and analysis. The aims of the study covered check if optimization of ready-to-use kit for determination of Tacrolimus (TAC), Sirolimus (SIR), Everolimus (EVR) and Cyclosporin A (CSA) in venous blood can be successfully applied for VAMS-collected blood and preliminary venous blood to Mitra® (VB/M) correlation study.<blockquote>Check the link for pdf of the original poster in the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Poster_Immuno_3000px_RGB_352b008e9d.pdf">LabRulezLCMS library</a>!</blockquote><h4 style="text-align:justify;">Methods</h4>Sample preparation for venous blood was done according to the procedure provided with the kit (Chromsystems kit: 93000). Sample preparation for Mitra®-collected blood included additional sonication, vortexing and combining of internal standards with extraction buffer. Only reagents supplied by the manufacturer were used. All samples were analyzed on <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=QTRAP+5500%2B">QTRAP 5500+</a> LC-MS/MS system (SCIEX). Data processing, quantitation and basic statistical analysis was done in SciexOS 3.3 software (SCIEX).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Venous_Blood_Chromsystems_b496c1d87a.jpg" alt="Bioanalytic: Venous blood (Chromsystems procedure)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Venous_Blood_Chromsystems_b496c1d87a.jpg 234w," sizes="100vw" width="1323" height="881"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Mitra_optimized_procedure_e671781241.jpg" alt="Bioanalytic: Mitra - optimized procedure based on Chromsystems kit." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Mitra_optimized_procedure_e671781241.jpg 211w," sizes="100vw" width="1340" height="994"><figure class="image"><img style="aspect-ratio:2064/674;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Data_processing_quantitation_8529b80c50.jpg" alt="Bioanalytic: Data processing, quantitation and basic statistical analysis was done in SciexOS 3.3 software (SCIEX)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Data_processing_quantitation_8529b80c50.jpg 245w," sizes="100vw" width="2064" height="674"></figure><h4 style="text-align:justify;">Results</h4>The optimized for 10µl and 20µl Mitra® samplers procedure met the validation criteria, including: reproducibility (%CV±15% and accuracy 85-115%), LLOQ (S/N≥10, calibrator 1), linearity (R≥0.995). LOD was not determined, however collected for 10 µl Mitra® sampler data shows that lower that 50 pg/ml concentration for each compound is achieved. The preliminary study of VB/M correlation, included 15 patients per compound where venous blood and 10 µl Mitra® samples were collected at the same time. Obtained VB/M factors were as follows: TAC=0.75, %CV=22.92%, SIR=0.77, %CV=36.10%, CSA=1.15, %CV=19.01%. No patients for EVR were tested during the study.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Tab_1_Validation_results_for_8c39ba1b30.jpg" alt="Bioanalytic: Tab. 1. Validation results for collected on 10 microL Mitra sampler lowest analytes concentrations provided with the kit (CAL 1, 4 replicates). LOD was not determined, however assitional analyses showed that lower that 50-100 pg/ml concentration for each compound can be achieved." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Tab_1_Validation_results_for_8c39ba1b30.jpg 245w," sizes="100vw" width="2679" height="925"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Tab_Results_of_preliminary_1d5b77648e.jpg" alt="Bioanalytic: Tab. 2. Results of preliminary study for VB/M correlation included 15 paptients par compound where venous blood and 10 microL Mitra samples were collected at the same time. No patients for EVR were tested during this study." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_From_venous_blood_to_VAMS_adaptation_of_a_commercial_CE_IVD_assay_kit_for_the_determination_of_Tacrolimus_Sirolimus_Everolimus_and_Cyclosporin_A_Tab_Results_of_preliminary_1d5b77648e.jpg 245w," sizes="100vw" width="2168" height="1117"><h4 style="text-align:justify;">Conclusions</h4>Optimized for VAMS procedure allowed to achieve validation criteria with the use of kit reagents. Additional steps required for Mitra® sample preparation are simple, robust and automation friendly. The VB/M correlations revealed that the capillary blood concentration of TAC and SIR are lower than in venous blood contrary to concentration of CSA. This data may support the fact that TAC and SIR enters erythrocytes in about 90%, while CSA only in 50% and the capillary blood has higher amount of serum fraction. However, due to relatively small number of patients included in the study it needs further investigation on larger group of patients and deeper statistical analysis.<blockquote><h4 style="text-align:justify;">Authors</h4>Rafał Szewczyk1,4; Arkadiusz Kocur2,3, Agnieszka Czajkowska3; Karina Izdebska4; Maciej Stopa1,4. 1LabExperts Sp. z o.o., Gdańsk, Poland; 2Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Faculty of Pharmacy, Medical University of Warsaw, Warsaw, Poland; 3Therapeutic Drug Monitoring, Clinical Pharmacokinetics and Toxicology Laboratory Unit, Department of Clinical Biochemistry, The Children’s Memorial Health Institute, Warsaw, Poland; 4Bioanalytic Sp. z o.o., Gdańsk, Poland</blockquote>

From venous blood to VAMS – adaptation of a commercial CE-IVD assay kit for the determination of Tacrolimus, Sirolimus, Everolimus and Cyclosporin A

<blockquote>Maciej Stopa1, 2; Rafał Szewczyk1, 2; Adrianna Radulska3, Anna Lenartowicz2; Julia Mironenka2; Adrian Soboń1, 2; Katarzyna Krupczyńska-Stopa1, 2; Tomasz Borkowski3; Ewelina Marciniak3, Leszek Kalinowski31<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/companies/315">Bioanalytic</a> Sp. z o.o., Gdansk, Poland; 2LabExperts Sp. z o.o., Gdansk, Poland; 3 Medical University of Gdansk, Gdansk, Poland</blockquote><h4>Abstract</h4>Cardiac arrhythmia affects approximately 12.6% of people over the age of 65. Ventricular arrhythmias are considered as responsible for 75% to 80% of sudden cardiac deaths. Therapy of the disease include ablation, use of cardioverter or defibrillator and pharmacological treatment where the key for successful therapy lays in maintaining the necessary concentration of drugs. The goal can be achieved by therapeutic drug monitoring (TDM). Developed assay named “CardioCarePack” is based on the use of capillary blood collected and dried at home by a patient with volumetric absorptive microsampling (VAMS), quantitative analysis of selected drugs and their metabolites and a telemedical system that integrates all data between a doctor, patient and laboratory supporting the therapy process. CardioCarePack is ready for commercialization.<h4>Methods</h4>Blood collected with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?distributorItems=315&amp;search=MITRA">MITRA® microsampling device</a> (20 µl) is prepared with full validated protocol that include sonication with 0.1% formic acid in water followed by acetonitrile extraction, evaporation under nitrogen stream and resuspension in LC mobile phase. Sample is subjected to LC-MS/MS analysis in positive ionization MRM mode with QTRAP 5500+ mass spectrometer (SCIEX) or similar one. Analysis covers simultaneous quantitation of 15 drugs: Sotalol, Metoprolol, Bisoprolol, Propafenone, Carvedilol, Nebivolol, Amiodarone, Digoxin, Perindopril, Ramipril, Eplerenone, Spironolactone, Zofenopril and 2 metabolites: 5-hydroxypropafenone and Desethylamiodarone. Results are placed in a server-based telemedical system where depending on accession rights history, doses, therapeutic index flagging, cardiograms, sample tracking and many other data are available for patient, doctor and laboratory staff, respectively.<h4>Preliminary Data</h4>The project involved more than 300 patients who had been monitored for 2 years during regular pharmacological therapy. Every half a year during a visit in medical facility venous blood for serum testing and capillary blood on the MITRA® sorbent were collected. Between the visits patients were collecting samples by VAMS themselves at home. All samples were analyzed by fully validated procedure where among the others linearity (r ≥ 0.995), accuracy (80 – 120%), reproducibility (%CV ≤ 15%) for both MITRA® and serum samples were calculated. The LC-MS/MS method developed for 17 compounds covers therapeutic range of the tested drugs: 0.25 – 25 ng/ml (Digoxin and Nebivolol) 2.5 – 250 ng/ml (Metoprolol, Bisoprolol, Propafenone, Carvedilol, Perindopril, Ramipril, Spironolactone, Zofenopril and 25 – 2500 ng/ml (Sotalol, Desethylamiodarone, Eplerenone, Amiodarone, 5-hydroxypropafenone). Results generated during LC-MS/MS analysis were analyzed with SCIEX OS 1.7 software.On the basis of samples collected during 4 visits in medical facility a correlation between drugs concentration in venous blood (serum) and capillary blood (MITRA®) (S/M) was calculated. For some compounds S/M ratio was close to 1 (ex. Sotalol – 0.95, %CV – 5.12%, Nebivolol – 1.03, %CV – 3.17%) or moderately to more than 2-fold different (ex. Digoxin – 0.69, %CV – 1.46%, Ramipril – 1.18, %CV – 11.1, Perindopril – 1.86, %CV – 4.73%, Amiodarone – 2.17, %CV – 10.78). The correlation factors are statistically significant (p &lt; 0.05) and can be used for accurate concentration estimation in venous blood.Developed telemedicine system integrate CardioCarePack service – a patient, doctor and LC-MS/MS laboratory. The system helps in Doctor’s supervision over the patient’s condition on the basis of collected within the system data ex.: autonomous verification of the therapeutic index with flagging, visualization of results, tracking samples on the patient-doctor line, schedule of visits, results history and many more.<h4>Novel aspect</h4>A modern telemedicine system for TDM of cardiological drugs based on LC-MS/MS analysis of samples collected at home with VAMS.

CardioCarePack – personalized medicine system to improve live quality of patients suffering from cardiac arrhythmias

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