Results From a Proficiency Testing Pilot for Immunosuppressant Microsampling Assays.

BACKGROUND: Therapeutic drug monitoring (TDM) of immunosuppressive drugs is important for the prevention of allograft rejection in transplant patients. Several hospitals offer a microsampling service that provides patients the opportunity to sample a drop of blood from a fingerprick at home that can then be sent to the laboratory by mail. The aim of this study was to pilot an external quality control program. METHODS: Fourteen laboratories from 7 countries participated (fully or partly) in 3 rounds of proficiency testing for the immunosuppressants tacrolimus, ciclosporin, everolimus, sirolimus, and mycophenolic acid. The microsampling devices included the following: Whatman 903 and DMPK-C, HemaXis, Mitra, and Capitainer-B. All assays were based on liquid chromatography with tandem mass spectrometry. In round 2, microsamples as well as liquid whole blood samples were sent, and 1 of these samples was a patient sample. RESULTS: Imprecision CV% values for the tacrolimus microsamples reported by individual laboratories ranged from 13.2% to 18.2%, 11.7%-16.3%, and 12.2%-18.6% for rounds 1, 2, and 3, respectively. For liquid whole blood (round 2), the imprecision CV% values ranged from 3.9%-4.9%. For the other immunosuppressants, the results were similar. A great variety in analytical procedures was observed, especially the extraction method. For the patient sample, the microsample results led to different clinical decisions compared with that of the whole blood sample. CONCLUSIONS: Immunosuppressant microsampling methods show great interlaboratory variation compared with whole blood methods. This variation can influence clinical decision-making. Thus, harmonization and standardization are needed. Proficiency testing should be performed regularly for laboratories that use immunosuppressant microsampling techniques in patient care.

Volumetric absorptive microsampling and dried blood spot microsampling vs. conventional venous sampling for tacrolimus trough concentration monitoring.

Objectives Monitoring tacrolimus blood concentrations is important for preventing allograft rejection in transplant patients. Our hospital offers dried blood spot (DBS) sampling, giving patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. In this study, both a volumetric absorptive microsampling (VAMS) device and DBS sampling were compared to venous whole blood (WB) sampling. Methods A total of 130 matched fingerprick VAMS, fingerprick DBS and venous WB samples were obtained from 107 different kidney transplant patients by trained phlebotomists for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. A multidisciplinary team pre-defined an acceptance limit requiring >80% of all matched samples within 15% of the mean of both samples. Sampling quality was evaluated for both VAMS and DBS samples. Results 32.3% of the VAMS samples and 6.2% of the DBS samples were of insufficient quality, leading to 88 matched samples fit for analysis. Passing-Bablok regression showed a significant difference between VAMS and WB, with a slope of 0.88 (95% CI 0.81-0.97) but not for DBS (slope 1.00; 95% CI 0.95-1.04). Both VAMS (after correction for the slope) and DBS showed no significant bias in Bland-Altman analysis. For VAMS and DBS, the acceptance limit was met for 83.0% and 96.6% of the samples, respectively. Conclusions VAMS sampling can replace WB sampling for tacrolimus trough concentration monitoring, but VAMS sampling is currently inferior to DBS sampling, both regarding sample quality and agreement with WB tacrolimus concentrations.

Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring.

Dried blood spot (DBS) analysis has been introduced more and more into clinical practice to facilitate Therapeutic Drug Monitoring (TDM). To assure the quality of bioanalytical methods, the design, development and validation needs to fit the intended use. Current validation requirements, described in guidelines for traditional matrices (blood, plasma, serum), do not cover all necessary aspects of method development, analytical- and clinical validation of DBS assays for TDM. Therefore, this guideline provides parameters required for the validation of quantitative determination of small molecule drugs in DBS using chromatographic methods, and to provide advice on how these can be assessed. In addition, guidance is given on the application of validated methods in a routine context. First, considerations for the method development stage are described covering sample collection procedure, type of filter paper and punch size, sample volume, drying and storage, internal standard incorporation, type of blood used, sample preparation and prevalidation. Second, common parameters regarding analytical validation are described in context of DBS analysis with the addition of DBS-specific parameters, such as volume-, volcano- and hematocrit effects. Third, clinical validation studies are described, including number of clinical samples and patients, comparison of DBS with venous blood, statistical methods and interpretation, spot quality, sampling procedure, duplicates, outliers, automated analysis methods and quality control programs. Lastly, cross-validation is discussed, covering changes made to existing sampling- and analysis methods. This guideline of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology on the development, validation and evaluation of DBS-based methods for the purpose of TDM aims to contribute to high-quality micro sampling methods used in clinical practice.

Quality Assessment of Dried Blood Spots from Patients With Tuberculosis from 4 Countries.

BACKGROUND: Dried blood spot (DBS) sampling is a blood collection tool that uses a finger prick to obtain a blood drop on a DBS card. It can be used for therapeutic drug monitoring, a method that uses blood drug concentrations to optimize individual treatment. DBS sampling is believed to be a simpler way of blood collection compared with venous sampling. The aim of this study was to evaluate the quality of DBSs from patients with tuberculosis all around the world based on quality indicators in a structured assessment procedure. METHODS: Total 464 DBS cards were obtained from 4 countries: Bangladesh, Belarus, Indonesia, and Paraguay. The quality of the DBS cards was assessed using a checklist consisting of 19 questions divided into 4 categories: the integrity of the DBS materials, appropriate drying time, blood volume, and blood spot collection. RESULTS: After examination, 859 of 1856 (46%) blood spots did not comply with present quality criteria. In 625 cases (34%), this was due to incorrect blood spot collection. The DBS cards from Bangladesh, Indonesia, and Paraguay seemed to be affected by air humidity, causing the blood spots not to dry appropriately. CONCLUSIONS: New tools to help obtain blood spots of sufficient quality are necessary and environmental specific recommendations to determine plasma concentration correctly. In addition, 3% of the DBS cards were rejected because the integrity of the materials suggesting that the quality of plastic ziplock bags currently used to protect the DBS cards against contamination and humidity may not be sufficient.

Performance of a web-based application measuring spot quality in dried blood spot sampling.

Background The dried blood spot (DBS) method allows patients and researchers to collect blood on a sampling card using a skin-prick. An important issue in the application of DBSs is that samples for therapeutic drug monitoring are frequently rejected because of poor spot quality, leading to delayed monitoring or missing data. We describe the development and performance of a web-based application (app), accessible on smartphones, tablets or desktops, capable of assessing DBS quality at the time of sampling by means of analyzing a picture of the DBS. Methods The performance of the app was compared to the judgment of experienced laboratory technicians for samples obtained in a trained and untrained setting. A robustness- and user test were performed. Results In a trained setting the app yielded an adequate decision in 90.0% of the cases with 4.1% false negatives (insufficient quality DBSs incorrectly not rejected) and 5.9% false positives (sufficient quality DBSs incorrectly rejected). In an untrained setting this was 87.4% with 5.5% false negatives and 7.1% false positives. A patient user test resulted in a system usability score of 74 out of 100 with a median time of 1 min and 45 s to use the app. Robustness testing showed a repeatability of 84%. Using the app in a trained and untrained setting improves the amount of sufficient quality samples from 80% to 95.9% and 42.2% to 87.9%, respectively. Conclusions The app can be used in trained and untrained setting to decrease the amount of insufficient quality DBS samples.

Clinical application of a dried blood spot assay for sirolimus and everolimus in transplant patients.

Background Monitoring of immunosuppressive drugs such as everolimus and sirolimus is important in allograft rejection prevention in transplant patients. Dried blood spots (DBS) sampling gives patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. Methods A total of 39 sirolimus and 44 everolimus paired fingerprick DBS and whole blood (WB) samples were obtained from 60 adult transplant patients for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. Two validation limits were pre-defined: limits of analytical acceptance were set at >67% of all paired samples within 20% of the mean of both samples and limits of clinical relevance were set in a multidisciplinary team at >80% of all paired samples within 15% of the mean of both samples. Results For both sirolimus and everolimus, Passing-Bablok regression showed no differences between WB and DBS with slopes of 0.86 (95% CI slope, 0.72-1.02) and 0.96 (95% CI 0.84-1.06), respectively. Only everolimus showed a significant constant bias of 4%. For both sirolimus and everolimus, limits of analytical acceptance were met (76.9% and 81.8%, respectively), but limits or clinical relevance were not met (77.3% and 61.5%, respectively). Conclusions Because pre-defined limits of clinical relevance were not met, this DBS sampling method for sirolimus and everolimus cannot replace WB sampling in our center at this time. However, if the clinical setting is compatible with less strict limits for clinical relevance, this DBS method is suitable for clinical application.

Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis.

In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[(2)H3] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 µmol/L (seven-point calibration curve), 116 to 7000 µmol/L (1-point calibration curve), and 1.00 to 400.0 µmol/L for creatinine-[(2)H3] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0% and a maximum bias of -5.9%. Creatinine in DBS was found stable at ambient temperature and 32 °C for 1 week and at -20 °C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement.

Analysis of remifentanil with liquid chromatography-tandem mass spectrometry and an extensive stability investigation in EDTA whole blood and acidified EDTA plasma.

BACKGROUND: Remifentanil is a µ-opioid receptor agonist that was developed as a synthetic opioid for use in anesthesia and intensive care medicine. Remifentanil is rapidly metabolized in both blood and tissues, which results in a very short duration of action. Even after blood sampling, remifentanil is unstable in whole blood and plasma through endogenous esterases and chemical hydrolysis. The instability of remifentanil in these matrices makes sample collection and processing a critical phase in the bioanalysis of remifentanil. METHODS: We have developed a fast and simple sample preparation method using protein precipitation followed by liquid chromatography-tandem mass spectrometry analysis. To improve the stability of remifentanil, citric acid, ascorbic acid, and formic acid were investigated for acidification of EDTA plasma. The stability of remifentanil was investigated in stock solution, EDTA whole blood, EDTA plasma, and acidified EDTA plasma at ambient temperature, 4 °C, 0 °C, and at -20 °C. RESULTS: The analytical method was fully validated based on the Food and Drug Administration guidelines for bioanalytical method validation with a large linear range of 0.20 to 250 ng/mL remifentanil in EDTA plasma acidified with formic acid. The stability results of remifentanil in EDTA tubes, containing whole blood placed in ice water, showed a decrease of approximately 2% in 2 hours. EDTA plasma acidified with citric acid, formic acid, and ascorbic acid showed 0.5%, 4.2%, and 7.2% remifentanil degradation, respectively, after 19 hours at ambient temperature. Formic acid was chosen because of its volatility and thus liquid chromatography-tandem mass spectrometry compatibility. The use of formic acid added to EDTA plasma improved the stability of remifentanil, which was stable for 2 days at ambient temperature, 14 days at 4 °C, and 103 days at -20 °C. CONCLUSIONS: The analytical method we developed uses a simple protein precipitation and maximal throughput by a 2-point calibration curve and short run times of 2.6 minutes. Best sample stability is obtained by placing tubes containing EDTA whole blood in ice water directly after sampling, followed by centrifugation and transfer of the EDTA plasma to tubes with formic acid. The stability of remifentanil in EDTA plasma was significantly improved by the addition of 1.5 µL formic acid per milliliter of EDTA plasma. This analytical method and sample pretreatment are suitable for remifentanil pharmacokinetic studies.

Application of sweat patch screening for 16 drugs and metabolites using a fast and highly selective LC-MS/MS method.

BACKGROUND: To facilitate the monitoring of drug abuse by patients, a method was developed and validated for fast and highly selective screening for amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylphenidate, cocaine, benzoylecgonine, morphine, codeine, heroin, 6-monoacteylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, nicotine, and cotinine in PharmCheck sweat patches. The analysis of sweat patches would provide a noninvasive alternative matrix to urine or blood samples. METHODS: The sweat patches were extracted during vigorous shaking for 10 minutes with 1.5 mL of 20 mmol/L ammonium formate, pH 7, and methanol (50%:50% vol/vol). The extracts were cleaned up by filtering through Whatman mini-Uniprep syringeless filter vials before injection. The method uses a single injection to detect and confirm all 16 drugs and metabolites within 9.6 minutes. RESULTS: The validated substances have a linear range of 3.0-300 nanograms per patch, except for nicotine which has a linear range of 30-3750 nanograms per patch. Stabilities of all substances in worn sweat patches were validated at room temperature for 7 days and as a processed sample in the autosampler at 10°C for 5 days. Only heroin was unstable, with high individual variability and reported bias and coefficient of variation of, respectively, -30.6% and 22.1% in worn sweat patches at room temperature. The monitoring of ion ratios was added to the validation criteria. This resulted in analytical cutoff concentrations of 3.0 and 60 nanograms per patch for nicotine with validated qualifier/quantifier ratios. All analytical cutoff concentrations were lower than the cutoff concentrations proposed by the Substance Abuse and Mental Health Services Administration. CONCLUSIONS: The method uses validated cutoff concentrations, qualifier/quantifier ratios, and a simple extraction without extensive sample treatment for the analysis of 16 drugs and metabolites with a runtime of 9.6 minutes. This method was successfully applied for the analysis of 96 worn sweat patches to monitor patients for drug abuse. The results provided the physician or health-care professional with information about drug abuse and could be used to improve patient care with patient-specific therapy.

Fast and highly selective LC-MS/MS screening for THC and 16 other abused drugs and metabolites in human hair to monitor patients for drug abuse.

BACKGROUND: To facilitate the monitoring of drug abuse by patients, a method was developed and validated for the analysis of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylphenidate, cocaine, benzoylecgonine, morphine, codeine, heroin, 6-monoacteylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), delta-9-tetrahydrocannabinol (THC), nicotine, and cotinine in human hair. METHODS: The hair preparation method contains a 3-step wash procedure with dichloromethane followed by a simultaneous hair pulverization and extraction procedure with disposable metal balls. The developed liquid chromatography tandem mass spectrometry method uses a single injection to detect and confirm all 17 abused drugs, including THC, within 4.8 minutes. RESULTS: Nicotine was validated with a linear range of 800-25,000 pg/mg hair, and all other substances were validated with a linear range of 30.0-2500 pg/mg hair. For inaccuracy and imprecision, the overall bias did not exceed -8.2% and the overall coefficient of variation did not exceed 17.7%. Autosampler stability was proven for 48 hours at 10°C for all substances. Analytical cutoff concentrations were defined for each substance at the lowest validated inaccuracy and imprecision concentration with a bias and coefficient of variation within 15% and qualifier/quantifier ratios within 20% of the set ratio. The analytical cutoff concentrations were 200 pg/mg for codeine and 80.0 pg/mg for 6-MAM, heroin, EDDP, and THC. The analytical cutoff concentration for nicotine was 800 pg/mg and for all other validated substances 30.0 pg/mg. This method was successfully applied to analyze hair samples from patients who were monitored for drug abuse. Hair samples of 47 subjects (segmented into 129 samples) showed 3,4-methylenedioxymethamphetamine, methylphenidate, cocaine, benzoylecgonine, codeine, methadone, EDDP, THC, nicotine, and cotinine above the analytical cutoff. CONCLUSIONS: The method was fully validated, including the validation of the qualifier/quantifier ratios. The analysis of real hair samples proved the efficacy of the developed method for monitoring drug abuse. The results obtained by this method provide the physician or health-care professional with extensive information about actual drug abuse or relapse and can be used for patient-specific therapy.

Everolimus decreases [U-13C]glucose utilization by pyruvate carboxylase in breast cancer cells in vitro and in vivo.

Reprogrammed metabolism is a hallmark of cancer, but notoriously difficult to target due to metabolic plasticity, especially in response to single metabolic interventions. Combining mTOR inhibitor everolimus and mitochondrial complex 1 inhibitor metformin results in metabolic synergy in in vitro models of triple-negative breast cancer. Here, we investigated whether the effect of this drug combination on tumor size is reflected in changes in tumor metabolism using [U-13C]glucose labeling in an MDA-MB-231 triple negative breast cancer xenograft model. The in vitro effects of everolimus and metformin treatment on oxidative phosphorylation and glycolysis reflected changes in 13C-labeling of metabolites in MDA-MB-231 cells. Treatment of MDA-MB-231 xenografts in SCID/Beige mice with everolimus resulted in slower tumor growth and reduced tumor size and tumor viability by 35%. Metformin treatment moderately inhibited tumor growth but did not enhance everolimus-induced effects. High serum levels of everolimus were reached, whereas levels of metformin were relatively low. Everolimus decreased TCA cycle metabolite labeling and inhibited pyruvate carboxylase activity. Metformin only caused a mild reduction in glycolytic metabolite labeling and did not affect pyruvate carboxylase activity or TCA cycle metabolite labeling. In conclusion, treatment with everolimus, but not metformin, decreased tumor size and viability. Furthermore, the efficacy of everolimus was reflected in reduced 13C-labeling of TCA cycle intermediates and reduced pyruvate carboxylase activity. By using in-depth analysis of drug-induced changes in glucose metabolism in combination with measurement of drug levels in tumor and plasma, effects of metabolically targeted drugs can be explained, and novel targets can be identified.

The Patient Motivation Pyramid and Patient-Centricity in Early Clinical Development.

BACKGROUND: It has been recognized that patients should be involved in the design of clinical trials. However, there is a lack of agreement on what patient-centricity means. METHODS: In this article, a Patient Motivation Pyramid based on Maslow's theory of human motivation is introduced as a tool to identify patient needs. This pyramid is used to make a comprehensive overview of options to implement a patient-centric trial design. The Pyramid with the described options can help to identify patient-centric activities suitable for given drug development. The current article further describes the potential benefits of patient-centric trial designs with an emphasis on early clinical development. Especially in early clinical development, during which trials have many assessments per patient, and the safety and clinical efficacy are uncertain, patient-centric trial design can improve feasibility. Finally, we present three case examples on patient-centric trial design. The first example is seeking patient input on the trial design for a First-in-Human trial which includes patients with Immune Thrombocytopenic Purpura. The second example is the use of a video-link for home dosing. The final example is the use of digital medicine in a decentralized trial in heart failure patients. RESULTS: A comprehensive overview of patients' needs can be accomplished by building a Patient Motivation Pyramid as a tool. Patient input can lead to improved endpoints, improved feasibility, better recruitment, less dropout, less protocol amendments, and higher patient satisfaction. The use of digital medicine can lead to a trial design with much less visits to the clinical research center in early clinical development and in a later development phase, even to a complete virtual trial. CONCLUSION: We recommend using the Patient Motivation Pyramid as a structural approach for identifying elements of patient-centricity. Secondly, we recommend starting using patient-centric approaches in an early phase of the medicine's lifecycle.

Corrigendum to "Mass spectrometry for therapeutic drug monitoring of anti-tuberculosis drugs" [Clin. Mass Spectrom. 14(Part A) (2019) 34-45].

[This corrects the article DOI: 10.1016/j.clinms.2018.10.002.].

Have we got 'patient-centric sampling' right?

RA Koster currently works as Associate Director of Bioanalytical Science at the LC-MS/MS department at PRA Health Sciences in the Laboratory in Assen, The Netherlands. He is responsible for the LC-MS/MS analytical method development and leads a team of method development analysts and scientists. As global microsampling specialist within PRA he is interested in all developments regarding microsampling and aims to continuously improve microsampling techniques. He has been working in the field of bioanalysis for 19 years, in which he performed and supervised numerous analytical method developments using LC-MS/MS. He started his career in 2001 at Pharma Bio-Research (before it was acquired by PRA) as an LC-MS/MS analyst. In 2005, he moved to the University Medical Center Groningen where he focused on the development and validation of analytical methods for drugs and drugs of abuse in matrices like blood, plasma, hair, saliva, dried blood spots and volumetric absorptive microsampling with LC-MS/MS. In 2015 he obtained his PhD on the subject 'The influence of the sample matrix on LC-MS/MS method development and analytical performance'. In 2017, he started as Senior Scientist at PRA Health Sciences and in 2019, he accepted his current role of Associate Director of Bioanalytical Science. He is a (co-)author of more than 35 publications.

UHPLC-MS/MS method for iohexol determination in human EDTA and lithium-heparin plasma, human urine and in goat- and pig EDTA plasma.

Aim: Iohexol plasma clearance is used as an indicator of kidney function in clinical and preclinical settings. To investigate the pharmacokinetic profile of iohexol, a rapid, simple method for measurement of iohexol in different matrices and species was needed. Materials & methods: Iohexol was separated on an Accucore C18 column (Thermo Fisher Scientific, CA, USA). Detection was performed on a Thermo Scientific Quantiva tandem quadrupole mass spectrometer. The method was validated according to the requirements for bioanalytical methods issued by the US FDA and European Medicines Agency. Conclusion: We developed and validated a fast and efficient analytical method, suitable for analyzing iohexol in human EDTA plasma, human lithium-heparin plasma, human urine and goat- and pig EDTA plasma, using only one calibration line prepared in human EDTA plasma.

Robust, high-throughput LC-MS/MS method for therapeutic drug monitoring of cyclosporine, tacrolimus, everolimus, and sirolimus in whole blood.

The authors describe a fast, robust, and straightforward liquid chromatography and tandem mass spectrometry (LC-MS/MS) method with the use of a single LC-MS/MS system for cyclosporine A, tacrolimus, sirolimus, and everolimus in whole blood. The purpose of this method was to replace the immunoassay (IA) methods used in the laboratory of a hospital performing most organ transplantations (including heart, lung, liver, kidneys, bone marrow, and intestinal tract). Several LC-MS/MS methods have been described so far; however, most of them require complicated online extraction procedures. The described LC-MS/MS method uses a chromatographic gradient in combination with protein precipitation as sample preparation. The chromatographic method is capable of separating otherwise interfering peaks, with an analysis time of 2.6 minutes. Analyses were performed on a triple quadrupole LC-MS/MS system, with a C18 column held at 60 degrees C. Sample preparation required only 1 precipitation/dilution step. Sirolimus and everolimus are prepared and measured separately from tacrolimus and cyclosporine. During method development, it was found that the use of zinc sulfate provides process efficiency results of about 100% for tacrolimus and cyclosporine A, but only 81% and 87% for sirolimus and everolimus, respectively. With the developed sample preparation without zinc sulfate for sirolimus and everolimus, process efficiencies were 99% and 108%, respectively. The methods have been fully validated, and in a comparative study, patient samples were analyzed with IA and our developed LC-MS/MS methods. In the comparative studies, correlations (R2 values) of more than 0.85 were found between the IA and the new LC-MS/MS patient blood levels. There was a systematic deviation in blood levels measured by LC-MS/MS compared with IAs for cyclosporine A (17% lower than with immunoassay) and everolimus (30% lower than with IA). There seemed to be little or no systematic deviation for sirolimus and tacrolimus. The controls determined by the LC-MS/MS method over the past 10 months showed coefficient of variations of no more than 8.0% for each of the 4 immunosuppressants. In conclusion, the authors found the developed methods to be cost saving, more flexible, and more sensitive and that these methods have larger linear ranges than the previously used IA methods. The methods are already used for more than 20,000 patient samples in the daily routine, analyzing approximately 70 patient samples per day.

Very complex internal standard response variation in LC-MS/MS bioanalysis: root cause analysis and impact assessment.

Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC-MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency. A decision tree is proposed to help find the underlying root cause for extreme IS variability.

A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects.

Aim: The aim of this study was to develop and validate a LC-MS/MS assay for tacrolimus, sirolimus, everolimus, cyclosporin A and mycophenolic acid using volumetric absorptive microsampling tips as a sampling device and to investigate the effect on the recoveries of the analyte concentration in combination with the hematocrit (HT), which included temsirolimus (a structural analog). Results: The maximum observed overall bias was 9.6% for the sirolimus LLOQ, while the maximum overall coefficient of variation was 8.3% for the everolimus LLOQ. All five immunosuppressants demonstrated to be stable in the volumetic absorbtive microsampling tips for at least 14 days at 25°C. Biases caused by HT effects were within 15% for all immunosuppressants between HT levels of 0.20 and 0.60 l/l, except for cyclosporin A, which was valid between 0.27 and 0.60 l/l. Reduced recoveries were observed at high analyte concentrations in combination with low HT values for sirolimus, everolimus and temsirolimus. Conclusion: A robust extraction and analysis method in volumetric absorptive microsampling tips was developed and fully validated. HT- and concentration-related recovery effects were observed but were within requirements of the purpose of the analytical method.

Mass spectrometry for therapeutic drug monitoring of anti-tuberculosis drugs.

Therapeutic drug monitoring (TDM) uses drug concentrations, primarily from plasma, to optimize drug dosing. Optimisation of drug dosing may improve treatment outcomes, reduce toxicity and reduce the risk of acquired drug resistance. The aim of this narrative review is to outline and discuss the challenges of developing multi-analyte assays for anti-tuberculosis (TB) drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) by reviewing the existing literature in the field. Compared to other analytical methods, LC-MS/MS offers higher sensitivity and selectivity while requiring relatively low sample volumes. Additionally, multi-analyte assays are easier to perform since adequate separation and short run times are possible even when non-selective sample preparation techniques are used. However, challenges still exist, especially when optimizing LC separation techniques for assays that include analytes with differing chemical properties. Here, we have identified seven multi-analyte assays for first-line anti-TB drugs that use various solvents for sample preparation and mobile phase separation. Only two multi-analyte assays for second-line anti-TB drugs were identified (including either nine or 20 analytes), with each using different protein precipitation methods, mobile phases and columns. The 20 analyte assay did not include bedaquiline, delamanid, meropenem or imipenem. For these drugs, other assays with similar methodologies were identified that could be incorporated in the development of a future comprehensive multi-analyte assay. TDM is a powerful methodology for monitoring patient's individual treatments in TB programmes, but its implementation will require different approaches depending on available resources. Since TB is most-prevalent in low- and middle-income countries where resources are scarce, a patient-centred approach using sampling methods other than large volume blood draws, such as dried blood spots or saliva collection, could facilitate its adoption and use. Regardless of the methodology of collection and analysis, it will be critical that laboratory proficiency programmes are in place to ensure adequate quality control. It is our intent that the information contained in this review will contribute to the process of assembling comprehensive multiplexed assays for the dynamic monitoring of anti-TB drug treatment in affected individuals.