Evaluation of five multisteroid LC‒MS/MS methods used for routine clinical analysis: comparable performance was obtained for nine analytes.

OBJECTIVES: A mass spectrometry (LC‒MS/MS)-based interlaboratory comparison study was performed for nine steroid analytes with five participating laboratories. The sample set contained 40 pooled samples of human serum generated from preanalyzed leftovers. To obtain a well-balanced distribution across reference intervals of each steroid, the leftovers first underwent a targeted mixing step. METHODS: All participants measured a sample set once using their own multianalyte protocols and calibrators. Four participants used in-house developed measurement platforms, including IVD-CE certified calibrators, which were used by three participants; the 5th lab used the whole LC‒MS kit from an IVD manufacturer. All labs reported results for 17-hydroxyprogesterone, androstenedione, cortisol, and testosterone, and four labs reported results for 11-deoxycortisol, corticosterone, cortisone, dehydroepiandrosterone sulfate (DHEAS), and progesterone. RESULTS: Good or acceptable overall comparability was found in Bland‒Altman and Passing‒Bablok analyses. Mean bias against the overall mean remained less than ±10 % except for DHEAS, androstenedione, and progesterone at one site and for cortisol and corticosterone at two sites (max. -18.9 % for androstenedione). The main analytical problems unraveled by this study included a bias not previously identified in proficiency testing, operator errors, non-supported matrix types and higher inaccuracy and imprecision at lower ends of measuring intervals. CONCLUSIONS: This study shows that intermethod comparison is essential for monitoring the validity of an assay and should serve as an example of how external quality assessment could work in addition to organized proficiency testing schemes.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

OBJECTIVES: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 µg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

OBJECTIVES: Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. METHODS: A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 µg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. CONCLUSIONS: A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine-10,11-epoxide in human serum and plasma.

OBJECTIVES: A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma. METHODS: To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. RESULTS: The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400-12.0 µg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %. CONCLUSIONS: In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making it suitable for standardizing routine assays and assessing clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of primidone in human serum and plasma.

OBJECTIVES: Primidone is an anticonvulsive drug used in the treatment of epilepsy and essential tremor. It offers beneficial effects in controlling seizures, but its usage is also associated with possible side effects. To ensure optimal therapy, it is crucial to measure its concentration through accurate quantification methods. Therefore, our main goal was to develop and validate a new reference measurement procedure (RMP) for accurately measuring primidone levels in human serum and plasma. METHODS: In our study, we focused on the separation of primidone from both known and unknown interferences using a C18 column. To achieve accurate sample preparation, we developed a protocol involving protein precipitation followed by a high dilution step. The validation of the assay and determination of measurement uncertainty were carried out following guidelines from organizations such as the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. These rigorous validation processes ensure the reliability and accuracy of our method for quantifying primidone levels in human serum and plasma samples. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interference. It can be used to quantify primidone in the range of 0.150-30.0 µg/mL. Intermediate precision was less than 4.0 %, and repeatability CV ranged from 1.0 to 3.3 % across all concentration levels. The relative mean bias ranged from 0.1 to 3.9 % for native serum levels, and from -2.6 to 2.8 % for lithium-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were 1.5-4.1 % and 0.9-1.0 %, respectively. CONCLUSIONS: In this study, we introduce an innovative LC-MS/MS-based candidate RMP specifically designed for primidone in human serum and plasma. Our RMP offers a traceable platform, facilitating the standardization of routine assays and enabling the evaluation of clinically relevant samples. With this novel approach, we aim to enhance the accuracy and reliability of primidone measurements, ultimately benefiting the field of clinical research and patient care.

Establishing metrological traceability for small molecule measurands in laboratory medicine.

For molecules that can be well described metrologically in the sense of the definition of measurands, and which can also be recorded analytically as individual substances, reference measurement service traceability to a metrologically sound foundation is a necessity. The establishment of traceability chains must be initiated by National Metrology Institutes (NMIs) according to applicable standards; they are at the top and leading position in this concept. If NMIs are not in the position to take up this task, alternative approaches must be sought. Traceability initiatives established by in vitro device industry or academia must meet the quality standards of NMIs. Adherence to International Organization for Standardization (ISO) procedure 15193 must be a matter of course for the establishment of reference measurement procedures (RMPs). Certified reference material (CRM) characterization must be thorough, e.g., by the application of quantitative nuclear magnetic resonance measurements and by adherence to ISO 15194. Both for RMPs and CRMs Joint Committee for Traceability in Laboratory Medicine (JCTLM) listing must be the ultimate goal. Results must be shared in a transparent manner to allow other stakeholders including NMIs to reproduce and disseminate the reference measurement procedures.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of gabapentin in human serum and plasma.

OBJECTIVES: To describe and validate a reference measurement procedure (RMP) for gabapentin, employing quantitative nuclear magnetic resonance (qNMR) spectroscopy to determine the absolute content of the standard materials in combination with isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) to accurately measure serum and plasma concentrations. METHODS: A sample preparation protocol based on protein precipitation in combination with LC-MS/MS analysis using a C8 column for chromatographic separation was established for the quantification of gabapentin. Assay validation and determination of measurement uncertainty were performed according to guidance from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. ID-LC-MS/MS parameters evaluated included selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement. RESULTS: The use of qNMR provided traceability to International System (SI) units. The chromatographic assay was highly selective, allowing baseline separation of gabapentin and the gabapentin-lactam impurity, without observable matrix effects. Variability between injections, preparations, calibrations, and days (intermediate precision) was <2.3%, independent of the matrix, while the coefficient of variation for repeatability was 0.9-2.0% across all concentration levels. The relative mean bias ranged from -0.8-1.0% for serum and plasma samples. Passing-Bablok regression analysis indicated very good inter-laboratory agreement; the slope was 1.00 (95% confidence interval [CI] 0.98 to 1.03) and the intercept was -0.05 (95% CI -0.14 to 0.03). Pearson's correlation coefficient was ≥0.996. Expanded measurement uncertainties for single measurements were found to be ≤5.0% (k=2). CONCLUSIONS: This analytical protocol for gabapentin, utilizing traceable and selective qNMR and ID-LC-MS/MS techniques, allows for the standardization of routine tests and the reliable evaluation of clinical samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of lamotrigine in human serum and plasma.

OBJECTIVES: We developed an isotope dilution (ID)-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for lamotrigine in human serum and plasma, using quantitative nuclear magnetic resonance-characterized reference standards to ensure traceability to the International System of Units. METHODS: A sample preparation protocol based on protein precipitation combined with LC-MS/MS analysis using a C18 column for chromatographic separation was established for the quantification of lamotrigine in human serum and plasma. Assay validation was performed according to current guidelines. Spiked serum and plasma samples were used to assess selectivity and specificity; a post-column infusion experiment and comparison of standard line slopes were performed to ascertain possible matrix effects. Precision and accuracy were determined in a 5 days validation experiment. Measurement uncertainty was determined per the Guide to the Expression of Uncertainty in Measurement. RESULTS: The method allowed the quantification of lamotrigine in serum and plasma in a range of 0.600-24.0 µg/mL without any observable matrix effects. The relative mean bias (n=6) ranged from 1.7 to 3.7%; intermediate precision, including variances in between-day, -calibration, and -injection, was ≤2.4%, independent of the level and matrix. Total measurement uncertainty for a single measurement was ≤2.6%; expanded uncertainty was ≤5.2% (coverage factor k=2). CONCLUSIONS: This candidate RMP based on ID-LC-MS/MS provides a traceable and reliable platform for the standardization of routine assays and the evaluation of clinical samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of topiramate in human serum and plasma.

OBJECTIVES: Topiramate is an antiepileptic drug (AED) used for the monotherapy or adjunctive treatment of epilepsy and for the prophylaxis of migraine. It has several pharmacodynamic properties that contribute to both its clinically useful properties and observed adverse effects. Accurate measurement of its concentration is therefore essential for dose adjustment/optimisation of AED therapy. Our aim was to develop and validate a novel reference measurement procedure (RMP) for the quantification of topiramate in human serum and plasma. METHODS: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method in combination with a protein-precipitation-based sample preparation allows for quantification of topiramate in human serum and plasma. To assure traceability to SI units, quantitative nuclear magnetic resonance (qNMR) was applied to characterize the reference material used as primary calibrator for this RMP. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Accuracy and precision was evaluated performing an extensive five day precision experiment and measurement uncertainty was evaluated according Guide to the Expression of Uncertainty in Measurement (GUM). RESULTS: The method enabled topiramate quantification within the range of 1.20-36.0 µg/mL without interference from structurally related compounds and no evidence of a matrix effect. Intermediate precision was ≤3.2 % and repeatability was 1.4-2.5 % across all concentration levels. The relative mean bias was -0.3 to 3.5 %. Expanded measurement uncertainties for target value assignment (n=6) were found to be ≤2.9 % (k=2) independent of the concentration level and the nature of the sample. CONCLUSIONS: In human serum and plasma, the RMP demonstrated high analytical performance for topiramate quantification and fulfilled the requirements on measurement uncertainty. Traceability to SI units was established by qNMR content determination of the topiramate, which was used for direct calibration of the RMP. This RMP is, therefore, fit for purpose for routine assay standardization and clinical sample evaluation.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine in human serum and plasma.

OBJECTIVES: An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of carbamazepine. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of carbamazepine from potential interferences, whether known or unknown, was achieved using a C18 column. A protein precipitation protocol followed by a high dilution step was established for sample preparation. Assay validation and determination of measurement uncertainty were performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization (ICH), and the Guide to the Expression of Uncertainty in Measurement (GUM). In order to demonstrate equivalence to the already existing RMP a method comparison study was performed. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of carbamazepine within the range of 0.800-18.0 µg/mL. Intermediate precision and repeatability (n=60 measurements) was found to be <1.6 % and <1.3 % over all concentration levels and independent from the matrix. The relative mean bias ranged from -0.1 to 0.6 % for native serum and from -0.3 to -0.1 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <1.8 % and <1.3 %, respectively. Method comparison showed a good agreement between the Joint Committee of Traceability in Laboratory Medicine (JCTLM) listed RMP and the candidate RMP resulting in a Passing-Bablok regression equation with a slope of 1.01 and an intercept of -0.01. The bias in the patient cohort was found to be 0.9 %. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for carbamazepine in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of levetiracetam in human serum and plasma.

OBJECTIVES: To develop an isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for levetiracetam quantification in human serum and plasma. METHODS: Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to characterize the RMP material to ensure traceability to SI units. To quantify levetiracetam, an LC-MS/MS method was optimized using a C8 column for chromatographic separation following protein-precipitation-based sample preparation. Spiked matrix samples of serum and plasma were used to test selectivity and specificity. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Precision and accuracy were evaluated over 5 days. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of levetiracetam within the range of 1.53-90.0 µg/mL. Intermediate precision was <2.2% and repeatability was 1.1-1.7% across all concentrations. The relative mean bias ranged from -2.5% to -0.3% across all levels and matrices within the measuring range. Diluted samples were found with a mean bias ranging from -0.1 to 2.9%. The predefined acceptance criterion for measurement uncertainty was met and determined for individual measurements independently of the concentration level and sample type to be ≤4.0% (k=2). CONCLUSIONS: We present a novel LC-MS/MS)-based candidate RMP for levetiracetam in human serum and plasma. Its expanded measurement uncertainty of ≤4.0% meets the clinical needs in levetiracetam monitoring. Utilizing qNMR to characterize levetiracetam reference materials allowed metrological traceability to SI units.

Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample Preparation with Phospholipid Removal and Comparison to Immunoassays.

Steroid analysis in clinical laboratories is dominated by immunoassays (IAs) that have a high sample turnover but are inherently limited in trueness, precision, and sensitivity. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has proved to be a far more capable tool, delivering better sensitivity, specificity, and the possibility of parallel analysis of multiple steroids and metabolites, providing the endocrinologist with more reliable and comprehensive diagnostic information. An LC-MS/MS assay with gradient elution over less than eight minutes and a one-step sample preparation combining protein precipitation with phospholipid removal of off-line solid-phase extraction was developed and validated. It allowed the quantification of 11-deoxycorticosterone (11-DOC), 11-deoxycortisol (11-DF), 17-OH-progesterone (17P), 21-deoxycortisol (21-DF), androstenedione (ANDRO), aldosterone (ALDO), corticosterone (CC), cortisol (CL), cortisone (CN), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol (E2), progesterone (PROG), and testosterone (TES) in human serum. Interday imprecision was generally better than 15%, trueness was proven by recovery experiments with ISO 17034-certified reference materials, proficiency testing (UK NEQAS), and measuring serum reference standards. In-house comparison against IVD-CE-certified immunoassays (IA) for 17P, ANDRO, CL, DHEAS, E2, PROG, and TES was conducted by assessing leftover routine patient samples and purpose-built patient serum pools. None of the compared routine IAs were meeting the standards of the LC-MS/MS. Insufficient overall comparability was found for ANDRO and 17P (mean bias > +65%). Accuracy limitations at lower concentrations were present in IAs for PROG, E2, and TES.

Personalized Therapy for Mycophenolate: Consensus Report by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.

ABSTRACT: When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.

Diagnostic Characteristics of 3-Parameter and 2-Parameter Equations for the Calculation of a Combined Indicator of Vitamin B12 Status to Predict Cobalamin Deficiency in a Large Mixed Patient Population.

BACKGROUND: A combined indicator for the determination of vitamin B12 status (4cB12) that employs four markers of vitamin B12 status (i.e., holotranscobalamin, HoloTC; vitamin B12, B12; methyl malonic acid, MMA; and homocysteine, Hcy) has been proposed for the comprehensive assessment of B12 status. We aimed to compare recently published 2- (2cB12) and 3-parameter (3cB12) cB12 equations missing one or two markers of B12 status with the established four-parameter cB12 (4cB12). METHODS: In 3,614 routine samples in which HoloTC, B12, MMA, Hcy and serum folate were measured, cB12 was assessed with 4cB12, as well as with four 3cB12 and six 2cB12 equations. Diagnostic accuracy (AUC) curves were calculated by receiver operating characteristic (ROC) curve analysis with the four-parameter equation (4cB12) as an index. Furthermore, we investigated whether calculating cB12 in addition to a 2-step algorithm employing the same parameters would add diagnostic value for the diagnosis of vitamin B12 deficiency. RESULTS: HoloTC showed the highest diagnostic accuracy among the single markers (AUC = 0.94). The cB12 equation using HoloTC and MMA (2cB12HoloTC/MMA) had the highest AUC among the 2-parameter equations (0.98). Among the 3-parameter equations, 3cB12HoloTC/MMA/Hcy and 3cB12HoloTC/B12/MMA revealed an AUC of 0.99, which was significantly higher than that of 2cB12HoloTC/MMA (p < 0.01). Calculating 2cB12HoloTC/MMA in addition to using a stepwise algorithm employing HoloTC and MMA for diagnosis of vitamin B12 deficiency increased the positive likelihood ratio from 12.1 to 42.6. CONCLUSIONS: cB12 calculated with two or three markers of B12 status provides a good approximation of the 4cB12 equation. A 2cB12 equation employing the same parameters improved diagnostic accuracy compared to the use of a 2-step diagnostic algorithm alone. Our results suggest, that laboratories should consider enriching their reports by additionally reporting a corresponding 2cB12 or 3cB12 to results obtained in stepwise diagnostic algorithms.

Towards eco-friendly secondary plant metabolite quantitation: Ultra high performance supercritical fluid chromatography applied to common vervain (Verbena officinalis L.).

This report presents the first ultra high performance supercritical fluid chromatography diode array detector based assay for simultaneous determination of iridoid glucosides, flavonoid glucuronides, and phenylpropanoid glycosides in Verbena officinalis (Verbenaceae) extracts. Separation of the key metabolites was achieved in less than 7 min on an Acquity UPC2 Torus Diol column using a mobile phase gradient comprising subcritical carbon dioxide and methanol with 0.15% phosphoric acid. Method validation for seven selected marker compounds (hastatoside, verbenalin, apigenin-7-O-glucuronide, luteolin-7-O-glucuronide, apigenin-7-O-diglucuronide, verbascoside, and luteolin-7-O-diglucuronide) confirmed the assay to be sensitive, linear, precise, and accurate. Head-to-head comparison to an ultra high performance liquid chromatography comparator assay did prove the high orthogonality of the methods. Quantitative result equivalence was evaluated by Passing-Bablok-correlation and Bland-Altman-plot analysis. This cross-validation revealed, that one of the investigated marker compound peaks was contaminated in the ultra high performance liquid chromatography assay by a structurally related congener. Taken together, it was proven that the ultra high performance supercritical fluid chromatography instrument setup with its orthogonal selectivity is a true alternative to conventional reversed phase liquid chromatography in quantitative secondary metabolite analysis. For regulatory purposes, assay cross-validation with highly orthogonal methods seems a viable approach to avoid analyte overestimation due to coeluting, analytically indistinguishable contaminants.

Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report.

Ten years ago, a consensus report on the optimization of tacrolimus was published in this journal. In 2017, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicity (IATDMCT) decided to issue an updated consensus report considering the most relevant advances in tacrolimus pharmacokinetics (PK), pharmacogenetics (PG), pharmacodynamics, and immunologic biomarkers, with the aim to provide analytical and drug-exposure recommendations to assist TDM professionals and clinicians to individualize tacrolimus TDM and treatment. The consensus is based on in-depth literature searches regarding each topic that is addressed in this document. Thirty-seven international experts in the field of TDM of tacrolimus as well as its PG and biomarkers contributed to the drafting of sections most relevant for their expertise. Whenever applicable, the quality of evidence and the strength of recommendations were graded according to a published grading guide. After iterated editing, the final version of the complete document was approved by all authors. For each category of solid organ and stem cell transplantation, the current state of PK monitoring is discussed and the specific targets of tacrolimus trough concentrations (predose sample C0) are presented for subgroups of patients along with the grading of these recommendations. In addition, tacrolimus area under the concentration-time curve determination is proposed as the best TDM option early after transplantation, at the time of immunosuppression minimization, for special populations, and specific clinical situations. For indications other than transplantation, the potentially effective tacrolimus concentrations in systemic treatment are discussed without formal grading. The importance of consistency, calibration, proficiency testing, and the requirement for standardization and need for traceability and reference materials is highlighted. The status for alternative approaches for tacrolimus TDM is presented including dried blood spots, volumetric absorptive microsampling, and the development of intracellular measurements of tacrolimus. The association between CYP3A5 genotype and tacrolimus dose requirement is consistent (Grading A I). So far, pharmacodynamic and immunologic biomarkers have not entered routine monitoring, but determination of residual nuclear factor of activated T cells-regulated gene expression supports the identification of renal transplant recipients at risk of rejection, infections, and malignancy (B II). In addition, monitoring intracellular T-cell IFN-g production can help to identify kidney and liver transplant recipients at high risk of acute rejection (B II) and select good candidates for immunosuppression minimization (B II). Although cell-free DNA seems a promising biomarker of acute donor injury and to assess the minimally effective C0 of tacrolimus, multicenter prospective interventional studies are required to better evaluate its clinical utility in solid organ transplantation. Population PK models including CYP3A5 and CYP3A4 genotypes will be considered to guide initial tacrolimus dosing. Future studies should investigate the clinical benefit of time-to-event models to better evaluate biomarkers as predictive of personal response, the risk of rejection, and graft outcome. The Expert Committee concludes that considerable advances in the different fields of tacrolimus monitoring have been achieved during this last decade. Continued efforts should focus on the opportunities to implement in clinical routine the combination of new standardized PK approaches with PG, and valid biomarkers to further personalize tacrolimus therapy and to improve long-term outcomes for treated patients.

Azole-resistant and -susceptible Aspergillus fumigatus isolates show comparable fitness and azole treatment outcome in immunocompetent mice.

No data are available on the in vivo impact of infections with in vitro azole-resistant Aspergillus fumigatus in immunocompetent hosts. Here, the aim was to investigate fungal fitness and treatment response in immunocompetent mice infected with A. fumigatus (parental strain [ps]) and isogenic mutants carrying either the mutation M220K or G54W (cyp51A). The efficacy of itraconazole (ITC) and posaconazole (PSC) was investigated in mice, intravenously challenged either with a single or a combination of ps and mutants (6 × 105 conidia/mouse). Organ fungal burden and clinical parameters were measured. In coinfection models, no fitness advantage was observed for the ps strain when compared to the mutants (M220K and G54W) independent of the presence or absence of azole-treatment. For G54W, M220K, and the ps, no statistically significant difference in ITC and PSC treatment was observed in respect to fungal kidney burden. However, clinical parameters suggest that in particular the azole-resistant strain carrying the mutation G54W caused a more severe disease than the ps strain. Mice infected with G54W showed a significant decline in body weight and lymphocyte counts, while spleen/body weight ratio and granulocyte counts were increased. In immunocompetent mice, in vitro azole-resistance did not translate into therapeutic failure by either ITC or PSC; the immune system appears to play the key role in clearing the infection.

Irregular analytical errors in diagnostic testing - a novel concept.

BACKGROUND: In laboratory medicine, routine periodic analyses for internal and external quality control measurements interpreted by statistical methods are mandatory for batch clearance. Data analysis of these process-oriented measurements allows for insight into random analytical variation and systematic calibration bias over time. However, in such a setting, any individual sample is not under individual quality control. The quality control measurements act only at the batch level. Quantitative or qualitative data derived for many effects and interferences associated with an individual diagnostic sample can compromise any analyte. It is obvious that a process for a quality-control-sample-based approach of quality assurance is not sensitive to such errors. CONTENT: To address the potential causes and nature of such analytical interference in individual samples more systematically, we suggest the introduction of a new term called the irregular (individual) analytical error. Practically, this term can be applied in any analytical assay that is traceable to a reference measurement system. For an individual sample an irregular analytical error is defined as an inaccuracy (which is the deviation from a reference measurement procedure result) of a test result that is so high it cannot be explained by measurement uncertainty of the utilized routine assay operating within the accepted limitations of the associated process quality control measurements. SUMMARY: The deviation can be defined as the linear combination of the process measurement uncertainty and the method bias for the reference measurement system. Such errors should be coined irregular analytical errors of the individual sample. The measurement result is compromised either by an irregular effect associated with the individual composition (matrix) of the sample or an individual single sample associated processing error in the analytical process. OUTLOOK: Currently, the availability of reference measurement procedures is still highly limited, but LC-isotope-dilution mass spectrometry methods are increasingly used for pre-market validation of routine diagnostic assays (these tests also involve substantial sets of clinical validation samples). Based on this definition/terminology, we list recognized causes of irregular analytical error as a risk catalog for clinical chemistry in this article. These issues include reproducible individual analytical errors (e.g. caused by anti-reagent antibodies) and non-reproducible, sporadic errors (e.g. errors due to incorrect pipetting volume due to air bubbles in a sample), which can both lead to inaccurate results and risks for patients.

Assuring the Proper Analytical Performance of Measurement Procedures for Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee.

Monitoring immunosuppressive drugs (ISDs) in blood or plasma is still a key therapeutic drug monitoring (TDM) application in clinical settings. Narrow target ranges and severe side effects at drug underexposure or overexposure make accurate and precise measurements a must. This overview prepared by the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology is intended to serve as a summary and guidance document describing the current state-of-the-art in the TDM of ISDs.

Therapeutic Drug Monitoring of Everolimus: A Consensus Report.

In 2014, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology called a meeting of international experts to provide recommendations to guide therapeutic drug monitoring (TDM) of everolimus (EVR) and its optimal use in clinical practice. EVR is a potent inhibitor of the mammalian target of rapamycin, approved for the prevention of organ transplant rejection and for the treatment of various types of cancer and tuberous sclerosis complex. EVR fulfills the prerequisites for TDM, having a narrow therapeutic range, high interindividual pharmacokinetic variability, and established drug exposure-response relationships. EVR trough concentrations (C0) demonstrate a good relationship with overall exposure, providing a simple and reliable index for TDM. Whole-blood samples should be used for measurement of EVR C0, and sampling times should be standardized to occur within 1 hour before the next dose, which should be taken at the same time everyday and preferably without food. In transplantation settings, EVR should be generally targeted to a C0 of 3-8 ng/mL when used in combination with other immunosuppressive drugs (calcineurin inhibitors and glucocorticoids); in calcineurin inhibitor-free regimens, the EVR target C0 range should be 6-10 ng/mL. Further studies are required to determine the clinical utility of TDM in nontransplantation settings. The choice of analytical method and differences between methods should be carefully considered when determining EVR concentrations, and when comparing and interpreting clinical trial outcomes. At present, a fully validated liquid chromatography tandem mass spectrometry assay is the preferred method for determination of EVR C0, with a lower limit of quantification close to 1 ng/mL. Use of certified commercially available whole-blood calibrators to avoid calibration bias and participation in external proficiency-testing programs to allow continuous cross-validation and proof of analytical quality are highly recommended. Development of alternative assays to facilitate on-site measurement of EVR C0 is encouraged.