Rules for mass spectrometry applications in the clinical laboratory.

MS-based analytical methods now play an important role in medical laboratory analysis. Predominantly triple-stage mass spectrometry is used for the quantification of small molecule biomarkers and xenobiotics in blood and urine. The spectrum of applications ranges from completely in-house developed analytical methods, to industrially manufactured kit solutions used on generic equipment, to the first closed MS-based analysis systems. It is to be expected that the weights will shift in the coming years. Thus, operation and evaluation for most applications will remain very challenging and very different from the far more user-friendly and fully automated systems - mainly photometry-based - which are commonly used in clinical laboratories. General regulatory requirements for medical analysis differ significantly between countries globally. General requirements for in-house developed assay methods are valid in some countries, but concrete and methodology-specific rules for operation and quantification when using MS methods in the medical diagnostic laboratory are not applied. This differs significantly from other bio-analytical areas such as food monitoring, pharmaceutical research, or forensics, where legally binding, detailed rules exist in some cases, e.g., for substance identification. Internationally used relevant and helpful general standards with regard to mass spectrometric examination procedures in the clinical laboratory are in particular CLSI 62A and ISO 15189, while the IVDR in the EU primarily regulates the manufacture of diagnostic articles and not their application. In addition, from many years of application experience, some general advice can be recommended as rules that can contribute to robustness and patient safety in the clinical application of MS procedures; with emphasis on: reasonable method description, batch release, competence management, maintenance, and continuity management. This article also proposes some procedural basic requirements for the application of MS procedures in the clinical laboratory.

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS.

OBJECTIVES: Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. METHODS: Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. RESULTS: The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) "Guideline on bioanalytical method validation". To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. CONCLUSIONS: Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.

OBJECTIVES: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. METHODS: After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. RESULTS: Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. CONCLUSIONS: We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

Mass spectrometric sample identification with indicator compounds introduced via labeled sample tubes.

Objectives: The risk of sample confusion continues to be a challenge for the pre-analytical part of the overall testing process. We here describe a novel system to track samples based on a chemical code labeling of test tubes with unique combinations of indicator compounds, which are naturally not present in specimens of human origin. As part of the sample vessel filling, the liquid specimens are permanently labeled with the compound code that can be tracked back to the primary tube. Methods: As a proof of concept we used 10 stable-isotope-labeled derivates of medical drugs as indicator substances to create a combinatory 10-digit binary number ID for individual test tubes, i.e. presence/absence of the respective compound. For this purpose, combinations of indicator compounds were provided in evaporated form in polypropylene tubes prior to filling with anonymized patient whole blood and corresponding plasmas subjected to liquid chromatography tandem-mass spectrometry designed to detect the 10 indicator compounds. Results: In the blind analysis, we correctly identified 307 different whole blood samples by readout of a 10-digit binary number ID based on the detection of indicator compounds with respect to their presence and number. Conclusions: We have demonstrated the feasibility of an internal labeling procedure for diagnostic samples with mass spectrometry-based readout of dissolved indicator compound combinations as a binary number ID. With an increasing number of coding compounds (≫10) a vast number of combinations for sample labeling can be realized beyond the proof of concept setting studied herein.

Isotope dilution-LC-MS/MS method for quantification of the urinary cotinine-to-creatinine ratio.

Background: Appropriate monitoring of tobacco smoking is extremely important in several areas of medicine, e.g. management of chronic obstructive pulmonary disease (COPD), epidemiological surveys, and allocation of heart or lung transplants. The major metabolite of nicotine is cotinine that is increasingly used as a laboratory parameter for assessing tobacco smoke exposure. Methods: Creatinine and cotinine were analyzed simultaneously in urine by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in one run within 3 min using a biphenyl column. For quantification, the respective stable-isotope-labeled standards were used. Results: Detuning and measuring a natural isotope of creatinine as precursor and product ion allowed a simultaneous quantification of creatinine and cotinine. The method revealed robust validation results. For both analytes, inaccuracy and imprecision of the quality control and external quality assessment (EQA) samples were ≤-11.1%. Conclusions: One essential novelty of the method presented here is the simultaneous quantification of creatinine and cotinine covered by one analytical method. Despite the very different natural concentrations of creatinine and cotinine, this allows the immediate reporting of the cotinine-to-creatinine ratio without the need for a separate creatinine analysis.

Comparative Routine Therapeutic Drug Monitoring of Mycophenolic Acid in human Plasma with HPLC-UV and Isotope Dilution LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring (TDM) of the immunosuppressant mycophenolic acid (MPA) is especially recommended for the control of personalized immunosuppressive therapy. Various test systems are available for MPA monitoring, including high performance liquid chromatography combined with UV detection (HPLC-UV) and isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS). METHODS: In the present work, commercially available kits for MPA monitoring with HPLC-UV and ID-LC-MS/ MS were subjected to routine use TDM. Following method verification according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 105 native sample duplicates from patients under therapy with mycophenolate mofetil were assayed with both procedures for comparative testing. RESULTS: Using bi-level quality controls, the estimate of repeatability, within-laboratory imprecision and inaccuracy were ≤ 5.18%, ≤ 5.95% and ≤ 3.86% for all MPA measurements. Weighted Deming regression analysis yielded a slope of 0.93, an intercept of 0.04, and Pearson's correlation coefficient (r) of 0.99, while Bland-Altman analysis showed a combined relative bias of 4.93% (± 1.96 SD: -16.68 - 26.54%). Plasma samples taken from a patient re-peatedly showed the presence of an interferent only in HPLC-UV analysis. CONCLUSIONS: Based on these results, HPLC-UV testing can be considered suitable for routine TDM of MPA in the clinical setting with high precision. Due to the risk of unforeseen analytical interference in ever-increasing multimorbidity and polypharmacy, highly selective ID-LC-MS/MS methodology should be given preference over HPLC-UV analysis whenever feasible.

Multi-mycotoxin stable isotope dilution LC-MS/MS method for Fusarium toxins in cereals.

A multi-mycotoxin stable isotope dilution LC-MS/MS method was developed for 14 Fusarium toxins including modified mycotoxins in cereals (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT2-toxin, T2-toxin, enniatin B, enniatin B1, enniatin A1, enniatin A, beauvericin, fusarenone X, nivalenol, deoxynivalenol-3-glucoside, and zearalenone). The chromatographic separation of the toxins with particular focus on deoxynivalenol and deoxynivalenol-3-glucoside was achieved using a C18-hydrosphere column. An expedient sample preparation method was developed that uses solid-phase extraction for the purification of trichothecenes combined with zearalenone, enniatins, and beauvericin and provides excellent validation data. Linearity, intra-day precision, inter-day precision, and recoveries were ≥0.9982, 1-6%, 5-12%, and 79-117%, respectively. Method accuracy was verified by analyzing certified reference materials for deoxynivalenol, HT2-toxin, and T2-toxin with deviations below 7%. The results of this method found barley malt samples from 2012, 2013, and 2014 frequently contaminated with high concentrations of enniatin B, deoxynivalenol, and its modified mycotoxin deoxynivalenol-3-glucoside. Samples from 2012 were especially contaminated. Fusarenone X was not detected in any of the analyzed samples.

Chemical Synthesis of Deoxynivalenol-3-ß-d-[(13)C6]-glucoside and Application in Stable Isotope Dilution Assays.

Modified mycotoxins have been gaining importance in recent years and present a certain challenge in LC-MS/MS analysis. Due to the previous lack of a labeled isotopologue of the modified mycotoxin deoxynivalenol-3-glucoside, in our study we synthesized the first (13)C-labeled internal standard. Therefore, we used the Königs-Knorr method to synthesize deoxynivalenol-3-ß-d-[(13)C6]-glucoside originated from unlabeled deoxynivalenol and [(13)C6]-labeled glucose. Using the synthesized isotopically-labeled standard deoxynivalenol-3-ß-d-[(13)C6]-glucoside and the purchased labeled standard [(13)C15]-deoxynivalenol, a stable isotope dilution LC-MS/MS method was firstly developed for deoxynivalenol-3-glucoside and deoxynivalenol in beer. The preparation and purification of beer samples was based on a solid phase extraction. The validation data of the newly developed method gave satisfying results. Intra- and interday precision studies revealed relative standard deviations below 0.5% and 7%, respectively. The recoveries ranged for both analytes between 97% and 112%. The stable isotope dilution assay was applied to various beer samples from four different countries. In summary, deoxynivalenol-3-glucoside and deoxynivalenol mostly appeared together in varying molar ratios but were quantified in rather low contents in the investigated beers.

Spuriously low immunosuppressant results due to incomplete hemolysis - A pitfall in transplant patient therapeutic drug monitoring.

Objective: Therapeutic drug monitoring (TDM) plays a crucial role in transplantation medicine when it comes to immunosuppressants like Tacrolimus, Cyclosporine A, Sirolimus, and Everolimus. The analysis involves using immunometric or mass spectrometric methods on whole blood samples. Hemolysis of the samples is necessary for the assessment. Typically, this is accomplished through manual protein precipitation using pre-treatment reagents, followed by vigorous vortex mixing and subsequent centrifugation. It is important to note that omitting the vortex step in these manual procedures can be seen as a potential procedural error. Methods: To assess the potential impact of omitting the vortex step, an experiment was conducted. Clinical samples were divided into two aliquots, which were then analyzed comparatively. In one group of aliquots, the vortex step was intentionally omitted, while the other followed the correct execution of the test. Results: The non-vortex-mixed samples showed significantly erroneous low results for all analytes. Conclusion: Omitting or inadequately performing vortex mixing during the hemolysis procedure can be considered as a significant potential source of analytical error in TDM of immunosuppressants.

Can linezolid be validly measured in endotracheal aspiration in critically ill patients? A proof-of-concept trial.

BACKGROUND: Therapeutic drug monitoring (TDM) of anti-infectives such as linezolid is routinely performed in blood of intensive care unit (ICU) patients to optimize target attainment. However, the concentration at the site of infection is considered more important for a successful therapy. Until now, bronchoalveolar lavage (BAL) is the gold standard to measure intrapulmonary concentrations of anti-infective agents. However, it is an invasive method and unsuitable for regular TDM. The aim of this proof-of-concept study was to investigate whether it is possible to reliably determine the intrapulmonary concentration of linezolid from endotracheal aspiration (ENTA). METHODS: Intubated ICU patients receiving 600 mg intravenous linezolid twice daily were examined in steady state. First, preliminary experiments were performed in six patients to investigate which patients are suitable for linezolid measurement in ENTA. In a second step, trough and peak linezolid concentrations of plasma and ENTA were determined in nine suitable patients. RESULTS: Linezolid can validly be detected in ENTA with viscous texture and > 0.5 mL volume. The mean (SD) linezolid trough concentration was 2.02 (1.27) mg/L in plasma and 1.60 (1.36) mg/L in ENTA, resulting in a median lung penetration rate of 104%. The mean (SD) peak concentration in plasma and ENTA was 10.77 (5.93) and 4.74 (2.66) mg/L. CONCLUSIONS: Linezolid can validly be determined in ENTA with an adequate texture and volume. The penetration rate is comparable to already published BAL concentrations. This method might offer a simple and non-invasive method for TDM at the site of infection "lung". Due to promising results of the feasibility study, comparison of ENTA and BAL in the same patient should be investigated in a further trial.

Determination of tenuazonic acid in human urine by means of a stable isotope dilution assay.

The content of tenuazonic acid in human urine was determined by a stable isotope dilution assay (SIDA) that was recently developed for the analysis of food commodities and extensively re-validated for urine matrix in this study. Linearity of the response curve was proven between molar ratios n(labeled standard)/n(analyte) of 0.02-100. The limits of detection and determination were 0.2 and 0.6 µg/L, respectively. The mean recovery of the stable isotope dilution assay was 102 ± 3 % in the range between 1.0 and 100 µg/L. Interassay precision was 6.7 % (relative standard deviation of three triplicate analyses of a human urine sample during 3 weeks). The method was applied to two studies dealing with urinary excretion of tenuazonic acid: In the first study, tenuazonic acid was quantified in the 24-h urine of six volunteers from Germany (three female, three male) in a concentration range of 1.3-17.3 µg/L or 2.3-10.3 ng/mg(-1) creatinine, respectively. In the second study, two volunteers (one female, one male) ingested 30 µg tenuazonic acid by consumption of naturally contaminated whole meal sorghum infant cereals and tomato juice, respectively. The urinary excretion of the ingested tenuazonic acid was 54-81 % after 6 h, depending on matrix and volunteer. After 24 h, 87-93 % of the ingested amount of tenuazonic acid was excreted, but the fate of the remaining about 10 % is open. Thus, it is not possible to exclude potential health hazards for the consumer, completely.

A suggested protocol for value assignment using isotope-dilution-LC-MS/MS in reference measurement procedures - Exemplary application for ciprofloxacin in serum.

BACKGROUND: Reference measurement procedures for assigning values to calibration materials play a crucial role in the concept of metrological traceability in laboratory medicine. ISO standard 15,193 specifies the requirements for such measurement procedures, albeit in a very general terms. MATERIALS AND METHODS: A standard structure of analysis series for value assignment by LC-MS/MS was developed and tested,this structure was complemented by a spreadsheet file for result calculation, metadata evaluation, and finally validation and confirmation of individual analysis runs and individual results, and measurement uncertainty evaluation. This framework was applied to a procedure for the quantification of ciprofloxacin in serum as an example. RESULTS: The approach of a detailed description of the analytical procedures of isotope dilution LC-MS/MS reference measurement methods together with a highly standardized spreadsheet-based method for data processing was found to be practical and efficient. The described measurement procedure for the quantification of ciprofloxacin in serum was found to be fit for purpose. CONCLUSION: A standardized, detailed procedure for the application of isotope dilution LC-MS/MS in reference measurement procedures can complement the ISO 15193 standard with respect to this particular analytical technique, which is now widely used in the context of metrological traceability in laboratory medicine.

Therapeutic drug monitoring in breast cancer therapy - LC-MS/MS method for quantification of the CDK4/6 inhibitors abemaciclib, palbociclib, ribociclib, and major metabolites abemaciclib M20 and M2 in human serum.

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib were approved by the U.S. Food and Drug Administration (FDA) and European Medicine Agency for the treatment of breast cancer between 2015 and 2018. Oral tumor therapeutics extend the options for cancer therapy, but also challenge physicians and patients. The aim of the present work was to establish a semi-automated liquid-chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of abemaciclib, its active metabolites abemaciclib M20 and M2, palbociclib, and ribociclib in human serum. Detuning of ribociclib enabled the development of a simultaneous quantification method for abemaciclib, M20, M2, palbociclib, and ribociclib in the respective relevant concentration ranges based on semi-automated sample preparation with isotope dilution LC-MS/MS. The method was validated according to the guidance of the FDA. The LC-MS/MS method was successfully validated according to FDA and showed inaccuracies ≤ 10.7% and imprecisions ≤ 8.51%. Linearity was given from 20 to 800 ng/mL for abemaciclib, 15-600 ng/mL for M20, 10-400 ng/mL for M2 and palbociclib, and 100-4000 ng/mL for ribociclib. Normalized matrix factors and process efficiency showed no significant matrix effects regardless of the analytes. To demonstrate the applicability of the method, authentic samples were also analyzed. This novel semi-automated LC-MS/MS method covering all previously approved CDK4/6 inhibitors as well as the similarly pharmacologically active metabolites in human serum simultaneously was developed for potential future use in routine analysis in order to improve personalized therapy, patient safety, and treatment success.

Heat-Triggered Release of Dexamethasone from Thermosensitive Liposomes Using Prodrugs or Excipients.

Dexamethasone (DXM) is a potent glucocorticoid with an anti-inflammatory and anti-angiogenic activity which is widely clinically used. Systemic side effects limit the long-term use of DXM in patients requiring formulations which deliver and selectively release the drug to the diseased tissues. This in vitro study compares the suitability of DXM and commonly used prodrugs dexamethasone-21-phosphate (DXMP) and dexamethasone-21-palmitate (DP) as well as DXM complexed by 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) for the use in thermosensitive liposomes (TSL). DXM showed a poor retention and a low final drug:lipid ratio in a 1,2-dipalmitoyl-sn­glycero-3-phosphodiglycerol-based TSL (DPPG2-TSL) and a low-temperature sensitive liposome (LTSL). In contrast to DXM, DXMP and DP were stably retained at 37 °C in TSL in serum and could be encapsulated with high drug:lipid ratios in DPPG2-TSL and LTSL. DXMP showed a rapid release at mild hyperthermia (HT) from both TSL in serum, whereas DP remained incorporated in the TSL bilayer. According to release experiments with carboxyfluorescein (CF), HP-γ-CD and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) are suitable vehicles for the loading of DXM into DPPG2-TSL and LTSL. Complexation of DXM with HP-γ-CD increased the aqueous solubility of the drug leading to approx. ten times higher DXM:lipid ratio in DPPG2-TSL and LTSL in comparison to un-complexed DXM. Both DXM and HP-γ-CD showed increased release at HT in comparison to 37 °C in serum. In conclusion, DXMP and DXM complexed by HP-γ-CD represent promising candidates for TSL delivery.

Extracorporeal adsorption of protective and toxic bile acids and bilirubin in patients with cholestatic liver dysfunction: a prospective study.

BACKGROUND: The release of toxic bile acids (BAs) in the blood of critically ill patients with cholestatic liver dysfunction might lead to the damage of various organs. Their extracorporeal elimination using the cytokine adsorber Cytosorb® (CS) (adsorption of especially hydrophobic molecules < 60 kDa) might be promising, but data proving a potential adsorption are missing so far. METHODS: The prospective Cyto-SOVLE study (NCT04913298) included 20 intensive care patients with cholestatic liver dysfunction, continuous kidney replacement therapy, total bilirubin concentration > 10 mg/dl and the application of CS into the dialysis circuit. Bilirubin and different BAs were measured pre- and post-CS at defined timepoints (10 min, 1, 3, 6, and 12 h after initiation). Relative reduction (RR, %) was calculated with: [Formula: see text]. RESULTS: The median RR for total and conjugated bilirubin after initiation was - 31.8% and - 30.3%, respectively, and decreased to - 4.5% and - 4.8% after 6 h. A high initial RR was observed for the toxic BAs GCA (- 97.4%), TCA (- 94.9%), GCDCA (- 82.5%), and TCDCA (- 86.0%), decreasing after 6 h to - 32.9%, - 32.7%, - 12.8%, and - 14.3%, respectively. The protective hydrophilic BAs showed a comparable RR after initiation (UDCA: - 77.7%, GUDCA: - 83.0%, TUDCA: - 91.3%) dropping after 6 h to - 7.4%, - 8.5%, and - 12.5%, respectively. CONCLUSIONS: Cytosorb® can adsorb bilirubin and toxic as well as protective BAs. However, a fast saturation of the adsorber resulting in a rapid decrease of the RR was observed. Furthermore, no relevant difference between hydrophobic toxic and hydrophilic protective BAs was detected regarding the adsorption amount. The clinical benefit or harm of the BA adsorption needs to be evaluated in the future.

An UHPLC-MS/MS method for quantification of the CDK4/6 inhibitor abemaciclib in human serum.

BACKGROUND: Abemaciclib is a new oral targeted treatment option for patients with advanced breast cancer. The emerging field of oral antitumor therapeutics presents challenges for both patients and healthcare teams; non-adherence and high inter-individual pharmacokinetic variability can influence response rates. METHODS: For monitoring abemaciclib in human sera, a rapid novel ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated. Sample preparation was based on a protein precipitation step followed by on-line solid phase extraction. Chromatographic separation was achieved using a biphenyl column and the isotope labeled standard abemaciclib-d8 was used for quantification. RESULTS: The method showed linearity over a wide calibration range from 20.0 to 2500 ng/mL. With accuracies and precisions of ≤13.9% and ≤4.42%, respectively, the validation results were within the criteria of acceptance. The fitness of the method was tested by monitoring abemaciclib levels under compassionate use for a single individual. CONCLUSIONS: The novelty of the presented two dimensional isotope dilution UHPLC-MS/MS method is in the semi-automated sample preparation, which results in negligible matrix effects, thereby allowing the introduction of abemaciclib into robust routine therapeutic drug monitoring (TDM). This method provides an efficient tool to verify the usefulness of personalized anticancer therapy in clinical practice.

Estimation of inter-laboratory reference change values from external quality assessment data.

INTRODUCTION: It is common for patients to switch between several healthcare providers. In this context, the long-term follow-up of medical conditions based on laboratory test results obtained from different laboratories is a challenge. The measurement uncertainty in an inter-laboratory context should also be considered in data mining research based on routine results from randomly selected laboratories. As a proof-of-concept study, we aimed at estimating the inter-laboratory reference change value (IL-RCV) for exemplary analytes from publicly available data on external quality assessment (EQA) and biological variation. MATERIALS AND METHODS: External quality assessment data of the Reference Institute for Bioanalytics (RfB, Bonn, Germany) for serum creatinine, calcium, aldosterone, PSA, and of whole blood HbA1c from campaigns sent out in 2019 were analysed. The median CVs of all EQA participants were calculated based on 8 samples from 4 EQA campaigns per analyte. Using intra-individual biological variation data from the EFLM database, positive and negative IL-RCV were estimated with a formula based on log transformation under the assumption that the analytes under examination have a skewed distribution. RESULTS: We estimated IL-RCVs for all exemplary analytes, ranging from 13.3% to 203% for the positive IL-RCV and - 11.8% to - 67.0% for the negative IL-RCV (serum calcium - serum aldosterone), respectively. CONCLUSION: External quality assessment data together with data on the biological variation - both freely available - allow the estimation of inter-laboratory RCVs. These differ substantially between different analytes and can help to assess the boundaries of interoperability in laboratory medicine.

Effect of gravimetric correction and type of pipettes used in sample preparation on the precision of LC-MS/MS-based analyses.

BACKGROUND: Currently, manual pipetting of human sample material is still a key process in sample preparation for chromatographic and mass spectrometric analyses in the clinical laboratory. In most cases, however, pipettes used in clinical laboratories are only specified for handling water-like liquids. Actual pipetted liquid volumes can be verified by weighing within the sample preparation process, and the results can be corrected accordingly (gravimetric correction). The aim of our study was to evaluate and compare the effects of gravimetric correction in terms of accuracy and precision for an air cushion and direct replacement pipette. METHODS: Forty-fold serial determination of linezolid in a spiked serum sample by ID-LC-MS/MS was applied as an exemplary measurement procedure. Polypropylene tubes were weighed before the addition of 50 µL serum, after the addition, and after the addition of the internal standard solution. Coefficients of variation (CV) were calculated as an indicator of measurement precision. RESULTS: The use of a direct replacement pipette was associated with improved measurement imprecision than an air cushion pipette (CV 1.70% vs 2.49% for serum, uncorrected results). The results obtained after gravimetric correction showed improved precision with the use of an air cushion pipette compared to the conventional approach (CV 1.51% vs 1.61%). Using a direct replacement pipette, the impact of gravimetric correction on imprecision was negligible. CONCLUSION: Using direct replacement pipettes in sample preparation enables more precise ID-LC-MS/MS analyses than using air cushion pipettes. Gravimetric correction can be a useful tool to improve the precision of LC-MS/MS measurement procedures when complex matrices such as human serum are handled with commonly used air cushion pipettes.

Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum.

BACKGROUND: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19. METHODS: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol. RESULTS: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 µL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls. CONCLUSION: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.