Quality Control Concentrations Research Articles

A-133 Trusted Test Goes Awry: Investigating Dilution Aberrations for Total Bilirubin

Abstract Background A patient with acute decompensated cirrhosis was hospitalized due to lightheadedness and jaundice, undergoing evaluation for a liver transplant. The attending physician brought to the laboratory’s attention a perplexing pattern in the patient's total bilirubin results. Initially, the patient displayed a consistent trend of elevated total bilirubin levels above the upper limit of linearity, ULOL (> 30 mg/dL), which then plummeted to under the limit of quantification (< 0.1 mg/dL) before surging back to > 30 mg/dL. Methods The total bilirubin in heparinized plasma was measured on the AU5800 by Beckman Coulter. Samples > ULOL are diluted automatically 1:3 utilizing the instrument’s on-board dilution feature. The final test results are reported automatically to an electronic medical record. Following the reported incident, an investigation and troubleshooting plan was developed, encompassing the following steps: Repeat patient testing with total bilirubin concentration > 30 mg/dL and <0.1 mg/dL; Rule out the previously reported potential interferences, including Immunoglobin M and acetaminophen; Conduct a 90-day quality control (QC) review; Perform split test analysis on two instruments within the laboratory and at a reference laboratory; Conduct patient chart reviews to identify discrepancies based on previously reported values; Complete a root cause analysis and develop corrective/preventive action plan. Results Patient sample < 0.1 mg/dL was tested on an alternative instrument, resulting > 30 mg/dL. Patient with normal IgM of 161 mg/dL didn’t reveal any other potential drug interferences in chart review. The 3-month QC review found consistent with peer performance. Split comparison of 6 patients revealed discrepancies for samples > 30 mg/dL on one of the two instrument units. When total bilirubin > 30 mg/dL, the malfunctioning unit erroneously reported values < 0.1 mg/dL after dilution. Review of 34 patient charts showed the impact dates were spanned for 39 days. Since the on-board dilution feature is utilized by 10 other analytes, 892 diluted samples were analyzed for discrepancies. 36 discrepant results were identified among 4 affected assays including total bilirubin, direct bilirubin, creatine kinase, and alanine aminotransferase. These results were corrected, and providers were notified. Conclusions The root cause was traced to a malfunction in one instrument unit, despite no malfunction flags. The laboratory QC concentrations fell within the analytical measurement range, missing the dilution issue during the daily QCs. During an extensive repair period, the on-board dilution was disabled on the affected unit and samples were re-routed to alternative instrument for testing. Policy changes and operator training were initiated for a new result verification process, impacting the laboratory productivity. Autoverification of diluted sample results was disabled for all chemistry analyzers. The vendor identified the issue as sample transfer unit crane misalignment in the up/down direction. It is worth noting that proficiency testing or QCs often do not extend beyond the analytical measurement ranges, leading to undetected incidents. Additional quality measures like periodic patient chart reviews, patient sample comparisons, and other quality assurance tools can aid in on-going dilution performance verification. Open communication between the physicians and laboratories is crucial for prompt issue identification and resolution.

B-174 Quantification of vitamin C in serum as a biomarker using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract Background Ascorbic acid (AA) or vitamin C, is a crucial substance that is involved in numerous physiological processes in living beings, including collagen production and antioxidant activity. When ascorbic acid is exposed to moisture, air, heat, light and oxygen, it oxidizes and turns into dehydroascorbic acid (DHAA). Our objective is to describe the sample preparation procedure and total quantification of AA and DHAA in human serum sample based on LDTD-MS/MS, and demonstrate its efficiency in quantification, combined with analysis selectivity and rapidity. Methods The Luxon is a sample introduction and ionization source that used a fiber-coupled laser diode to thermally desorb neutral molecules and charge them using a corona needle so they can be analyzed by mass spectrometry. Due to the instability of ascorbic acid, plasma samples were enriched and stabilized with an acidic solution. Sample preparation utilized a phosphate-containing buffer to minimize interferents and help volatize the analyte, followed by a precipitation step using acetonitrile to further clean the extract. Stable isotope-labeled internal standard (13C6 ascorbic acid) and homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) negative mode. Calibration curves ranging from 1 to 100 μg/mL and QCs were prepared in ascorbic acid-depleted serum (kept at room temperature/exposed to light for 4 days). The peak area against the internal standard (IS) ratio was used to normalize the signal. The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. The following parameters were assessed for quantitative analysis: linearity, precision, accuracy, carryover and parallelism. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the correlation coefficient of calibration curves prepared in water to six curves prepared with biological matrixes. Intra and inter assay precision and accuracy were determined by measuring QC samples ranging from low to high concentration in five replicates for 3 consecutive days. Results Calibration curves (n=3) showed excellent linearity. The calibration curve resulted in a linear response throughout the concentration range, the correlation coefficient was 0.99. For each quality control concentrations CVs were <10% complying with desirable specifications for imprecision and accuracy. Intra-assay CVs were 3.0-8.89% and inter-assay CVs were 7.64-9.79% for precision which reflects the agreement between repeated measurements. The results for accuracy showed similar results, intra-assay CVs were 3.83-9.20% and inter-assay CVs were 5.0-9.7%. Conclusions The new LDTD-MS/MS method described here has been validated as an effective technique to reliably and rapidly quantify ascorbic acid. Simple sample preparation, rapid acid stabilization combined with high yield allow analysis without degradation responses.

B-173 Cystine Analysis in Urine Sample Using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract Background Cystine is a polar amino acid found in the urine of healthy individuals; however, a genetic defect in renal transport can lead to cystine crystals and stones. A usual laboratory approach for diagnosing and monitoring cystinuria is to measure cystine concentration in 24-hour urine. The disease is rare, affecting around 1 in every 10,000 live births. The objective of this study was to develop a rapid assay using Tandem Mass Spectrometry methodology with an internal standard of Cystine derivatized in a urine sample, Luxon Ion Source®, Laser Diode Thermal Desorption (LDTD) technology. Methods The Luxon is a sample introduction and ionization source with a coupled laser diode, providing thermal uniformity, precision, accuracy and speed. Thermally desorbed neutral molecules are analyzed by mass spectrometry (MS). Initially, the sample was derivatized to make the cystine thermally vaporize, followed by liquid-liquid extraction assisted by salting out (SALLE). The isotope-labeled internal standard (Cystine-d4), homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) positive mode. The ratio of peak area to internal standard (ISTD) was used to normalize the signal. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the (r2) of calibration curves prepared in water with six curves prepared with biological matrices. Intra- and interassay precision and accuracy were determined by measuring control samples ranging from low to high concentration in five replicates over 3 consecutive days. For urine analysis, aqueous calibration curves in the concentration range of 1 to 100 mg/L (n=3) showed excellent linearity. The following parameters were evaluated for quantitative analysis: linearity, precision, accuracy, carryover and parallelism.​ Results The calibration curve resulted in a linear response across the entire concentration range, the correlation coefficient (r) was 0.99. For each quality control concentration, CVs were <10%, meeting desirable imprecision and precision specifications. The intraassay CVs were 1.0-2.56% and the interassay CVs were 2.15-4.58% for precision, which reflects the agreement between repeated measurements. Precision results showed similar results, intra-assay CVs were 2.23-3.66% and inter-assay CVs were 1.0-2.77% Conclusions The new LDTD-MS/MS method for the determination of urinary Cystine was accepted after verification and validation of quality control parameters. The technique proved to be effective for reliably and quickly quantifying cystine in urinary samples from human patients, being a valuable tool in the diagnosis and prevention of cystine kidney stones. The assay developed, using the Luxon Ion Source®, based on LDTD (Laser Diode Thermal Desorption) technology, proved to be ultrafast for measuring derivatized cystine in urine

Development and Validation of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Ceftolozane and Tazobactam in Human Plasma Microsamples.

Ceftolozane/tazobactam (C-T) is a novel beta-lactam/beta-lactamase inhibitor combination approved for the treatment of various infections caused by difficult-to-treat Pseudomonas aeruginosa. In critically ill patients, C-T may exhibit significant pharmacokinetic variability, both between individuals and within individuals, warranting therapeutic drug monitoring for clinical purposes. We aim to develop and validate a novel and sensitive analytical method for concurrently determining C and T in human plasma microsamples (3μL). The method was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with positive electrospray ionization and multiple reaction monitoring (MRM) detection modes, employing specific mass transitions for both drugs. Sample preparation was simple, and the chromatographic run lasted only 4 minutes. Validation was conducted according to European Medicines Agency (EMA) guidelines, encompassing specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and drug stability. The validated method was applied to measure C and T in 32 plasma samples collected from critically ill patients with multidrug-resistant, gram-negative, bacterial infections. The method ensured accurate (BIAS% 2.1-9.6 for C and -2.2 to 15.2 for T) and precise intraday CV% for C: 6.7-5.5; for T: 1.3-8.9; interday CV% for C 6.0-10.8; for T 4.1-10.2) measurements of C-T over a wide concentration range (0.2-200.0 mg/L for C and 0.1-100.0 mg/L for T). Overall, the recovery at quality control concentration levels was high for both C and T (mean values: 90-91 for C and 89-92 for T). Analyte stability was satisfactory in both human plasma and extracts under various storage conditions. The clinical applicability of the assay was confirmed by the reliably quantifying C and T in clinical plasma samples. The developed and validated LC-MS/MS method is sensitive and suitable for monitoring C and T in human plasma microsamples.

Integrating Patient-Based Real-Time Quality Control (PBRTQC) in a New Field: Inter-Comparison between Biochemical Instrumentations with LDL-C.

Patient-based real-time quality control (PBRTQC) can be a valuable tool in clinical laboratories due to its cost-effectiveness and constant monitoring. More focus is placed on discovering and improving algorithms that compliment conventional internal control techniques. The practical implementation of PBRTQC with a biochemical instrument comparison is lacking. We aim to evaluate PBRTQC's efficacy and practicality by comparing low-density lipoprotein cholesterol (LDL-C) test results to ensure consistent real-time monitoring across biochemical instrumentations in clinical laboratories. From 1 September 2021 to 30 August 2022, the First Affiliated Hospital of Xi'an Jiaotong University collected data from 158,259 both healthy and diseased patients, including 84,187 male and 74,072 female patients, and examined their LDL-C results. This dataset encompassed a group comprising 50,556 individuals undergoing health examinations, a group comprising 42,472 inpatients (IP), and a group comprising 75,490 outpatients (OP) for the PBRTQC intelligent monitoring platform to conduct daily tests, parameter configuration, program development, real-time execution, and performance validation of the patients' data. Moreover 40 patients' LDL-C levels were assessed using two biochemical analyzers, designated as the reference and comparator instruments. A total of 160 LDL-C results were obtained from 40 both healthy and diseased patients, including 14 OP, 16 IP, and 10 health examination attendees, who were selected to represent LDL-C levels broadly. Two biochemical instruments measured LDL-C measurements from the same individuals to investigate consistency and reproducibility across patient statuses and settings. We employed exponentially weighted moving average (EWMA) and moving median (MM) methods to calculate inter-instrument bias and ensure analytical accuracy. Inter-instrument bias for LDL-C measurements was determined by analyzing fresh serum samples, different concentrations of quality control (QC), and commercialized calibrators, employing both EWMA and MM within two assay systems. The assessment of inter-instrumental bias with five different methods adhered to the external quality assessment standards of the Clinical Laboratory Center of the Health Planning Commission, which mandates a bias within ±15.0%. We calculated inter-instrument comparison bias with each of the five methods based on patient big data. The comparison of fresh serum samples, different concentrations of QC, commercialized calibrators, and EWMA were all in the permissive range, except for MM. MM showed that the bias between two biochemical instruments in the concentration ranges of 1.5 mmoL/L-6.2 mmoL/L exceeded the permissible range. This was mainly due to the small number of specimens, affected by variations among individual patients, leading to increased false alarms and reduced effectiveness in monitoring the consistency of the inter-instrumental results. Moreover, the inter-comparison bias derived from EWMA was less than 3.01%, meeting the 15% range assessment criteria. The bias result for MM was lower than 24.66%, which was much higher than EWMA. Thus, EWMA is better than MM for monitoring inter-instrument comparability. PBRTQC can complement the use of inter-comparison bias between biochemical analyzers. EWMA has comparable inter-instrument comparability monitoring efficacy. The utilization of AI-based PBRTQC enables the automated real-time comparison of test results across different biochemical instruments, leading to a reduction in laboratory operating costs, enhanced work efficiency, and improved QC. This advanced technology facilitates seamless data integration and analysis, ultimately contributing to a more streamlined and efficient laboratory workflow in the biomedical field.

Development of simultaneous quantitative analytical method for three active components of Korean mint (Agastache rugosa (Fisch. & C.A.Mey.) Kuntze) extract in human plasma using ultra-high-performance liquid chromatography–tandem mass spectrometry

Agastache rugosa contains phenolic compounds and flavonoids, and has been extensively used as a traditional herbal medicine. The major components in Agastache rugosa extract (ARE) are rosmarinic acid, tilianin, and acacetin, for which several analytical techniques have been reported. However, these substances have yet to be simultaneously quantified in human plasma. In this study, we aimed to simultaneously determine the three active components of ARE in human plasma by developing a reliable quantitative analytical method using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation of the plasma samples was achieved using an ACQUITY UPLC® BEH C18 column with a gradient mobile phase of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed using a triple quadrupole tandem mass spectrometer in negative electrospray ionization (ESI−) and multiple reaction monitoring (MRM) modes. The developed quantitative method was validated for the three active components. All three analytes exhibited a linear response over the ranges of 0.5–50 ng/mL for rosmarinic acid, 0.1–20 ng/mL for acacetin, and 0.5–20 ng/mL for tilianin with a weighting factor of 1/x (where x is the concentration). At three quality control (QC) concentration levels (low, medium, and high), including the lower limit of quantitation (LLOQ), acceptable accuracy (±15 %) was achieved in the intra- and interday validations. The concentration of rosmarinic acid was highest in plasma. Tilianin and acacetin appeared and were eliminated earlier in the plasma than rosmarinic acid. This study provides a successfully validated method that can be used in further clinical applications of Agastache rugosa extracts.

A-106 Sigma Metric Performance of Chemistries on the Abbott Alinity c Analyzer

Abstract Background The analytical performance of the Abbott Alinity c (Abbott Park, IL) was compared to our current Abbott Architect analyzers using sigma metrics. Methods Sigma metrics of 28 chemistry analytes on our six Abbott Alinity analyzers was compared to three Abbott Architect 16 000 analyzers and one Architect 8000 analyzer using Bio-Rad Multiqual quality control (QC) (Hercules, CA). Method imprecision was estimated for each assay using the cumulative coefficient of variation (CV) of 12 months data for each analyzer at three QC concentrations and calculating the average sigma. Method bias was estimated by comparing each analyzer QC mean to peer group QC means. Sigma values were calculated for each method as (TEa—Bias%)/CV% using allowable total error (TEa) from three sources: the CLIA proficiency testing evaluation limits, Australasian Association of Clinical Biochemistry allowable limits of performance (RCPA quality assurance programs Pty limited, Adelaide, Australia) and desirable biological variation (Ricos C et al, Scand J Clin Lab Invest. 1999, 59(7):491–500). Average sigma values were generated for each analyte and graded as optimal >6 sigma; good 5–6 sigma; marginal 3–5 sigma; or poor <3 sigma. Results Assay sigmas varied across QC concentrations and between analyzers of the same platform. Na, Cl, and CO2 had the lowest sigma performance. Since the sigma calculations are dependent on TEa, tighter TEa limits (Ricos and RCPA) generated lower sigmas compared to the wider CLIA TEa limits especially for Ca, Cl, total bilirubin, CO2, Na, total protein, and HDL. Conclusion None of the chemistries met optimal six sigma performance for all analytes, concentrations of QC and sources of TEa. However, the assay performance between analyzer platforms was comparable.

An HPLC-MS/MS method for determination of sulbactam in human plasma and its pharmacokinetic application in critically ill patients with augmented renal clearance.

A simple, rapid, and specific method has been developed and validated to measure sulbactam in human plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The pharmacokinetic characteristics of sulbactam in critically ill patients with augmented renal clearance were investigated after the repeated administration of cefoperazone-sulbactam (3 g, q8, IV drip, combination ratio of 2:1). Sulbactam plasma concentration was determined using LC-MS/MS with tazobactam used as an internal standard (IS). The method was fully validated with a sensitivity of 0.20 μg/mL, the linear concentration was ranged from 0.20 to 30.0 μg/mL. The intra-batch precision (RSD%) was less than 4.9%, and the accuracy deviation (RE%) ranged from -9.9 to 1.0%; the inter-batch precision (RSD%) was less than 6.2%, and the accuracy deviation (RE%) ranged from -9.2% to 3.7%. The value of the mean matrix factor at the low and high quality control (QC) concentration was 96.8 and 101.0%, respectively. The extraction recovery for QCL and QCH of sulbactam were 92.5 and 87.5%, respectively. Plasma samples and clinical data were collected at 0 (pre dose), 0.25, 0.5, 1, 2, 3, 6, and 8 hours (post dose) from 11 critically ill patients. Pharmacokinetic parameters were determined by non-compartmental analysis (NCA) using Phoenix WinNonlin software. This method was successfully applied to study the pharmacokinetics of sulbactam for critically ill patients. The main pharmacokinetic parameters of sulbactam in augmented renal function and normal renal function groups were summarized as follows: half-life, 1.45 ± 0.66 and 1.72 ± 0.58 hours, area under the concentration-time curve from 0 to 8 hours, 59.1 ± 20.1 and 111.4 ± 23.2 μg × h/mL, drug plasma clearance at steady state, 18.9 ± 7.5 and 9.32 ± 2 .03 L/h, respectively. These results suggested that a higher dose of sulbactam should be used in critically ill patients with augmented renal clearance.

Determination of olverembatinib in human plasma and cerebrospinal fluid by an LC-MS/MS method: Validation and clinical application

A sensitive and robust LC-MS/MS method has been developed and validated for olverembatinib quantification in human plasma and cerebrospinal fluid (CSF). The method involved liquid-liquid extraction with methyl tertiary butyl ether for plasma pretreatment and precipitation enrichment with methanol for CSF pretreatment. Separation was achieved on the C18 column with gradient elutions of 10 mM ammonium formate in water and methanol-acetonitrile (50:50,v/v). Analyte detection was conducted by electrospray ionization (ESI) in a positive ion mode using multiple reaction monitoring (MRM). The m/z transitions were 533.4→433.2 for olverembatinib and m/z 502.4→394.2 for the internal standard (IS, Imatinib-d8). Calibration curves ranged from 0.500 to 50.0 ng/mL for plasma and from 0.0100 to 1.00 ng/mL for CSF. The intra- and inter-day precision and accuracy were < 15% for both plasma and CSF with four different quality control concentrations. The relative matrix effect was < 10% in plasma and artificial CSF. This method was successfully utilized for the measurement of olverembatinib concentrations in plasma and CSF from chronic myeloid leukemia patients.

Analytical evaluation and Sigma metrics of 6 next generation chemistry assays on the Abbott Architect system.

We evaluated analytical and Sigma performance for 6 next generation chemistry assays on the Abbott Architect c8000 system. Albumin with bromocresol purple or green, amylase, cholesterol, total protein, and urea nitrogen were analyzed using photometric technology. Analytical performance goals were defined based on Accreditation Canada Diagnostics (ACD) and Clinical Laboratory Improvement Amendments (CLIA). Precision study consisted of testing 2 quality control concentrations and 3 patient serum sample pools, twice a day in quintuplicate over 5days. Linearity testing consisted of 5-6 concentrations of commercial linearity materials. We tested a minimum of 120 serum/plasma specimens on the new and current Architect methods for comparison. We assessed accuracy with reference materials for 5 assays, and a calibration standard for cholesterol. Bias from the reference standard target value was used for Sigma metric analysis. Observed total imprecision of the assays ranged from 0.5 to 4%, meeting pre-defined goals. Linearity was acceptable over the tested range. Measurements on the new and current Architect methods were comparable. Accuracy ranged from 0 to 2.0% absolute mean difference from target value. All 6 next generation clinical chemistry assays demonstrated Six Sigma quality, using CLIA standards. Applying ACD recommendations, 5 assays showed Six Sigma, while cholesterol showed Five Sigma performance.

Validation of a HS–GC–FID Method for the Quantification of Sevoflurane in the Blood, Urine, Brain and Lungs for Forensic Purposes

Background: Sevoflurane is a polyfluorinated compound extensively used as an inhalation anesthetic in patients undergoing surgery. If administered outside the operating room, sevoflurane is dangerous and potentially lethal, and toxicologists may be asked to investigate its presence in biological matrices for forensic purposes. The aim of the present study is to develop and validate a method for the detection and the quantification of sevoflurane in biological fluids and organs through gas chromatography coupled to flame ionization detection (GC–FID). Methods: The method was optimized based on the maximization of the signal-to-noise ratio. The GC–FID instrument was equipped with a Zebron capillary column ZB-624 (30 m, 0.32 mm ID, 1.80 µm film thickness). Results: The method was validated over a concentration range of 1.0–304.0 µg/mL (blood and urine) and µg/g (brain, lungs). The lower limit of quantitation was 1.0 µg/mL or µg/g. Both the intra- and interassay imprecision and inaccuracy were ≤15% at all quality control concentrations in all the matrices. The method was successfully applied to measure the sevoflurane concentrations for 20 negative controls and for a real forensic case. Conclusions: The present method is suitable for the identification and quantification of sevoflurane in fluids and organs and can be a reliable tool in forensic casework.

Calibration frequency and analytical variability of laboratory measurements

BackgroundThere is uncertainty whether increased frequency of calibrations may affect the overall analytical variability of a measurement procedure as reflected in quality control (QC) performance. In this simulation study, we examined the impact of calibration frequencies on the variability of laboratory measurements. MethodsA 5-point calibration curve was modeled with simulated concentrations ranging from 10 to 10,000 mmol/l, and signal intensities with CVs of 3 % around the mean, under a Gaussian distribution. 3 levels of QC (20, 150, 600 mmol/l) interspersed within the analytical measurement range were also simulated. ResultsThe CV of the 3 QC levels remained stable across the different calibration frequencies simulated (5, 10, 15 and 30 QC measurements per recalibration episode). The imprecision was greatest (18 %) at the lowest concentration of 20 mmol/l, when the calibration curve was derived using ordinary least squares regression, reducing to 3.5 % and 3.8 % at 150 and 600 mmol/l, respectively. The CV of all 3 QC concentrations remained constant at 3.4 % and close the predefined CV (3 %) when weighted least squares regression was used to derive the calibration model. Similar findings were observed with 2-point calibrations using WLS models at narrower concentration ranges (50 and 100 mmol/l as well as 50 and 500 mmol/l). DiscussionWithin the parameters of the simulation study, an increased frequency of calibration events does not adversely impact the overall analytical performance of a measurement procedure under most circumstances.

Practical Considerations for Translating Mesenchymal Stromal Cell-Derived Extracellular Vesicles from Bench to Bed.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown potential for the treatment of tendon and ligament injuries. This approach can eliminate the need to transplant live cells to the human body, thereby reducing issues related to the maintenance of cell viability and stability and potential erroneous differentiation of transplanted cells to bone or tumor. Despite these advantages, there are practical issues that need to be considered for successful clinical application of MSC-EV-based products in the treatment of tendon and ligament injuries. This review aims to discuss the general and tissue-specific considerations for manufacturing MSC-EVs for clinical translation. Specifically, we will discuss Good Manufacturing Practice (GMP)-compliant manufacturing and quality control (parent cell source, culture conditions, concentration method, quantity, identity, purity and impurities, sterility, potency, reproducibility, storage and formulation), as well as safety and efficacy issues. Special considerations for applying MSC-EVs, such as their compatibility with arthroscopy for the treatment of tendon and ligament injuries, are also highlighted.

Development and Validation of a Method of Liquid Chromatography Coupled with Tandem Mass Spectrometry for Quantification of ST-246 (Tecovirimat) in Human Plasma.

The aim of this work was to develop and validate a sensitive and robust method of liquid chromatography coupled with tandem mass spectrometry to quantitate ST-246 (tecovirimat) in plasma using an internal standard (2-hydroxy-N-{3,5-dioxo-4-azatetracyclo [5.3.2.02.6.08.10]dodec-11-en-4-yl}-5-methylbenzamide). The method was validated in negative multiple reaction monitoring mode following recommendations of the European Medicines Agency for the validation of bioanalytical methods. The calibration curve for the analyte was linear in the 10–2500 ng/mL range with determination coefficient R2 > 0.99. Intra- and inter-day accuracy and precision for three concentrations of quality control were <15%. Testing of long-term stability of ST-246 (tecovirimat) in plasma showed no degradation at −20 °C for at least 3 months. The method was applied to a clinical assay of a new antipoxvirus compound, NIOCH-14. Thus, the proposed method is suitable for therapeutic drug monitoring of ST-246 (tecovirimat) itself and of NIOCH-14 as its metabolic precursor.

Development and validation of a robust and sensitive HPLC-MS/MS method for the quantitation of MRTX849 in plasma and its application in pharmacokinetics.

MRTX849 is a novel, highly selective, targeted inhibitor of KRAS (G12C), which significantly improves the objective response rate in patients with advanced solid tumors. However, neither an analytical HPLC-MS/MS assay nor pharmacokinetics has been reported for MRTX849 in plasma. In the present study, chromatography was accomplished on a reversed phase C18 column (50 × 2.1 mm, 3.5 μm). The limit of detection of MRTX849 was 0.02 ng mL-1 at S/N ≥ 3. Only 20 μL of plasma was utilized for accurate quantitation. The optimized analytical assay was fully validated and verified in accordance with guidelines. The calibration curve for MRTX849 was linear with a correlation coefficient >0.99 in the range of 0.05-200 ng mL-1. The intra- and inter-day accuracy and precision were all within ±10%. The matrix effect and recovery were consistent and acceptable under several quality control concentrations. This HPLC-MS/MS method was successfully applied for a pharmacokinetic study of MRTX849 at a dose of 15 mg kg-1.

NOVEL STABILITY INDICATING LC-MS/MS METHOD FOR THE SIMULTANEOUS ESTIMATION OF REMOGLIFLOZIN ETABONATE AND VILDAGLIPTIN HUMAN PLASMA

The present study intended to develop an easy and novel Liquid Chromatography–Mass Spectrometry/Mass spectrometry (LC–MS/MS) method for simultaneous assay of remogliflozin etabonate and vildagliptin, a combined formulation used in treatment of type II diabetes in human plasma. Alogliptin drug was selected as internal standard and the analytes were isolated from the spiked plasma matrix using liquid-liquid extraction procedure and the extracts were chromatographed on Inertsil ODS (4.6 mm×100 mm, 5 μm) C18 column. The mobile phase comprises of methanol, acetonitrile and 0.1 % formic acid in 40:50:10 (v/v) at 0.5 mL/min flow rate and analysis was completed within 6 min run time. The method produces peaks with acceptable symmetry and resolution with acceptable system suitability at 2.6 min for remogliflozin etabonate, 2.7 min for vildagliptin, 1.2 min for alogliptin (internal standard). Multiple Reaction Monitoring (MRM) mode was used for the mass spectral characterization of column eluents using mass detector. In the mass spectral studies confirms the characteristic fragment ion transitions at m/z of 523 to m/z of 247 as MH+ ion for remogliflozin, m/z of 304 to m/z of 180 as MH+ ion for vildagliptin. The method can detect both the analyte up to 1.5 ng/mL and having lower limit of quantification (LLOQ) of 5 ng/mL. The method having broad calibration range of LLOQ to 300 ng/mL and was validated for precision, accuracy, stability studies such as freeze-thaw, short term and long-term stability in LLOQ, MQC (medium quality control concentration) and HQC (high quality control concentration) levels and produce acceptable results. Hence it can be confirmed that the method can be adopted for assay of remogliflozin etabonate and vildagliptin simultaneously in plasma samples.

BIO-ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF DECITABINE AND CEDAZURIDINE IN HUMAN PLASMA USING LC-MS/MS

Objective: The present work aimed to develop a novel, reliable and accurate Liquid Chromatography-Mass Spectrometry/Mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Decitabine and Cedazuridine a combined medication used for the treatment of chronic myelomonocytic leukemia in human plasma. Methods: Talazoparib drug is used as an internal standard in the study. Both the analytes and internal standard were isolated from 100 ml plasma samples by liquid-liquid extraction and then chromatographed on Zorbax SB-CN (4.6 mm×75 mm, 3.5 µm) column with a mobile phase consisting of 0.1 % ammonium formate and methanol in the ratio of 65:45 (v/v) pumped at 0.5 ml/min. The method had a chromatographic total run time of 5 min. Results: The developed method gave a symmetric peak at a retention time of 1.7 min for Decitabine, 2.2 min for Cedazuridine, 3.5 min for Talazoparib and satisfied all the peak properties as per USP guidelines. The mass spectral characterization of separated analytes in the LC method was performed using a mass detector operated at Multiple Reaction Monitoring mode with precursor-to-product ion transitions at m/z of 229 to m/z of 114 as MH+ion for Decitabine, m/z of 269 to m/z of 118 as MH+ion for Cedazuridine. A very sensitive limit of detection of 0.3 ng/ml was observed and showed a calibration curve linear over the concentration range of LLOQ (lower limit of quantification) to 500 ng/ml. The other validation parameters were found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction concentration was acceptable and very high for both the analytes in HQC (high-quality control concentration), MQC (medium quality control concentration) and LLOQ levels. The peak area response ratio of Decitabine and Cedazuridine with the internal standard in freeze-thaw, short term and long term stability studies was found to be acceptable confirms that the method is stable. Conclusion: It can be concluded that the proposed method is specific, accurate, and precise and could be used for the simultaneous estimation of Decitabine and Cedazuridine in human plasma.

Determination of Mertansine in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry and Pharmacokinetics of Mertansine in Rats

Mertansine, a thiol-containing maytansinoid, is a tubulin inhibitor used as the cytotoxic component of antibody-drug conjugates for the treatment of cancer. Liquid chromatography-tandem mass spectrometry was described for the determination of mertansine in rat plasma. 50-μL rat plasma sample was pretreated with 25 μL of 20 mM tris-(2-carboxyethyl)-phosphine, a reducing reagent, and further vortex-mixing with 50 μL of 50 mM N-ethylmaleimide for 3 min resulted in the alkylation of thiol group in mertansine. Alkylation reaction was stopped by addition of 100 μL of sildenafil in acetonitrile (200 ng/mL), and following centrifugation, aliquot of the supernatant was analyzed by the selected reaction monitoring mode. The standard curve was linear over the range of 1-1000 ng/mL in rat plasma with the lower limit of quantification level at 1 ng/mL. The intra- and inter-day accuracies and coefficient variations for mertansine at four quality control concentrations were 96.7-113.1% and 2.6- 15.0%, respectively. Using this method, the pharmacokinetics of mertansine were evaluated after intravenous administration of mertansine at doses of 0.2, 0.5, and 1 mg/kg to female Sprague Dawley rats.

Sample Multiplexing: Increased Throughput for Quantification of Total Testosterone in Serum by Liquid Chromatography-Tandem Mass Spectrometry.

For high-volume assays, optimizing throughput reduces test cost and turn-around time. One approach for liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays is sample multiplexing, wherein the analyte of interest is derivatized in different specimens with reagents of different molecular weight (differential mass tagging). Specimens can then be combined and simultaneously analyzed within a single injection to improve throughput. Here we developed and validated a quantitative, sample-multiplexed LC-MS/MS assay for serum total testosterone (TT) based on this approach. For the sample-multiplexed assay, calibrators, controls, and patient specimens were first extracted separately. After mass tagging with either methoxyamine or hydroxylamine, they were combined and injected into the LC-MS/MS system. To evaluate assay performance, we determined limit of quantification (LOQ), linearity, recovery, and imprecision. A method-comparison study was also performed, comparing the new assay with the standard LC-MS/MS assay in 1574 patient specimens. The method was linear from 2.5 to 2000 ng/dL, with accuracies from 93% to 104% for both derivatives. An LOQ of 1.0 ng/dL was achieved. Intra-assay and total CVs across 4 quality control concentrations were less than 10%. The assay demonstrated good agreement (Deming regression, 1.03x + 6.07) with the standard LC-MS/MS assay for the patient specimens tested (TT, 3 to 4862 ng/dL). Sample multiplexing by differential mass tagging of TT increases LC-MS/MS throughput 2-fold without compromising analytical accuracy and sensitivity.

A sensitive method for analyzing fluconazole in extremely small volumes of neonatal serum

BackgroundThe need for a large volume of serum sample significantly reduces the feasibility of neonatal pharmacokinetic studies in daily practice, which must often rely on scavenged or opportunistic sampling. This problem is most apparent in preterm newborns, where ethical and practical considerations prohibit the collection of large sample volumes. Most of the fluconazole analysis assays published thus far required a minimum serum sample of 50 to 100 μL for a single assay. The purpose of the present study was to develop and validate a sensitive method requiring a smaller sample volume (10 μL) to satisfy clinically relevant research requirements.MethodsFollowing simple protein precipitation and centrifugation, the filtrated supernatant was injected into a liquid chromatography system and separated with a C18 reverse-phase column. Fluconazole and the internal standard (IS, fluconazole-d4) were detected and quantified using tandem mass spectrometry. The method was validated with reference to the Food and Drug Administration’s Guidance for Industry. Accuracy and precision were evaluated at six quality control concentration levels (ranging from 0.01 to 100 μg/mL).ResultsInvestigated calibration curves were linear in the 0.01–100 μg/mL range. Intra- and inter-day accuracy (− 7.7 to 7.4%) and precision (0.3 to 6.0%) were below 15%. The calculated limit of detection and the lower limit of quantification (LLOQ) was 0.0019 μg/mL and 0.0031 μg/mL, respectively. Fluconazole in the prepared samples was stable for at least 4 months at − 20 °C and − 80 °C. This method was applied to analyze 234 serum samples from ten neonates who received fosfluconazole, a water-soluble phosphate prodrug of fluconazole which converts to fluconazole in the body, as part of a pharmacokinetic study using daily scavenged laboratory samples. The median (range) concentration up to 72 h after fosfluconazole administration was 2.9 (0.02 to 26.8 μg/mL) μg/mL, which was within the range of the calibration curve.ConclusionFluconazole was able to be detected in an extremely small volume (10 μL) of serum from neonates receiving fosfluconazole. The method presented here can be used to quantify fluconazole concentrations for pharmacokinetic studies of the neonatal population by using scavenged samples.