Simultaneous monitoring of seven antiepileptic drugs by dried blood spot and dried plasma spot sampling: method validation and clinical application of a LC-MS/MS-based technique.

Alternative blood sampling strategy can enhance the application of therapeutic drug monitoring (TDM), then improve precision therapy and medication compliance. In developing nations, alternative sampling strategy that allows self-sampling and room temperature transport is especially important. This study validates the use of dried blood spot (DBS) and dried plasma spot (DPS) sampling along with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analyzing seven common antiepileptic drugs (AEDs) (phenytoin, lamotrigine, levetiracetam, topiramate, carbamazepine, oxcarbazepine and its active metabolite 10,11-dihydro-10-hydroxy carbamazepine) and evaluates their applicability to clinical practice. Following simple protein precipitation with acetonitrile, the AEDs were separated on a C18 column by gradient elution with a mobile phase consisting of acetonitrile-water-0.1% formic acid at a flow rate of 0.65 mL/min. The method provided linear analysis over the tested concentration ranges, with a total run time of 7 min. Intra- and inter-assay precision for all quality controls were ≤12% with accuracies of 85.9%-113%. The average extraction efficiencies were 69.0%-92.4% for DBS and 65.9%-96.5% for DPS, and no significant matrix effects were observed. The AEDs were stable in all samples for seven days at room temprature and 40°C. There was good correlation between the dry and wet plasma concentrations with greater accuracy for DPS compared to DBS indicating that alternative sampling strategy using DBS and DPS are suitable for monitoring the concentrations of AEDs with satisfied performance and logistical advantages.

A deep-learning pipeline to diagnose pediatric intussusception and assess severity during ultrasound scanning: a multicenter retrospective-prospective study.

Ileocolic intussusception is one of the common acute abdomens in children and is first diagnosed urgently using ultrasound. Manual diagnosis requires extensive experience and skill, and identifying surgical indications in assessing the disease severity is more challenging. We aimed to develop a real-time lesion visualization deep-learning pipeline to solve this problem. This multicenter retrospective-prospective study used 14,085 images in 8736 consecutive patients (median age, eight months) with ileocolic intussusception who underwent ultrasound at six hospitals to train, validate, and test the deep-learning pipeline. Subsequently, the algorithm was validated in an internal image test set and an external video dataset. Furthermore, the performances of junior, intermediate, senior, and junior sonographers with AI-assistance were prospectively compared in 242 volunteers using the DeLong test. This tool recognized 1,086 images with three ileocolic intussusception signs with an average of the area under the receiver operating characteristic curve (average-AUC) of 0.972. It diagnosed 184 patients with no intussusception, nonsurgical intussusception, and surgical intussusception in 184 ultrasound videos with an average-AUC of 0.956. In the prospective pilot study using 242 volunteers, junior sonographers' performances were significantly improved with AI-assistance (average-AUC: 0.966 vs. 0.857, P < 0.001; median scanning-time: 9.46 min vs. 3.66 min, P < 0.001), which were comparable to those of senior sonographers (average-AUC: 0.966 vs. 0.973, P = 0.600). Thus, here, we report that the deep-learning pipeline that guides lesions in real-time and is interpretable during ultrasound scanning could assist sonographers in improving the accuracy and efficiency of diagnosing intussusception and identifying surgical indications.

Comparison of LC-MS3 and LC-MRM Methods for Quantifying Amantadine and Its Application in Therapeutic Amantadine Monitoring in Human Plasma.

A simple sample preprocessing method was developed for the quantitative determination of amantadine (AMT) in human plasma by liquid chromatography-tandem mass spectrometry cubed (LC-MS3). The LC-MS3 system comprised a Shimadzu Exion LC-20AD HPLC pump coupled with a QTRAP 5500 mass spectrometer. First, the plasma samples were pretreated using acetonitrile as the extracting solution to precipitate protein. Next, amantadine and amantadine-d15 (AMT-d15) were separated on an Agilent Poroshell 120 SB-C18 column (4.6 mm × 50 mm, 2.7 µm) using isocratic elution with solvent A (70% 0.1% formic acid) and solvent B (30% acetonitrile) at a flow rate of 0.8 mL/min. The total run time for each sample was 3 min. The system used triple-stage fragmentation transitions at m/z 152.2→135.3→107.4 for AMT quantification in the positive ion mode and m/z 167.0→150.3→118.1 for AMT-d15 quantification. The LC-MS3 assay was linear (r > 0.995) with a concentration range of 50−1500 ng/mL. The lower limit of quantification (LLOQ) was 50 ng/mL, and the intra-day and inter-day accuracies and precisions were less than 8.0% at all concentrations. In addition, the recoveries and matrix effect for AMT in human plasma were within acceptable limits. In terms of stability, AMT had no significant degradation under all conditions. All the results met the requirements of the guidelines of the Food and Drug Administration (FDA) for biological method validation. The novelty of the MS3 assay was that it presented a methodology with higher selectivity and sensitivity. This method was successfully applied to 44 human plasma samples, and the obtained quantitative results were compared with another liquid chromatography-multiple reaction monitoring (LC-MRM) method. The Passing-Bablok regression coefficients and Bland-Altman plot revealed no difference between the LC-MS3 and LC-MRM methods, implying that the developed LC-MS3 method is a reliable and accurate assay for AMT determination in human plasma. These results are also a proof of concept for determining chemicals in biological samples by the LC-MS3 strategy.

Dried Plasma Spot Based LC-MS/MS Method for Monitoring of Meropenem in the Blood of Treated Patients.

Meropenem (MER) is widely used to treat complicated and serious infections. Therapeutic drug monitoring (TDM) provides a valid clinical tool to avoid suboptimal concentrations and dose−related adverse reactions. However, TDM seems to face challenges since the limited stability of MER in plasma makes transport difficult between clinics and laboratories. Dried plasma spot (DPS) sampling is an attractive but underutilized method for TDM that has the desired features of easy collection, storage, and transport, and overcomes known hematocrit (HCT) issues in dried blood spot (DBS) analysis. This study was designed to investigate a DPS−based liquid chromatography−tandem mass spectrometry (LC−MS/MS) method for quantification of MER. The method was developed and validated for DPS and wet plasma samples. Calibration curves were linear (R2 > 0.995) over the concentration range of 0.5−50 µg/mL. Overall accuracy and precision did not exceed 15% and no significant matrix effect was observed. MER has been more stable in DPS than in wet plasma samples. A comparison of DPS and wet plasma concentrations was assessed in 32 patients treated with MER. The results showed that there was no significant difference between the two methods. So the DPS method developed in this study is appropriate and practical for the monitor of MER in the daily clinical laboratory practice.

A sensitive and selective HPLC-MS3 method for therapeutic drug monitoring of meropenem and its validation by comparison with HPLC-MS2 methods.

Meropenem, a representative ß-lactam antibiotic, is widely used to treat complicated and serious infections. Therefore, it is of great significance to monitor the plasma drug concentration for individualized antimicrobial therapy. This study first describes the development and validation of high-performance liquid chromatography-tandem mass spectrometry cubed method for monitoring meropenem in human plasma. Protein precipitation with methanol and a chromatographic analysis time of 7 min make this method simple and of high throughput. Meropenem was extracted from human plasma with recoveries >94.1%. Calibration curves were linear (R2  > 0.995) in the concentration range of 0.5-50 µg/mL. Overall accuracy and precision did not exceed 8.0% as well as no significant matrix effect was observed. The novelty of this method is that the triple-stage mass spectrometry technology improves the selectivity and sensitivity. A comparison of the presented method and traditional liquid chromatography-tandem mass spectrometry method was assessed in 44 patients treated with meropenem and Passing-Bablok regression coefficients and Bland-Altman plots showed that no significant difference between the two methods. So the triple-stage mass spectrometry method developed in this study is appropriate and practical for the monitor of meropenem in the daily clinical laboratory practice.

LC-MS3 Strategy for Quantification of Carbamazepine in Human Plasma and Its Application in Therapeutic Drug Monitoring.

This study developed a detection method based on the strategy of HPLC/MS3 and verified its suitability by quantifying carbamazepine in human plasma. The high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS3) system was performed using a Shimadzu UFLC XR liquid chromatography and a SCIEX QTRAP® 5500 linear ion trap triple quadrupole mass spectrometer. The specific operation was as follows: the sample protein was firstly precipitated using methanol, then carbamazepine and carbamazepine-D2N15 were separated on an ACQUITY UPLC HSS T3 column using the gradient elution with solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.25 mL/min. Each sample was run for 7 min. This method was validated for various parameters including accuracy, precision, selectivity, linearity, LLOQ, etc. Only 5 µL of sample plasma could obtain the result of LLOD 0.5 µg/mL. The intra-day and inter-day precision was <8.23%, and accuracy was between -1.74% and 2.92%. This method was successfully used for monitoring the blood concentration of epilepsy patients after carbamazepine treatment.

Dried plasma spot-based liquid chromatography-tandem mass spectrometry for the quantification of methotrexate in human plasma and its application in therapeutic drug monitoring.

Methotrexate, a folic acid antitumor drug, is widely used to treat childhood acute lymphoblastic leukemia. Therapeutic drug monitoring is crucial for adjusting the dosage of methotrexate according to its plasma concentration and reducing adverse effects. Micro-sampling strategies, like dried plasma spot, is an attractive but underutilized method that has the desired features of easy collection, storage, and transport, and overcomes known hematocrit issues in dried blood spot analysis. This study describes a dried plasma spot-based liquid chromatography-tandem mass spectrometry method for quantification of methotrexate. The assay showed good linearity over 30-2000 ng/mL (R2 ≥ 0.995) as well as excellent precision (0.6-9.3%) and accuracy (89.2-108.3%). Methotrexate was extracted from dried plasma spot and wet plasma samples with recoveries greater than 92.1%, and no significant matrix effect was observed. A comparison of dried plasma spot and wet plasma concentrations was assessed in 27 patients treated with methotrexate and Passing-Bablok regression coefficients showed that no significant difference between the two methods. The Bland-Altman plots showed similar agreement between the methods, indicating that the proposed dried plasma spot sampling method is an effective way to monitor the concentration of methotrexate in human plasma.

Comparison of LC-MS3 and LC-MRM strategy for quantification of methotrexate in human plasma and its application in therapeutic drug monitoring.

A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry cubed (LC/MS3) method was developed and validated for quantification of methotrexate in human plasma. The MS3 detection is a scanning mode of QTrap MS systems or ion trap MS systems. After simple protein precipitation with methanol, methotrexate and methotrexate-d3 were separated on an Agilent Poroshell 120 SB-C18 column (4.6 × 50 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 60% 0.1% formic acid in water and 40% 0.1% formic acid in acetonitrile. The flow rate is 0.8 mL/min. MS3 detection in positive ion mode used the MRM3 transitions at m/z 455.2→308.2→175.1 for quantification of methotrexate and m/z 458.2→311.2→175.1 for quantification of methotrexate-d3. The total run time was only 3 min for each sample. The LC/MS3 assay was linear in the concentration range 10-3000 ng/mL(R2 ≥ 0.995) and the intra- and inter-day accuracies were< 3.72% and precisions were< 7.78% at all concentrations. The absolute recoveries (%) and matrix effect (%) for methotrexate in human plasma were between 92.6 and 114.3. The novelty of the presented methodology is the MS3 technique resulting in enhanced selectivity and sensitivity. The application of this LC-MS3 method was successfully completed on 46 human plasma samples and the quantitative results of identical human plasma samples were compared with another LC-MRM based method. Passing-Bablok regression coefficients demonstrated that there is no significant difference between the LC-MS3 method and LC-MRM method. Bland-Altman plots showed a concordant results, supporting the developed LC-MS3 method is a reliable and accurate assay for determination of methotrexate in human plasma. This work is also a proof of concept for using LC-MS3 technique to determination of chemicals in biological samples.

Development and application of sequential window acquisition of all theoretical mass spectra data acquisition modes on ultra-high-performance liquid chromatography triple-quadrupole/time-of-flight mass spectrometry for metabolic profiling of amino acids in human plasma.

Accumulating evidence suggests that amino acids are important indicators of nutritional and metabolic status. A high-resolution mass spectrometry method based on sequential window acquisition of all theoretical mass spectra acquisition was developed for the simultaneous determination of 16 amino acids in human plasma. Sample preparation by protein precipitation using a mixture of acetonitrile and formic acid was followed by a BEH Amide column. The superiority of this method was investigated by comparing it to time-of-flight scan and multiple reaction monitoring modes. The limit of detection in sequential window acquisition of all theoretical mass spectra mode for threonine, methionine, histidine, citrulline, and tryptophan is 0.1 ng on the column; for lysine and asparagine is 0.2 ng; for alanine, pyroglutamic acid, leucine, ornithine, and aspartate is 0.5 ng, for arginine is 1.0 ng; for glutamate and serine is 2.0 ng; for glutamine is 10.0 ng. This method was linear in the range 0.8-40 µg/mL for arginine, citrulline, glutamate, histidine, leucine, methionine, pyroglutamic acid, threonine, tryptophan; 2-100 µg/mL for asparagine, aspartate, lysine, ornithine, serine; and 4-200 µg/mL for alanine, glutamine with good accuracy and precision. Significantly different levels in 11 amino acids were observed between childhood and adulthood, representing the growth and development of individuals relating to the level of amino acids.

Therapeutic drug monitoring of valproic acid using a dried plasma spot sampling device.

Valproic acid (VPA) dosing needs to be individualized for epilepsy patients through therapeutic drug monitoring (TDM). The patients must show up in the clinic at the therapeutic window time to venipuncture sample. Dried plasma spot (DPS) sampling is an alternative way to replace conventional venipuncture sampling. The aim of this study was to develop and validate a DPS-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to monitor VPA in a routine clinical laboratory setting. We compare the DPS with the wet plasma method of clinical samples by LC-MS/MS. The method was linear over the dynamic range of 10-200 µg/ml (covering entire therapeutic range) with a correlation coefficient r2 ≥ 0.995. Both the DPS and wet plasma methods were fully validated for the accuracy, precision, recovery, and matrix effect. The analyte stability was examined under conditions mimicking the sample storage, transport, and analysis procedures. A clinical study with epilepsy patients receiving VPA (n = 35) showed that, after correction for hematocrit (HCT), plasma concentrations can be successfully calculated from the DPS quantification results. Passing-Bablok regression coefficients showed no proportional bias between estimated and measured plasma concentrations. Similar agreement was found by Bland-Altman plots. The dried sample could be mailed to the clinical lab to test by regular mail service. So DPS can be used for drug monitoring with self-sampling strategy at the patient's convenient time and place specially for ambulatory patients not attending a clinic.

Development, validation and comparison of four methods for quantifying endogenous 25OH-D3 in human plasma.

To meet the increasing clinical needs for 25-hydroxyvitamin D3 (25OH-D3) detection, the development of an efficient and accurate high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for plasma 25OH-D3 quantitation is important. Since 25OH-D3 is an endogenous compound, the lack of a plasma blank increases the difficulty of accurately quantifying 25OH-D3. Selection of a method suitable for clinical monitoring among various methods for endogenous compound quantification is necessary. Methyl tert butyl ether was chosen for the sample treatment in a liquid-liquid extraction protocol. Water as a blank matrix, 5% human serum albumin in water as a blank matrix, surrogate analyte and background subtraction were designed to address the problem of a deficiency of a plasma blank. Four liquid chromatography-tandem mass spectrometry methods were fully validated to verify the advantages and limitations owing to regulatory deficiencies for endogenous compound validation. All four methods met the criteria and could be used to monitor clinical samples. Overall 30 human plasma samples were quantified in parallel using the four methods. The difference between any two methods was <12.6% and the total relative standard deviation was <5.2%. Background subtraction and 5% human serum albumin in water as a blank matrix may be better choices considering data quality, matrix similarity, cost and practicality.