Background: Linezolid (LZD) and voriconazole (VCZ) are commonly used antibiotics in critically ill patients. However, the complex clinical environment often leads to changes in the pharmacokinetic and pharmacodynamic parameters of these patients.

Aims: To develop a simple, rapid, and cost - effective method for the simultaneous determination of LZD, VCZ, and voriconazole N - oxide (VNO), the primary metabolite of VCZ in humans, and to apply it to routine therapeutic drug monitoring (TDM).

Methods: LZD, VCZ, VNO, and the internal standard (triazolam) were extracted from plasma using acetonitrile - precipitated protein. Chromatographic separation was completed within 4 minutes on an Agilent ZORBAX RRHD Eclipse XDB - C18 (2.1 mm × 100 mm, 1.8 μm) column with a mobile phase of methanol and water (containing 0.1% formic acid) by ultra - performance liquid chromatography - tandem mass spectrometry (UPLC - MS/MS).

Results: Good linearity was observed for LZD in the 0.1 - 20 μg/mL range and VCZ and VNO in the 0.05 - 10 μg/mL range. The intra - and inter - day precision of QC samples were below 6.16% and 9.24% respectively, and the accuracy ranged from - 11.77% to 10.17%. The method was successfully applied to 163 patients for routine TDM.

Conclusions: The developed UPLC - MS/MS method is suitable for the simultaneous determination of LZD, VCZ, and VNO in human plasma and can be effectively used for routine TDM in clinical practice.

Key Words: Linezolid, Voriconazole, Voriconazole N - oxide, Therapeutic drug monitoring, UPLC - MS/MS

Background: LM1010, a high-performance liquid chromatography system designed for therapeutic drug monitoring (TDM) of various drugs in clinical practice, is currently approved in Japan. LM1010 enables rapid measurement of drugs in serum specimens and performs fully automated data analysis based on the absolute calibration curve method. We evaluated the analytical performance of the LM1010 for determining serum concentrations of linezolid (LZD).

Aims: The present study aimed to evaluate the analytical performance of LM1010 and investigate the long-term stability of LZD in serum specimens stored at −30°C.

Methods: A total of 148 serum specimens were collected from 25 patients (17 males and 8 females) who were treated with LZD. Serum concentrations of LZD were measured using LM1010 and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The measured values were also compared among three LM1010 instruments at different institutions to validate the analytical performance.

Results: A Passing–Bablok regression analysis demonstrated a strong correlation in the measured values between LM1010 and LC-MS/MS (r = 0.991), and comparable results were observed among the three LM1010 instruments (r = 0.982–0.998). Bland–Altman analyses revealed that the difference in the average between LM1010 and LC-MS/MS methods was 1.7%, and similar results were observed among the three LM1010 instruments. The long-term stability of serum LZD was also confirmed in LZD-spiked serum specimens stored at −30°C for 48 weeks.

Conclusions: The present results confirm that LM1010 can be used for TDM of LZD in serum specimens even in those stored at −30°C.

Key Words: linezolid, therapeutic drug monitoring, high-performance liquid chromatography

Background: Antipsychotic drugs (APDs) are frequently used in combination with other drugs, leading to potential drug interactions.

Aim: Here we present a simple, cost-effective and robust protein precipitation and LC-MS/MS method for the simultaneous analysis of 35 APDs in human plasma for clinical research.

Methods: In brief, to 50µL of plasma, 100µL of protein precipitation solvent containing internal standards was added. The samples were mixed, centrifuged for five minutes and 100µL of the supernatant was diluted with water. Samples were injected onto a Waters XSelect™ HSS C18 SB XP Column using an ACQUITY™ UPLC™ I-Class PLUS FL System and eluted using a water/methanol/formic acid/ ammonium acetate gradient. APDs were quantified using a Xevo™ TQ-S micro Mass Spectrometer.

Results: The method was demonstrated to be linear across calibration range for all analytes, with analytical sensitivity investigations showing precise quantification (<20% CV, ≤15% bias). Non-linearity was less than 10% across all the analytes. Coefficients of variation (CV) for total precision and repeatability of QCs were ≤8.7% CV and ≤7.4%, respectively. Recoveries for all analytes were within ±15%. Mean extraction efficiencies for the 35 APDs ranged from 52.2%-96.9%.

Conclusions: A fast and inexpensive LC-MS/MS clinical research method was developed for 35 APDs, based on protein precipitation and requiring only 50µL of plasma. The method demonstrated acceptable analytical sensitivity, recovery, linearity and precision for all analytes.

For Research Use Only. Not for Use in Diagnostic Procedures.

Waters, ACQUITY, UPLC, Xevo, and XSelect are trademarks of Waters Technologies Corporation.

Key words: LC-MS/MS, Anti-psychotics, robust

Background: LC-MS/MS is the gold standard for quantifying small molecules. Categorizing drugs based on their log D for assay development in LC-MS/MS may facilitate development of combined drug assays.

Aims: To combine drugs in a single analytical run based on lipophilicity, therapeutic range and molecular weight.

Methods: This approach compares log D values of TDM drugs, categorizing them into low and high log D groups. From a list of TDM drugs, voriconazole, isavuconazole, posaconazole, itraconazole, hydroxy itraconazole, carbamazepine, amiodarone and warfarin were selected for combining into a single assay based on their comparable molecular weights and high log D values. A calibration range covering the therapeutic range of all drugs was selected. A protein precipitation method using non polar solvent MTBE to extract these drugs from 50 µl of plasma was developed with minimal use of deuterated isotopes as internal standards.

Results: Validation followed ICH M10 guidelines ensuring accuracy, precision, reproducibility, matrix effect, and stability across clinical concentration ranges (0.5 to 10 µg/mL). Reverse phase chromatography using CORTECS C8 column and a water/methanol gradient elution containing 0.2% formic acid separated all eight compounds within a 3-minute run time.

Conclusions: To our knowledge this is the first time log D concept was used to combine drug assays. We could successfully combine 8 drugs taking into consideration their log D, molecular weight and therapeutic range for TDM. This approach can reduce analytical run time, turnaround time, and cost, enabling more frequent TDM reporting.

Key Words: TDM, assay development, lipophilicity, log D

Background:Simplifications in LCMS instrumentation have made MS a viable option for clinical research. The ability to support various analysis methods on a single system is a key feature. However, the automation of sample preparation, automatic transfer to LCMS, and standardization of analytical conditions have been considered challenging.

Aims:The purpose is the development and the validation of a unified methods set for LC/MS/MS, for 24/7 therapeutic drug analysis, with a fully automated platform.

Methods:The evaluated analytical system was a fully automated platform, CLAM-LC/MS/MS (Shimadzu, Japan). HL-7 interface standards were used for bidirectional communication between LIS (Dedalus, Germany) and the platform. Target applications were Antibiotics, DOAC, Antiepileptics, Neuroleptics, Tricyclic Antidepressants, Other Antidepressants, Benzodiazepines, Antifungals, and several other drugs. All methods use similar analytical conditions. The fitness for purpose for 24/7 use was evaluated by requesting measurements for several methods in random alternance.

Results:Each method was evaluated based on the following validation items and met the criteria: isobars resolution, accuracy, repeatability, intermediate precision, mobile phase stability, LLOQ confirmation, carryover and ring trial analysis. Also, repeated measurements for several methods in random alternance showed results within the acceptance criteria.

Conclusion:This strategy proved its fitness for purpose. The fast LCMS methods which can alternate smoothly, and the automated sample extraction enable robust therapeutic drug analysis with a high throughput and at low cost, without compromising the user comfort. For research use only.

Key words:24/7, automated, TDM, analysis, RUO, LCMS

Background: Benzodiazepines are important forensic toxicology drugs which are prescribed for neurological and psychiatric disorders but also have a high potential for abuse.

Aims: The aim was to develop a forensic toxicology research method for confirmatory analysis of 26 traditional and designer benzodiazepines via UHPLC-MS/MS.

Methods: Urine samples (100µL) were added to a Waters Oasis™ MCX µElution™ Plate followed by 100 µL of a solution containing recombinant beta-glucuronidase enzyme, hydrolysis buffer and internal standards. The sample was loaded, washed and then eluted using 50:50 acetonitrile:methanol containing 5% ammonia. Samples were diluted with 2% acetonitrile: 1% formic acid prior to analysis.
Samples were injected onto an ACQUITY™ UPLC™ BEH™ C18 Column using an ACQUITY UPLC I-Class PLUS System and eluted using a water/formic acid/acetonitrile gradient with detection on a Xevo™ TQ-S micro Mass Spectrometer.

Results: The panel of benzodiazepines were well separated, with baseline resolution for most compounds. The extraction recovery was within 80-120% for the majority of compounds. The matrix effects were mostly minimized to ±20% after internal standard correction. Calibration curves were linear with r2≥0.99 and coefficients of variation were less than 15% for all analytes. The data for proficiency samples showed excellent agreement to existing research methods.

Conclusion: Leveraging SPE sample preparation and UHPLC-MS/MS with the Xevo TQ-S micro Mass Spectrometer, a rapid, accurate and precise method was demonstrated for analyzing benzodiazepines in urine.

For Forensic Toxicology Use Only

Waters, Oasis, µElution, ACQUITY, UPLC, Xevo, and BEH are trademarks of Waters Technologies Corporation.

Keywords: Benzodiazepines, Urine, LC-MS/MS

Background: 2-Benzylbenzimidazole ‘nitazene’ opioids pose a growing threat to public health, and are increasingly mixed with or (mis)sold as heroin and present in falsified (non-)opioid medications. Lateral flow immunoassay nitazene test strips (NTS; BTNX Rapid ResponseTM) became commercially available in Q1-2024, aiming to enable rapid detection of nitazene analogues in drug samples.

Aims: This lab-based study aimed at evaluating the potential of NTS for drug checking applications.

Methods: Following dilution of drug standards in water, the NTS readouts were analyzed independently by two individuals and by ImageJ. The limit of detection for isotonitazene was determined using two manufacturing lots of NTS. Cross-reactivity with 32 additional nitazene analogues was evaluated. Six sourced drug samples were tested to explore the ability of NTS to detect the presence of a nitazene analogue in authentic samples.

Results: The limits of detection for isotonitazene were 2000 or 3000 ng/mL, depending on the lot. 24/33 nitazene analogues cross-reacted with the NTS at concentrations ≤ 9000 ng/mL. Structural analysis indicated that either substitution or removal of the 5-nitro group, or lengthening the linker between the two aromatic rings, generally hampered detection. All six authentic drug samples consistently tested positive.

Conclusions: Our findings indicate that NTS can theoretically alert to the presence of most nitazene analogues that have emerged on recreational drug markets. However, in practice, factors like specificity, lot-to-lot variability, nitazene content in drug samples, solubility, and different testing conditions should be considered.

Keywords: New synthetic opioids, Nitazenes, Harm reduction, Drug checking, Immunoassay test strips

Background: Conventional assays measure total valproic acid (VPA) concentrations to guide epilepsy therapy, but only unbound VPA is pharmacologically active. Free VPA concentrations correlate better with efficacy and toxicity, especially since VPA is highly protein-bound (90–95%). Hypoalbuminemia or drug-drug interactions can elevate unbound VPA levels despite normal or low total VPA levels, highlighting the need for free VPA measurement.

Aims: Determining ultrafiltration variables and setting up an optimized protocol to measure unbound VPA concentrations.

Methods: Since no IVDR-compliant method exists for free VPA concentrations, an in-house ultrafiltration protocol was developed at Ghent University Hospital using Centrifree devices (500µL serum, incubation and centrifugation at 37°C, 1990g, 30 minutes). Ultrafiltrate and total VPA concentrations were analyzed using Chemiluminescent Microparticle ImmunoAssay (Abbott Architect i2000SR). Validation included assessing matrix effects across a broad VPA range (±4–125mg/L) and evaluating filter device type, temperature, serum volume, centrifugation time, and buffer (pH 7.5).

Results: A ~15% matrix effect was observed, with higher recoveries in ultrafiltrate versus serum. Key centrifugation variables affecting results included: filter membrane type (10kD vs. 30kD), temperature (concentrations 37°C > 22°C), serum volume (400µL vs. 500µL), centrifugation time, and phosphate buffer use. Vivacon® filters yielded different results than Centrifree, underlining the importance of consistent device selection.

Conclusions: Free VPA measurements are influenced by multiple variables, necessitating standardized ultrafiltration protocols. Variability in filter devices and processing conditions emphasize the importance of robust guidelines and external quality controls to improve accuracy and comparability.

Key words: Hypoalbuminemia, drug-drug interactions, ultrafiltration, unbound concentration, valproic acid

Background:
Successful biomarker quantitation in complex matrices requires sensitivity and specificity. The Stellar™ mass spectrometer (MS) is a hybrid quadrupole, dual-pressure linear ion trap MS that offers alternative fragmentation mechanisms and/or MS3 fragmentation as an efficient solution to reduce background interferences and provide greater specificity.

Aims:
To demonstrate the advantages of utilizing multiple collision activations and MS3 provided by Stellar MS to the selective and sensitive quantification of 18 steroids in serum.

Method:
Commercially available 18 steroids standards and their corresponding internal standards were serially diluted in blank human serum to generate calibrator samples over 4-orders of magnitude concentration range. Quality control human serum samples were purchased from Chromsystem. Steroids were extracted with protein precipitation for the LCMS analysis. Multiple fragmentation schemes were evaluated and optimized on the Stellar MS, and the data was analyzed in TraceFinder™ software for the quantitative performance.

Results:
The detection of 18 steroids were optimized in Stellar MS under multiple fragmentation mechanisms (beam-type CID, resonance-type CID, and MS3 in different combinations) with polarity switching. Excellent analytical performance of the calibration curves were achieved for all steroids. Comparing to the MS2 beam-type CID commonly used in contemporary triple quadruple MS, steroids such as cortisone, estradiol, and dihydrotestosterone benefited from the alternative, resonance-type CID and MS3 acquisition modes, resulting in a 2-10 fold increase of the LOQ values.

Conclusions:
Stellar MS provides unique selectivity allied to fast acquisition for high-throughput and sensitive biomarker quantitation optimal for routine analysis

Key words:
Alternative fragmentation mechanism, Stellar MS, steroids, serum

Background: The internal standard (IS) is key in LC-MS/MS for drug analysis. There is no commercially available isotope-labeled internal standards for surufatinib due to the challenges of chemical synthesis and high cost. Thus, it is very important to select an appropriate IS for the accurate quantitative detection of surufatinib.

Aims: The optimization of various parameters was conducted to obtain the optimal IS of surufatinib.

Methods: The 15 IS candidates include 10 TKIs and 5 other drugs. Parameter optimization consists of signal crossing in the MS ion channels, the influence of IS concentration, and the influence of the matrix effect. The resolution (R), correlation coefficient (r²), and matrix effect were used as evaluation indexes.

Results: In the ion signal channel of surufatinib, only imatinib showed a weak response. The r² values could reach 0.990, except for gefitinib. Zanbrutinib, ritonavir, nirmatrelvir and gabapentin were most significantly inhibited in hemolytic plasma. The IS with the least R is PRA (0.08). Regorafenib exhibited high variability (CV% > 30%) in different standard curves. Only osimertinib, crizotinib, praseltinib and olaparib fell within the acceptable ranges of 15% in QC. In short, the praseltinib was the most optimal choice as IS for surufatinib.

Conclusions: The praseltinib was selected as the best IS for surufatinib, as it exhibited smaller R and As, better stability and linearity, and less affected by the matrix.

Key Words: Surufatinib, Internal standard, Selection