Chromatography-tandem Mass Spectrometry Method Research Articles

A study of the pharmacokinetics of clozapine and its metabolites by the dynamics of its distribution in the oral fluid of healthy volunteers.

Clozapine (CLZ) -related accidents or crimes are common in the world. Oral fluid drug detection is a convenient measure of dealing with things like that. There has not been any literature reported detailedly the representation rule of clozapine and its metabolites in oral fluid so far. The study aimed to describe the pharmacokinetics of CLZ and its metabolites N-desmethylclozapine and clozapine-N-oxide in human oral fluid after a single 12.5mg oral dose of CLZ. Twenty-nine volunteers, including 20 males and 9 females, were recruited, and 2mL oral fluid was collected from each participant at post-consumption time-points of prior (zero), 0.5, 1.5, 3, 5, 8, 12, 24, 36, 51, 82, and 130h, respectively. Analytes of interest were extracted with solid-phase extraction and analyzed with liquid chromatography tandem mass spectrometry method. Pharmacokinetic parameters were calculated using the pharmacokinetic software DAS according to the non-compartment model. The maximum concentration, the time of maximum concentration, oral clearance, and the elimination half-life of clozapine were 16.57 ± 9.63ng/mL, 4.53 ± 3.61h, 57.65 ± 23.77 L/h and 53.58 ± 52.28h, respectively. The maximum concentration, the time of maximum concentration, and the elimination half-life of the metabolite N-desmethylclozapine were 3.08 ± 1.19ng/mL, 9.38 ± 9.33h and 62.67 ± 82.57h, respectively; of clozapine-N-oxide were 1.15 ± 0.36ng/mL, 4.53 ± 2.19h and 19.15 ± 23.11h, respectively. It was the first study on the pharmacokinetics of CLZ and its metabolites in the oral fluid of Chinese healthy volunteers, and it provided a basis for the therapeutic drug monitoring and toxicological interpretation in clozapine-related cases.

Development and validation of a novel 7α-hydroxy-4-cholesten-3-one (C4) liquid chromatography tandem mass spectrometry method and its utility to assess pre-analytical stability.

7α-Hydroxy-4-cholesten-3-one (C4) is the common intermediary of both primary bile acids. C4 is recommended by the British Society of Gastroenterology for the investigation of bile acid diarrhoea (BAD) in patients with chronic diarrhoea. This project aimed to develop and validate an assay to quantitate C4 in serum and assess the stability of C4 in unseparated blood. Accuracy was underpinned by calibrating to quantitative nuclear magnetic resonance analysis. C4 was analysed in a 96-well plate format with a deuterated C4 internal standard and liquid-liquid extraction. Validation followed the 2018 Food and Drug Administration guidelines. To assess C4 stability, healthy volunteers (n=12) donated 8 fasted samples each. Samples were incubated at 20 °C for up to 72 h and retrieved, centrifuged, aliquoted and frozen for storage at different time points prior to C4 analysis. The C4 method demonstrated excellent analytical performance and passed all validation criteria. The method was found to be accurate, precise, free from matrix effects and interference. After 72 h of delayed sample separation, C4concentration gradually declined by up to 14 % from baseline. However, the change was not significant for up to 12 h. We present a robust method of analysing serum C4, offering a convenient alternative to 75SeHCAT for BAD investigation. C4 was found to decline in unseparated blood over time; however, after 12 h the mean change was <5 % from baseline. Our results suggest C4 is suitable for collection from both primary and secondary care prior to gastroenterology referral.

Targeted proteomic approach for quantification of collagen type I and type III in formalin-fixed paraffin-embedded tissue

Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC–MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.

Pre-analytical stability and physiological fluctuations affect plasma steroid hormone outcomes: A real-world study

Since steroids are crucial for diagnosing endocrine disorders, the lack of research on factors that affect hormone levels makes interpreting the results difficult. Our study aims to assess the stability of the pre-analytical procedure and the impact of hormonal physiological fluctuations using real-world data. The datasets were created using 12,418 records from individuals whose steroid hormone measurements were taken in our laboratory between September 2019 and March 2024. 22 steroid hormones in plasma by a well-validated liquid chromatography tandem mass spectrometry method were measured. After normalization transformation, outlier removal, and z-score normalization, generalized additive models were constructed to evaluate preanalytic stability and age, sex, and sample time-dependent hormonal fluctuations. Most hormones exhibit significant variability with age, particularly steroid hormone precursors, sex hormones, and certain corticosteroids such as aldosterone. 18-hydroxycortisol, 18-oxocortisol. Sex hormones varied between males and females. Levels of certain hormones, including cortisol, cortisone, 11-deoxycortisol, 18-hydroxycortisol, 18-oxocortisol, corticosterone, aldosterone, estrone, testosterone, dihydrotestosterone, dehydroepiandrosterone sulfate, 11-ketotestosterone, and 11-hydroxytestosterone, fluctuated with sampling time. Moreover, levels of pregnenolone and progesterone decreased within 1 hour of sampling, with pregnenolone becoming unstable with storage time at 4 degrees after centrifugation, while other hormone levels remained relatively stable for a short period of time without or after centrifugation of the sample. This is the first instance real-world data has been used to assess the pre-analytic stability of plasma hormones and to evaluate the impact of physiological factors on steroid hormones.

Disturbed carnitine metabolism is independently correlated with sarcopenia and prognosis in patients on hemodialysis

Background & AimsSarcopenia is frequent in hemodialysis patients and associated with an increased likelihood of adverse outcomes. Early identification of the risk of sarcopenia and effective intervention are of great importance for dialysis patients. However, little research has been carried out on potential biomarkers of sarcopenia in hemodialysis patients. The aim of this study was to investigate whether serum carnitine or acylcarnitine levels are biomarkers of sarcopenia in hemodialysis patients, and whether these are prognostic factors for occurrence of complications. MethodsThis prospective clinical pilot study enrolled patients (n=259) who were treated in the Blood Purification Center from May 2021 to July 2022, all participants were followed-up for 1- year. Serum carnintine and acylcarnitine (AC) were measured using our previously reported targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The correlations between carnitine or acylcarnitine levels with sarcopenia and prognosis in patients were analysed. ResultsThe C0 (Free carnitine, FC) and total carnitine (TC) levels were significantly lower in the sarcopenia group than in the nonsarcopenia group (nonsarcopenia vs. sarcopenia: 20.97 (16.96, 25.83) vs. 17.77 (14.30, 22.78); p =0.002) and (nonsarcopenia vs. sarcopenia: 30.12 (24.76, 36.62) vs. 26.03 (21.30, 32.01); p = 0.003). Besides, significant difference between the groups were noted in low free carnitine (C0 < 20 μmol/L) patients (nonsarcopenia vs. sarcopenia: 72 (42.4%) vs. 56 (62.9%); p = 0.002) and high C2/ C0 ratio (> 0.4) patients (nonsarcopenia vs. sarcopenia: 36 (21.2%) vs. 30 (33.7%); p = 0.028). By multivariable analysis, the disturbed CM defined as C0 deficient and/or C2/C0 carnitine ratio abnormal rise was independently and significantly correlated with the prevalence of sarcopenia after adjusting for some confounding factors, such as age, gender and dialysis duration (P values for trend <0.05).Hemodialysis patients with sarcopenia [OR: 3.214 (1.307,7.904)] and disturbed CM [OR: 3.217 (1.112,9.305)] both had a 3-fold increased risk of falling and fracture after one year follow up. In addition, age and sarcopenia [OR: 2.883 (1.321, 6.289)] were independently and positively associated with incidence of Cardio- and cerebro-vascular events. ConclusionDisturbed carnitine metabolism is independently correlated with sarcopenia and prognosis in patients with hemodialysis. Serum carnitine level and C0/C2 ratio has the potential to be a simple, objective, and quick test for sarcopenia assessment whether such an intervention should be carried out for dialysis patients.

Elimination patterns of dimetridazole in egg of laying hens and tissues of broiler after oral administration.

Dimetridazole (DMZ) is a broad-spectrum anti-anaerobic and antiprotozoal drug extensively used for the control of blackhead disease in poultry (especially turkeys). The presence of DMZ and its metabolites in animal food poses potential risks to human health. In this study, we developed a high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) method for the precise detection of DMZ and its metabolite 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI). Our results demonstrate a strong linear relationship (r2 > 0.99) between the concentrations of DMZ and HMMNI in tissues and egg within the range of 1~100 ng/g. The limits of detection (LOD) were determined to be 0.5 ng/g, with corresponding limits of quantification (LOQ) at 1.0 ng/g. Furthermore, average recoveries in tissues and egg fell within the range of 84.90% to 103.01%, with coefficients of variation below 15% for both intra-day and inter-day analyses. To investigate the residue elimination pattern of DMZ and HMMNI, diets containing 500 mg/kg DMZ were fed to healthy SanHuang chicken and Hy-line Gray laying hens for 10 consecutive days. The results indicated that the concentration of HMMNI consistently exceeded that of DMZ during the same period, in both broiler tissues and egg. Sebum showed the slowest elimination of DMZ and HMMNI, becoming undetectable after 168 h of withdrawal. In egg, residues of both substances peaked on the first day after drug withdrawal, followed by slow elimination with half-lives of 0.45 days for DMZ and 0.66 days for HMMNI. Based on these findings, WT1.4 software was used to calculate a withdrawal time of 11 days for broilers and an egg abandonment period of 14 days after withdrawal for laying hens, providing a scientific basis for the safe and rational use of DMZ in poultry farming.

Improved high-performance liquid chromatography tandem mass spectrometry method for quantification of infliximab in pediatric plasma and its application in therapeutic drug monitoring.

The application of infliximab (IFX) to immune-mediated disease is limited by the significant individual variability and associated clinical nonresponse, emphasizing the importance of therapeutic drug monitoring (TDM). Because of the cross-reactivity, limited linear range, and high costs, the clinical application of the previous reported methods was limited. Here, an improved high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to address the issues. This study developed an improved bioanalytical HPLC-MS/MS method coupling nanosurface and molecular-orientation limited proteolysis technology. The commercially available compound P14R was selected as the internal standard. This method was developed with fewer volume of reagents and was thoroughly validated. The validated method was applied to TDM in pediatric inflammatory bowel disease (IBD). Chromatography was performed using a Shim-pack GISS-HP C18 metal-free column (3 μm, 2.1 × 100 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile at 0.4 mL/min. Detection and quantitation were performed using electrospray ionization (ESI) and multiple reaction monitoring in the positive ion mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability. The method exhibited a linear dynamic range of 0.3-100 μg/mL, with intra- and inter-day precision and relative errors below 15%. The recovery and matrix effect were measured as 87.28%-89.72% and 41.98%-67.17%, respectively, which were effectively compensated by the internal standard. A total of 32 samples collected from 24 pediatric patients with IBD were analyzed using the validated method, and only 46.9% achieved the reported targeted trough level. This study developed an improved HPLC-MS/MS method for the quantitative determination of IFX concentration in human plasma. The accurate, reliable, and cost-effective method was validated and utilized in the analysis of clinical samples. The results confirmed the importance of TDM on IFX and the clinical application prospects of the improved method.

Development and validation of a sensitive and simultaneous liquid chromatography tandem mass spectrometry method for the determination of eight phytocannabinoids in various CBD products

In this study, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of eight phytocannabinoids in various cannabidiol (CBD) products from Japanese market. This method was combined with electrospray ionization in positive mode and sample preparation with QuEChERS. Three types of commercial products such as honey, chocolate, and gummies were used to perform accurate quantification with unified protocol of LC-MS/MS and QuEChERS. The limit of detection and quantification were 5–20 µg g−1 and 10–40 µg g−1, respectively. Reproducibility was ensured using matrices free of target foods, resulting in an accuracy within ±10 % and a precision with a relative standard deviation of less than 5 % for all targets. Finally, this analytical method was applied to 8 series of commercial samples from the Japanese market. This unified protocol will serve as a reference as an official method in Japan.

Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method

Psilocybin is a psychedelic compound found in some hallucinogenic “magic mushrooms”. Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.

Comparative Label-Free Proteomics Study on Celiac Disease-Active Epitopes in Common Wheat, Spelt, Durum Wheat, Emmer, and Einkorn.

Wheat species with various ploidy levels may be different regarding their immunoreactive potential in celiac disease (CD), but a comprehensive comparison of peptide sequences with known epitopes is missing. Thus, we used an untargeted liquid chromatography tandem mass spectrometry method to analyze the content of peptides with CD-active epitope in the five wheat species common wheat, spelt, durum wheat, emmer, and einkorn. In total, 494 peptides with CD-active epitope were identified. Considering the average of the eight cultivars of each species, spelt contained the highest number of different peptides with CD-active epitope (193 ± 12, mean ± SD). Einkorn showed the smallest variability of peptides (63 ± 4) but higher amounts of certain peptides compared to the other species. The wheat species differ in the presence and distribution of CD-active epitopes; hence, the entirety of peptides with CD-active epitope is crucial for the assessment of their immunoreactive potential.

Establishment and clinical application of a candidate reference measurement procedure for quantification of urinary vanillylmandelic acid and homovanillic acid using ID-LC-MS/MS method

Neuroblastoma (NB), an embryonic tumor of the autonomous nervous system, poses a significant threat to the health and lives of children. Accurate measurement of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in human urine is crucial for screening and diagnosis of NB. Although various laboratories have developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect VMA and HVA, the comparability between the results obtained from different laboratories and methods was poor. The absence of reference method for VMA and HVA hinders the standardization of their measurements. Therefore, a candidate reference measurement procedure (cRMP) based on isotope dilution LC-MS/MS (ID-LC-MS/MS) for the detection of VMA and HVA in human urine was established. Urine samples were spiked with VMA-d3 and HVA-d5 as internal standards and extracted using a protein precipitation method. The cRMP exhibited desirable precision with the total imprecision below 5 %. The accuracy of this cRMP was demonstrated by the high analytical recovery (98.64 % - 102.22 % and 98.41 % - 100.97 % for VMA and HVA, respectively), and comparability between different reference systems. The limit of detection for HVA and VMA were 15.625 ng/mL and 3.906 ng/mL, respectively; the quantification limits were 62.5 ng/mL and 7.813 ng/mL, respectively, which can meet the clinical detection requirements. The linear range was from 78.125 ng/mL to 20 μg/mL. Specificity evaluations showed no corresponding interference from structurally similar analogs. In conclusion, we have established a cRMP based on ID-LC-MS/MS for the measurement of VMA and HVA in urine samples, demonstrating well-defined method performance including accuracy, precision, and specificity. This newly established cRMP is suitable for routine assay standardization and evaluation of clinical samples. Furthermore, this method has the potential to significantly enhance the diagnostic accuracy for neuroblastoma.

Effect of spring runoff on 2,6-dichloro-1,4-benzoquinone formation during water treatment

This study investigated the impacts of spring runoff on the formation of halobenzoquinones (HBQs) and their correlation with common water quality parameters (WQPs) and aromatic amino acids (AAs) in source water. Source water and treated water samples were collected at two drinking water treatment plants in 2021, 2022, and 2023. HBQs and aromatic AAs were analyzed using solid phase extraction with high performance liquid chromatography–tandem mass spectrometry methods. The only HBQs detected in treated water were 2,6-dichloro-1,4-benzoquinone (DCBQ) and hydroxy‑DCBQ (OH-DCBQ). The concentration of DCBQ was 3–4 times higher during spring runoff events than during non-spring-runoff periods, suggesting the impact of spring runoff on the formation of DCBQ. The DCBQ concentrations in finished water positively correlated with the color, dissolved organic carbon, total organic nitrogen, and specific ultraviolet absorbance WQPs of source water in 2021 and 2022. The temporal trend of the total aromatic AAs determined in source water was strongly and positively correlated to DCBQ in finished water. Finally, there was a significant positive correlation between the concentration of DCBQ determined immediately following the addition of chlorine and the presence of its transformation product, OH-DCBQ, in finished water. The results also showed that powdered activated carbon can remove some of the HBQ precursors in the source water to reduce DCBQ formation. This study demonstrated that WQPs and aromatic AAs are useful indicators for the removal of precursors to reduce HBQ formation during drinking water treatment.

Determination of six volatile fatty acids in human serum, urine and faeces by low temperature derivatisation combined with HPLC–MS/MS

A stable isotope dilution-liquid chromatography tandem mass spectrometry method based on a low-temperature derivatization strategy with 3-nitrophenylhydrazine (3-NPH) was developed for the determination of six volatile fatty acids (VFAs) in serum, urine, and feces. Ice acetonitrile was used to precipitate proteins and extract the target analytes. The extract was derivatized with 3-NPH methanol solution at 4 °C. BEH C8 (1.7 μm, 2.1 × 100 mm) column was used for chromatographic separation, and acetonitrile–water (both containing 0.01 % formic acid) were used as the mobile phase with a gradient elution of 10 min. Electrospray ionization source (ESI) in negative ion multiple reaction monitoring (MRM) mode were used for analyte detection. The regression coefficients R2 of the calibration curves for the six VFAs were in the range of 0.9963–0.9994, and the LOQs were in the range of 0.02–0.5 μg mL−1, with the recoveries in the range of 85.3–104.3 %, and the intra- and inter-day precision in the range of 1.8–9.1 %. The method is simple, accurate and reliable, and has been applied in the sensitive determination of VFAs in complex biological samples.

A simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method for the quantitative analysis of VX-548 in monkey plasma: Method validation and application to pharmacokinetic study.

VX-548 is an orally active and highly selective NaV 1.8 inhibitor that is undergoing development for the treatment of acute pain. The aim of this study was to develop a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the measurement of VX-548 in monkey plasma. VX-548 was extracted from the plasma using acetonitrile-mediated protein precipitation. The quantitative analysis was performed on a Thermo Vantage TSQ mass spectrometer with ibrutinib as an internal standard. Chromatography was performed on a Waters ACQUITY UPLC BEH C18 column with 0.1% aqueous formic acid and acetonitrile as mobile phase. The precursor-to-product ion transitions were m/z 474.2 > 165.0 and m/z 441.2 > 138.1 for VX-548 and internal standard, respectively. This developed method was successfully validated in the concentration range of 1-1000 ng/mL. The calibration curve showed excellent linearity with a correlation coefficient of >0.999. The precision expressed as relative standard deviation (RSD) was <8.4%, whereas the accuracy denoted as relative error (RE) ranged from -5.0% to 9.1%. The mean recovery was >84%. VX-548 was stable in monkey plasma after storage under certain conditions. The validated method was successfully applied to the pharmacokinetic study of VX-548 in monkey plasma after single oral (2 mg/kg) and intravenous (1 mg/kg) administrations.

Development and application of a sensitive liquid chromatography-tandem mass spectrometry method for the quantitative analysis of 11 free fatty acids in human serum using a derivatisation strategy

A stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water–acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL−1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL−1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL−1; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng mL−1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples.

Rapid and reliable quantification of urinary malondialdehyde by HILIC-MS/MS: A derivatization-free breakthrough approach

BackgroundThe development of fast analytical methods is crucial for the research, discovery, and confirmation of crucial biomarkers. Furthermore, the implementation of fast analytical strategies contributes to efficient and time-effective procedures. In this sense, analysis of malondialdehyde (MDA) has become an important tool for understanding the role of oxidative stress in various diseases and for evaluating the efficacy of therapeutic interventions. ResultsA rapid and robust liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) has been developed to determine endogenous amounts of malondialdehyde (MDA) in human urine without any associated derivatization reaction. MDA was separated in 4 min through a Urea-HILIC column and was analyzed using a triple quadrupole mass spectrometer in negative electrospray ionization mode. With a 50-fold dilution as the only sample pretreatment after alkaline hydrolysis, no matrix effect was present, which allowed for a fast and simple quantification by means of an external standard calibration with a limit of detection of 0.20 ng mL−1. The whole methodology was validated by analyzing unspiked and spiked urine samples from ten healthy individuals and comparing with the results obtained by the standard addition method. MDA was detected in all cases, with natural concentrations varying from 0.11 ± 0.03 to 0.31 ± 0.03 mg g−1 creatinine. Accuracies were found to be satisfactory, ranging from 95 % to 101 %. The proposed method also exhibited good repeatability and reproducibility (RSD<15 %) for four quality control levels. SignificanceThe main significance of this method is the avoidance of a derivatization reaction for the determination of urinary MDA, this constituting a step forward when compared with previous literature. This breakthrough not only streamlines time analysis to less than 5 min per sample but also results in a more robust procedure. Consequently, the method here developed could be applied to subsequent future research involving the determination of MDA as a lipid peroxidation biomarker, where simple, rapid, and reliable methods could represent a significant improvement.

Effects of two commonly used antidepressants amitriptyline and fluoxetine on the pharmacokinetics of abrocitinib in rats

Abrocitinib is approved to treat moderate-to-severe atopic dermatitis and eliminated mainly through cytochrome P450 (CYP450) enzyme. Two commonly used antidepressants, amitriptyline and fluoxetine, could inhibit the activities of CYP2C19 and CYP3A4. In this study, we developed a new and quick ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantitatively analyzing the plasma concentration of abrocitinib, and further investigated the effects of amitriptyline or fluoxetine on the pharmacokinetics of abrocitinib in rats. The selectivity, linearity, recovery, accuracy, precision, matrix effect and stability of UPLC-MS/MS assay were satisfied according to the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. Our result showed that when co-administered with amitriptyline and fluoxetine, the CLz/F of abrocitinib was reduced by 44.4 % and 33.3 %, respectively, while the AUC(0–t) of abrocitinib was increased by 77.7 % and 49.4 %, respectively. It indicated that amitriptyline and fluoxetine could significantly increase the plasma concentration of abrocitinib in rats. Thus, dose adjustment of abrocitinib may be required when it is combined with amitriptyline or fluoxetine in ongoing clinical practice.

Determine the pharmacokinetics (half-life, volume of distribution and clearance) of AMB-FUBINACA in rats plasma using GC–MS / MS

BackgroundSeveral novel synthetic cannabinoids, including methyl 2-(1-(4-fluorobenzyl)-1Hindazole-3-carboxamido)-3-methylbutanoate (AMB-FUBINACA), have recently surfaced on the illicit drug market. To determine the pharmacokinetic properties (half-life, volume of distribution, and clearance) of AMB-FUBINACA in rats plasma, a straightforward, quick, and highly sensitive analytical approach was developed. MethodsEighteen Wistar rats were divided into two groups: one control (saline vehicle) and one treatment group (AMB-FUBINACA at 50 mg/kg). Blood samples (400 μL) were withdrawn via catheters immediately before (t = 0) and at 30, 60, 90, 120, and 240 min following injection. Samples were collected into 1 mL tuberculin syringes, then transferred to 1.5 mL plastic tubes containing 5 μL of 1000 IU/mL K3-EDTA (Thomas Scientific). Place the EDTA tubes containing samples in a centrifuge and spin at 1000 g for 10 min at 4 °C. The top layer is the plasma fraction, which is decanted into cryovials and stored at −20 °C until analysis. The gas chromatography tandem mass spectrometry (GC–MS/MS) method was optimized and validated, combined with liquid-liquid extraction, to analyze AMB-FUBINACA in rat plasma. ResultsThe research method successfully met the validation requirements set by the FDA, demonstrating selectivity and linear calibration curves within a concentration range of 0.5–1000 ng/ml. The correlation coefficient (r2) was determined to be 0.99, indicating a strong linear relationship. The analyte's limit of quantitation (LOQ) was determined to be 1–5 ng/mL. Subsequently, the method was successfully applied to investigate the pharmacokinetics of AMB-FUBINACA in rats' blood samples. Following oral administration, AMB-FUBINACA was rapidly absorbed, with a plasma half-life (t1/2) of 5.91 h, a volume of distribution (Vd) of 203.13 l, and a plasma clearance of 23.81122 L/h. ConclusionThese findings contribute to the understanding of AMB-FUBINACA's pharmacokinetics and pharmacodynamics.

Development and validation of an HILIC/MS/MS method for determination of nusinersen in rabbit plasma

A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80 %. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits.

Determination of nine components of fried Ziziphi Spinosae Semen extract in beagle dog plasma by UPLC-MS/MS and its application in pharmacokinetic research

This study established a convenient, rapid, and sensitive ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of magnoflorine,(R)-coclaurine, vicenin Ⅱ, isospinosin, spinosin, swertisin, N-nornuciferine, 6-feruloylspinosin, and jujuboside B in beagle dog plasma after oral administration of fried Ziziphi Spinosae Semen(FZSS) extract. The Waters HSS-T3 C_(18) column(2.1 mm×100 mm, 1.8 μm) was used. The methanol-aqueous solution(containing 0.01% formic acid) was adopted as the mobile phase for gradient elution. The nine components and two internal standards were completely separated within 8 min. The mass spectrometry detection was performed in multiple reaction monitoring(MRM) mode by positive and negative ion switching of electrospray ionization. The analytical method was validated in terms of specificity, selectivity, linear range, accuracy, precision, recovery, matrix effect, and stability. It could meet the requirement of pharmacokinetic research after oral administration of FZSS extract to beagle dogs. The results showed that the time to reach the peak concentration(T_(max)) of magnoflorine,(R)-coclaurine, vicenin Ⅱ, isospinosin, spinosin, 6-feruloylspinosin, and jujuboside B was 2.40-3.20 h, and the elimination halflife(t_(1/2)) was 2.08-6.79 h after a single-dose oral administration of FZSS to beagle dogs. The exposure of magnoflorine and spinosin was high, with a peak concentration(C_(max)) of 76.7 and 31.5 ng·mL~(-1) and an area under the curve(AUC_(0-∞)) of 581 and 315 ng·h·mL~(-1), respectively. The exposure of the remaining five compounds was lower, with a C_(max) of 0.81-13.0 ng·mL~(-1) and an AUC_(0-∞) of 6.00-106 ng·h·mL~(-1). This study provides a reference for the follow-up research of FZSS.