Liquid Chromatography-tandem Mass Spectrometry Method Research Articles

Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis.

Systemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue. The quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters. The method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17β-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003-10 ng/ml for serum (250 µl) and 0.038-125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively). The method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Screening Potential Citrate Lyase Inhibitors from a Library of Marine Compounds.

Tuberculosis, a persistent illness caused by Mycobacterium tuberculosis, remains a significant global public health challenge. The widespread use of anti-tuberculosis drugs has resulted in the emergence of drug-resistant strains, which complicates treatment efforts. Addressing this issue is crucial and hinges on the development of new drugs that can effectively target the disease. This involves identifying novel therapeutic targets that can disrupt the bacterium's survival mechanisms in various environments such as granulomas and lesions. Citrate lyase, essential for the survival of Mycobacterium species at lesion sites and in granulomatous conditions, is a potential target for the treatment of tuberculosis. This manuscript aimed to construct an efficient enzyme inhibitor screening platform using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF MS). This system can accurately identify compounds with enzyme inhibitory activity from a library of marine terpenoids and phenolic compounds. Utilizing the screened herbal enzyme inhibitors as a starting point, we analyzed their chemical structures and skillfully built a library of marine compounds based on these structures. The results showed that all of the tested compounds from the phenolics library inhibited citrate lyase by more than 50%, and a significant portion of terpenoids also demonstrated inhibition, with these active terpenoids comprising over half of the terpenoids tested. The study underscores the potential of marine-derived phenolic and terpenoid compounds as potent inhibitors of citrate lyase, indicating a promising direction for future investigations in treating tuberculosis and associated disorders.

A LC-MS/MS method for the simultaneous determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma and its clinical application in patients with chronic kidney disease.

The aim of this study was to develop a high-performance liquid chromatography-tandem mass spectrometry method for the determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma samples. Strata-X 33 μm solid-phase extraction cartridges were used for the extraction of the catecholamines from human plasma samples. The catecholamines were separated in a 150 × 3 mm Shim-pack GIST C18-AQ column with 3μm particle size, placed in an oven at 40°C and perfused with 82% mobile phase A (acetonitrile-H2O; 90:10, v/v) + 0.4% acetic acid and 18% mobile phase B (deionized H2O) + 0.2% formic acid at a flow rate of 340 μl/min in isocratic mode. The injected volume was 4 μl and the run lasted 4 min. The method was linear from 0.1 to 20 ng/ml and the lower limit of quantification was 0.1 ng/ml for all analytes. The method was applied to evaluate the plasma levels of catecholamines in plasma of patients with chronic kidney disease and allowed the detection for the first time of circulating levels of the novel catecholamines 6-bromodopamine and 6-cyanodopamine.

Simultaneous quantification of five antiretrovirals in human tissues using ultra-high performance liquid chromatography-tandem mass spectrometry methods for therapeutic drug monitoring at the sites of action

Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05–50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5–107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts.

Exploring the relationships between exposure levels of bisphenols and phthalates and prostate cancer occurrence

We established an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously analyzing the metabolites of bisphenols and phthalates in urine to identify the associations between these exposure levels and prostate cancer (PCa) based on a case–control study. After purifying urine samples with SPE, 18 metabolites were separated on a C18 column, and MS detection was performed. The UPLC-MS/MS method has been proven effective at evaluating bisphenol and phthalate exposure (0.020–0.20 μg/L of the limits of detection, 71.8 %∼119.4 % of recoveries, 0.4 %∼8.2 % of precision). Logistic regression explored the association between exposure level and PCa in 187 PCa cases and 151 controls. The detection rates of bisphenol A (BPA) and most phthalate metabolites were 100 % ranging from 0.06–46.74 and 0.12–899.92 μg/g creatinine, respectively, while the detection rates of other bisphenols and mono-benzyl phthalate (MBzP) are low, ranging from 0 % to 21.85 %. Correlation analysis of the metabolite levels indicated that the exposure sources of BPA, di-ethyl phthalate (DEP), and di(2-ethylhexyl) phthalate (DEHP) were different, and that the exposure sources of di-n-butyl phthalate (DnBP) and di-isobutyl phthalate (DiBP) may differ between two groups. Logistic regression analysis revealed that BPA (OR<0.45 vs ≥1.43 =10.02) and DEHP exposure (OR<21.75 vs ≥45.42 =48.26) increased the risk of PCa.

#2850 Elevation of serum indoxyl-sulfate in mice with normal kidney function triggers cardiac and vascular dysfunctions

Abstract Background and Aims Acute kidney disease (AKD) is associated with a long-term risk of cardiovascular mortality and major cardiovascular events, particularly heart failure and acute myocardial infarction. To date, the underlying mechanisms responsible for this elevated cardiovascular risk remain poorly understood. Patients with AKD show increased circulating levels of indoxyl-sulfate (IS), a uremic toxin known to promote oxidative stress, inflammation and apoptosis in cultured vascular and cardiac cells. In this context, we hypothesized that IS elevation during AKD might promote acute myocardial and vascular lesions, which over time favour the onset of cardiovascular events. In a first attempt to test this hypothesis, we aimed to verify whether IS elevation in mice with normal renal function induces myocardial and vascular dysfunctions similar to that observed in AKD. Method Thirty female C57BL6J mice were divided into three groups: control (CT, n = 10), IS (n = 10) and AKD (n = 10). AKD was induced by 5/6 nephrectomy. Blank surgeries were performed in the CT and IS groups. IS was administered continuously for 15 days via drinking water (2.5 g/L) to reach, in IS mice, the IS serum concentrations found in AKD mice, as monitored by a validated high-performance liquid chromatography-tandem mass spectrometry method. Cardiac and vascular functions were assessed by echocardiography at baseline and at D15. Expression of markers of contractility (SERCA-2a for myocardium, αSMA for aorta), inflammation (TNF-α, IL-1β), and endothelial dysfunction (VCAM-1, ICAM-1, iNOS) were assessed by qRT-PCR and western blot. The presence of cardiac lesions was assessed histologically. Results Elevation of serum IS concentrations was similar in IS and AKD mice (serum IS: 10.7 ± 3.4 µg/mL in IS-group and 10.9 ± 5.8 µg/mL in AKD-group vs. 4.0 ± 2.1 µg/mL in CT-group (p &amp;lt; 0.001 in CT vs IS and AKD group). Compared with CT mice, IS and AKD mice showed systolic and diastolic dysfunctions as evidenced by decreased ejection fraction (EF: 62.4% [58.0-72.3] in CT vs. 50.1% [45.0-57.0] in IS (p &amp;lt; 0.05 vs CT) and 51.3% [45.6-57.1] in AKD (p &amp;lt; 0.01 vs CT)), decreased fractional shortening (FS: 33.6% [30.1-40.4] in CT vs. 24.8% [21.9-29.7] in IS (p &amp;lt; 0.05 vs CT) and 25.8% [22.4-29.6] in AKD (p &amp;lt; 0.01 vs CT)) and increased isovolumic relaxation time (IVRT: 17.2 ± 1.6 ms in CT vs. 20.7 ± 1.3 ms in IS (p &amp;lt; 0.001 vs CT) and 19.0 ± 1.7 ms in AKD (p &amp;lt; 0.05 vs CT)), respectively. For all echocardiographic parameters assessed, there were no significant differences observed between IS and AKD mice. Myocardium from IS and AKD mice showed decreased expression of SERCA-2a and elevated expression of both IL-1β and TNF-α at the mRNA and protein levels compared to CT mice. Aortas from IS and AKD mice also showed decreased mRNA expression of α-SMA and increased mRNA expression of IL-1β, VCAM-1, and iNOS compared to aortas from CT mice. The mRNA and protein expression of these inflammatory and contractile markers were not statistically different in the hearts and aortas from IS compared to AKD mice. No signs of vascular dysfunction were observed in either of the groups on ultrasound. Conclusion Systemic exposure to IS in mice without renal failure leads to significant cardiac and vascular dysfunctions that were similar to those observed in mice with AKD. Additional studies are needed to confirm the causal role of IS in the cardiovascular impact of AKD, and to determine whether IS could represent a valuable prognostic tool and therapeutic target for preventing AKD-associated cardiovascular complications.

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.

LC-MS-MS method for mitragynine and 7-hydroxymitragynine in hair and its application in authentic hair samples of suspected kratom abusers.

Kratom is a natural psychoactive product known primarily in Southeast Asia, including Thailand, Malaysia etc. It is also known as krathom, kakuam, ithang, thom (Thailand), biak-biak, ketum (Malaysia) and mambog (Philippines), and is sometimes used as an opium substitute. It is stimulant at doses of 1-5g, analgesic at doses of 5-15g, and euphoric and sedative at doses above 15g. Mitragynine is the most abundant indole compound in kratom (Mitragyna speciosa) and is metabolized in humans to 7-hydroxymitragynine, the more active metabolite. Adverse effects include seizures, nausea, vomiting, diarrhoea, tachycardia, restlessness, tremors, hallucinations and death. There are few studies on the analytical method for the detection of mitragynine and 7-hydroxymitragynine in hair. Therefore, this study proposes a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the analysis of kratom in hair. Hair samples were first weighed to approximately 10 mg and washed with methanol. Then the washed hair samples were cut into pieces and incubated in methanol with stirring and heating (16h/38℃). Extracts were then analysed by LC-MS-MS. This method was validated by determining the limit of detection (LOD), limit of quantification (LOQ), linearity, intra- and inter-day accuracy and precision, recovery and matrix effects. The intra- and inter-day precision (CV%) and accuracy (bias%) were within ±20%, which was considered acceptable. Using this newly developed LC-MS-MS method, the simultaneous detection of mitragynine and 7-hydroxymitragynine in six authentic hair samples was achieved to provide the direct evidence of kratom use in the past. Mitragynine concentrations ranged from 16.0 to 2,067 pg/mg (mean 905.3 pg/mg), and 7-hydroxymitragynine concentrations ranged from 0.34 to 15 pg/mg (mean 7.4 pg/mg) in six authentic hair samples from kratom abusers. This may be due to the higher sensitivity of the LOD in this study, with values of 0.05 pg/mg for mitragynine and 0.2 pg/mg for 7-hydroxymitragynine in hair, respectively.

Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.

To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 μL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Fosfomycin Concentrations in Human Prostatic Tissue.

The aim of this study was to develop and validate a fast and sensitive bioanalytical method for the accurate quantification of fosfomycin concentrations in human prostatic tissue. The sample preparation method only required milligrams of tissue sample. Each sample was mixed with two times its weight of water and homogenized. A methanol solution that was three times the volume of the internal standard (fosfomycin-13C3) was added, followed by vortex mixing and centrifugation. After its extraction from the homogenized prostatic tissue, fosfomycin was quantified by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) triple quadrupole system operating in negative electrospray ionization and multiple reaction monitoring detection mode. The analytical procedure was successfully validated in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability, according to EMA guidelines. The validation results, relative to three QC levels, were 9.9% for both the within-day and inter-day accuracy (BIAS%); 9.8% for within-day precision; and 9.9 for between-day precision. A marked matrix effect was observed in the measurements but was corrected by normalization with the internal standard. The average total recovery was high (approximatively 97% at the three control levels). The dynamic range of the method was 0.1-20 μg/g (R2 of 0.999). Negligible carry-over was observed after the injection of highly concentrated samples. F in the sample homogenate extracts was stable at 10 °C and 4 °C for at least 24 h. In the tissue sample freeze-thaw experiments, a significant decrease in F concentrations was observed after only two cycles from -80 °C to room temperature. The novel method was successfully applied to measure fosfomycin in prostatic tissue samples collected from 105 patients undergoing prostatectomy.

A New and Rapid LC-MS/MS Method for the Determination of Cysteamine Plasma Levels in Cystinosis Patients.

Cystinosis is a rare lysosomal storage disorder caused by autosomal recessive mutations in the CTNS gene that encodes for the cystine transporter cystinosin, which is expressed on the lysosomal membrane mediating the efflux of cystine. Cysteamine bitartrate is a cystine-depleting aminothiol agent approved for the treatment of cystinosis in children and adults. In this study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cysteamine levels in plasma samples. This LC-MS/MS method was validated according to the European Medicines Agency (EMA)'s guidelines for bioanalytical method validation. An ultra-performance liquid chromatograph (UPLC) coupled with a 6470 mass spectrometry system was used for cysteamine determination. Our validated method was applied to plasma samples from n = 8 cystinosis patients (median, interquartile range (IQR) = 20.5, 8.5-26.0 years). The samples were collected before cysteamine oral administration (pre-dose) and 1 h after (post-dose). Our bioanalytical method fulfilled the regulatory guidelines for method validation. The cysteamine plasma levels in pre-dose samples were 2.57 and 1.50-3.31 μM (median and IQR, respectively), whereas the post-dose samples reported a cysteamine median concentration of 28.00 μM (IQR: 17.60-36.61). Our method allows the rapid determination of cysteamine plasma levels. This method was successfully used in cystinosis patients and, therefore, could be a useful tool for the evaluation of therapy adherence and for future pharmacokinetic (PK) studies involving a higher number of subjects.

Method development, residue level analysis, and dietary risk assessment of six morpholine pesticide residues in plant-derived foods based on modified QuEChERS–liquid chromatography–tandem mass spectrometry

A modified QuEChERS purification–liquid chromatography-tandem mass spectrometry method was developed to simultaneously detect six morpholine pesticide residues in plant-derived foods. The samples were extracted using acetonitrile and purified using three adsorbents, namely, graphitized carboxyl MWCNTs, C18, and SAX. The limit of quantitation was 0.07–0.75 µg/kg, and the average recoveries ranged from 71.3 % to 113.8 %. The precision ranged from 1.2 % to 13.8 % at four spiked concentrations of 0.5, 1, 5, and 10 µg/kg. The method was applied to analyze 540 samples obtained from 17 types of plant-derived foods collected in Yining, China. Dimethomorph and flumorph were detected in nine types of matrices, and the residue levels did not exceed the maximum residue limits (MRLs) set by the national standard GB 2763–2021. Furthermore, we assessed the risk of acute and chronic exposure to the tested pesticides. The pesticide residue levels were acceptable in all the samples.

Preclinical pharmacokinetic studies of villocarine A, an active Uncaria alkaloid.

Villocarine A is a bioactive indole alkaloid isolated from the Uncaria genus. It has demonstrated vasorelaxation activity and potential to protect the central nervous system. To identify the pharmacokinetic properties of villocarine A, a series of in vitro and in vivo studies have been performed. Villocarine A was found to be highly permeable (15.6 ± 1.6*10-6cm/s) across human colorectal adenocarcinoma cell monolayer with high protein binding (>91%) in both rat and human plasma. Hepatic extraction ratio of villocarine A was 0.1 in pooled rat liver and 0.2 in human liver microsomes and was found stable in rat plasma at 37°C. Due to the high permeability and low rate of metabolism properties, villocarine A was initially considered suitable for preclinical development and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification (linearity: 1-150 ng/ml) in rat plasma was developed and validated for in vivo studies. Essential pharmacokinetic parameters included the volume of distribution and clearance of villocarine A, which were found to be 100.3 ± 15.6L/kg and 8.2 ± 1.1L/h/kg, respectively, after intravenous administration in rats. Following oral dosing, villocarine A exhibited rapid absorption as the maximum plasma concentration (53.2 ± 10.4ng/ml) occurred at 0.3 ± 0.1h, post-dose. The absolute oral bioavailability of villocarine A was 16.8 ± 0.1%. To our knowledge, this was the first pharmacokinetic study of villocarine A, which demonstrated the essential pharmacokinetic properties of villocarine A: large volume distribution, high clearance, and low oral bioavailability in rats.

Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC-MS/MS.

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95-99.97 (norvancomycin), 725.90-100.04 (vancomycin), 384.16-67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5-50 μg/ml for serum and 0.5-100 μg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.

A developed and validated centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method for rapid quantification of unbound lenvatinib in human plasma

In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (Vu). The results indicated that the centrifugal force and centrifugal time have an influence on Vu that is significantly positive (P < 0.05). This developed method with good linearity (r2 = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %−106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice.

Tramadol/Diclofenac Fixed-Dose Combination for Acute Pain Management: Bioavailability Assessment of a Generic Product.

The multimodal analgesia strategy for acute pain involves using 2 or more analgesic medications with distinct mechanisms of action. This study assessed the bioavailability and tolerability of 2 tramadol hydrochloride (50mg)/diclofenac sodium (50mg) fixed-dose combination formulations under fed conditions to attend the Brazilian regulatory requirements for generic product registration. An open-label, randomized, single-dose, 2-period, 2-way crossover trial was conducted, including healthy subjects of both sexes. Subjects received a single dose of either the test or reference formulation of tramadol/diclofenac fixed-dose combination tablets with a 7-day washout period. Blood samples were collected up to 36hours after dosing for tramadol and 12hours for diclofenac and quantified using a validated liquid chromatography-tandem mass spectrometry method. Of 56 subjects enrolled, 53 completed the study. The 90% confidence intervals for maximum plasma concentration and area under the concentration-time curve from time 0 to the last quantifiable concentration were within acceptable bioequivalence limits of 80%-125%. Considering the results presented in this study, the test formulation is bioequivalent to the reference formulation and could be interchangeable in medical practice.

Adherence to low-dose methotrexate in children with juvenile idiopathic arthritis using a sensitive methotrexate assay

BackgroundLow-dose weekly methotrexate (MTX) is the mainstay of treatment in juvenile idiopathic arthritis. Unfortunately, a substantial part of patients has insufficient efficacy of MTX. A potential cause of this inadequate response is suboptimal drug adherence. The aim of this study was to assess MTX adherence in juvenile idiopathic arthritis patients by quantification of MTX concentrations in plasma. Secondly, the association between MTX concentrations and either self-reported adherence issues, or concomitant use of biologics was examined.MethodsThis was a retrospective, observational study using plasma samples from juvenile idiopathic arthritis patients. An ultrasensitive liquid chromatography-tandem mass spectrometry method was developed for quantification of MTX and its metabolite 7-hydroxy-MTX in plasma. The determined MTX plasma concentrations in juvenile idiopathic arthritis patients were compared with corresponding adherence limits, categorising them as either adherent or possibly non-adherent to MTX therapy.ResultsPlasma samples of 43 patients with juvenile idiopathic arthritis were analysed. Adherence to MTX in this population was 88% shortly after initiation of MTX therapy and decreased to 77% after one year of treatment. Teenagers were more at risk for non-adherence (p = 0.002). We could not find an association between MTX adherence with either self-reported adherence issues, nor with the use of concomitant biological treatment (p = 1.00 and p = 0.27, respectively; Fisher’s Exact).ConclusionsQuantification of MTX in plasma is a feasible and objective method to assess adherence in patients using low-dose weekly MTX. In clinical practice, the use of this method could be a helpful tool for physicians to refute or support suspicion of non-adherence to MTX therapy.

A green analytical method for the simultaneous determination of 17 perfluoroalkyl substances (PFAS) in human serum and semen by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)

The ubiquity of perfluoroalkyl substances has raised concerns about the unintended consequences of PFAS exposure on human health. In the present study, an eco-friendly ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of 17 PFAS in human serum and semen samples. QuEChERS salts MgSO4:NaCl 4:1 (w/w) were used for the extraction. The separation of analytes was performed on an ACQUITY BEH C18 column (100 × 2.1 mm, 1.7 μm), using water:methanol 95:5 and methanol as mobile phases A and B, respectively, both containing 2 mM ammonium acetate. Multiple reaction monitoring (MRM) in negative ion mode was used, selecting two transitions for each analyte, except for perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA). The analytical method was validated according to the Organization of Scientific Area Committees (OSAC) for Forensic Sciences guidelines and AGREE approach software was used to evaluate the greenness of the method. The developed procedure was applied to the analysis of 10 paired human serum and semen samples, proving the suitability in high throughput laboratories due to the easy preparation and the reduced volume of toxic solvents. Moreover, it allows to perform further investigation on the correlation between serum and semen PFAS concentration, focusing on male reproductive system correlated pathologies, such as male infertility.

Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis.

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography-tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of Caenorhabditis elegans

Multiple isozymes are encoded in the Caenorhabditis elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM) from the same MS run. Validating this sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are both required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3, but not CGT-1 and CGT-2, plays a major role in producing GlcCers. Deletion of sms-5 hardly affected SM levels. RNAi of sms-1, sms-2, and sms-3 all lowered the abundance of certain SMs with an odd-numbered N-acyl chains (mostly C21 and C23, with or without hydroxylation). Unexpectedly, sms-2 RNAi and sms-3 RNAi elevated a subset of SM species containing even-numbered N-acyls. This suggests that sphingolipids containing even-numbered N-acyls could be regulated separately, sometimes in opposite directions, from those containing odd-numbered N-acyls, which are presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of GlcCers and SMs. These findings underscore the effectiveness of this RPLC-MS/MS method in studies of C. elegans sphingolipid biology.