UPLC-MS/MS Method for Simultaneous Determination of 14 Antimicrobials in Human Plasma and Cerebrospinal Fluid: Application to Therapeutic Drug Monitoring

Pharmacokinetics/pharmacodynamics is the foundation for guiding the rational application of antibiotics in clinical practice, so it is necessary to establish quantitative methods for accurate drug concentration determination. This study aimed to develop a rapid and simple ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 14 antibiotics (amikacin, etimicin, ceftazidime, cefepime, cefoperazone, ceftriaxone, daptomycin, latamoxef, linezolid, meropenem, biapenem, ampicillin, norvancomycin, and vancomycin) in human plasma and cerebrospinal fluid. Antibiotics were chromatographically separated on a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) via gradient elution within 3 minutes and were monitored using positive ion fitted with multiple reaction monitoring. The lower limit of quantification was 0.05–2.0 μg·mL−1. The method was verified according to the FDA bioanalysis method validation guidelines, which showed excellent accuracy (from 86.75% to 110.85%) and precision (from 0.46% to 10.97%). At last, this method was successfully applied to therapeutic drug monitoring in 113 patients under antibiotics treatment.

Development and Validation of a Bioanalytical UHPLC-MS/MS Method Applied to Murine Liver Tissue for the Determination of Indocyanine Green Loaded in H-Ferritin Nanoparticles

Indocyanine green (ICG) is one of the most commonly used fluorophores in near-infrared fluorescence-guided techniques. However, the molecule is prone to form aggregates in saline solution with a limited photostability and a moderate fluorescence yield. ICG was thus formulated using protein-based nanoparticles of H-ferritin (HFn) in order to generate a new nanostructure, HFn-ICG. In this study, an ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system was employed to develop and validate the quantitative analysis of ICG in liver tissue samples from HFn-ICG-treated mice. To precipitate HFn, cold acetone in acidic solution at pH 5.0 was used. The processed liver samples were injected into the UHPLC-MS/MS system for analysis using the positive electrospray ionization mode. Chromatographic separation was achieved on a Waters Acquity UPLC® HSS T3 Column (1.8 μm, 2.1 × 100 mm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. The selected reaction monitoring transitions of m/z 753 →m/z 330 and m/z 827 →m/z 330 were applied for ICG and IR-820 (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 50–1,500 ng/ml. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in liver tissue homogenates. ICG extraction recoveries ranged between 85 and 108%. The intra- and inter-day precisions were less than 6.28%. The method was applied to a bio-distribution study to compare the amount of ICG levels from mice treated with HFn-ICG and free ICG. The analyses of the homogenate samples from the two types of treatment showed that the concentration levels of ICG is approximately six-fold higher than those of free ICG (1,411 ± 7.62 ng/ml vs. 235 ± 26.0 ng/ml) at 2 h post injection.

The Influences of Perfluoroalkyl Substances on the Rheumatoid Arthritis Clinic

Objective. Perfluoroalkyl substances (PFASs) are a class of synthetic organic fluorine chemicals, which have been mass-produced and widely used in the past 60 years, and also have been shown to be one of the major pollutants affecting human health. The impact of fluoride on the development of RA is unclear. The objective of this study was to assess the relationship between perfluoroalkyl substances and the clinic of RA. Methods. A cohort of 155 patients with RA in Second Affiliated Hospital of Zhejiang University School of Medicine were investigated. Demographic and clinical data of these patients were recorded. The concentration of perfluoroalkyl substances were measured by ultra-performance liquid chromatography system (ACQUITY, UPLC) coupled to a tandem mass spectrometer that operated in negative ionization mode. The correlations between the clinical data and the concentration of perfluoroalkyl substances were analyzed.Results. There were 43 male patients and 112 female patients in the cohort. Some of perfluoroalkyl substances (PFOA, PFNA, PFTrA, PFOS) were correlated negatively with the Body Mass Index (BMI); some of them (PFOA, PFNA, PFTrA, PFOS, 8:2Cl-PFESA) were correlated positively with the Disease Activity Score 28 (DAS28); two (PFOA, PFOS) of them were correlated positively with the white blood cell count, and one (PFUnA) of them was correlated negatively with the hemoglobin; two (PFDA, PFUnA) of them were correlated negatively with the presence of interstitial lung disease. Conclusion. These data suggest that exposure to perfluoroalkyl substances may promote the disease activity of rheumatoid arthritis and the visceral lesions.

FORCED INDICATING UPLC-DAD METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF APALUTAMIDE IN BULK AND IN PHARMACEUTICAL DOSAGE FORM

A new analytical method was developed for the estimation of apalutamide in bulk and its pharmaceutical formulation. The sensitive, précise and accurate method was developed by using Waters Acquity UPLC system equipped with quaternary gradient pump. The column used was Waters C18 150 X 2.1 mm X 1.7 µm and mobile phase was 0.2 % OPA buffer in water : acetonitrile in the ratio of 25:75 V/V. The buffer pH was maintained at 4.5. The fl ow rate of mobile phase was 0.5 mL min-1 and detection was at 272 nm by using PDA detector. The method was performed at ambient temperature. The retention time of the apalutamide was 1.27 min. The % RSD value in precision was >2 %. The accuracy of the method was found to be between 99.54 % - 100.01 %. The limit of detection and limit of quantifi cation values were found to be 0.14 µg mL-1 and 0.48 µg mL-1, respectively. The linearity concentration range was found to be 11.25 - 67.5 µg mL-1, it show wide linearity concentration range. The method was proved to have good robustness after changing parameters of fl ow rate, temperature and mobile phase composition. The method showed good ability towards different stress conditions of acidity, alkalinity, peroxide and UV-light. The method can be used for the routine analysis of apalutamide in bulk and its pharmaceutical dosage form by using UPLC.

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

Simultaneous Determination of Albendazole and Its Three Metabolites in Pig and Poultry Muscle by Ultrahigh-Performance Liquid Chromatography-Fluorescence Detection

A fast, simple and efficient ultrahigh-performance liquid chromatography-fluorescence detection (UPLC-FLD) method for the determination of residues of albendazole (ABZ) and its three metabolites, albendazole sulfone (ABZ-SO2), albendazole sulfoxide (ABZ-SO), and albendazole-2-aminosulfone (ABZ-2NH2-SO2), in pig and poultry muscle (chicken, duck and goose) was established. The samples were extracted with ethyl acetate, and the extracts were further subjected to cleanup by utilizing a series of liquid–liquid extraction (LLE) steps. Then, extracts were purified by OASIS® PRiME hydrophilic-lipophilic balance (HLB) solid-phase extraction (SPE) cartridges (60 mg/3 mL). The target compounds were separated on an ACQUITY UPLC® BEH C18 (2.1 mm × 100 mm, 1.7 μm) chromatographic column, using a mobile phase composed of 31% acetonitrile and 69% aqueous solution (containing 0.2% formic acid and 0.05% triethylamine). The limits of detection (LODs) and limits of quantification (LOQs) of the four target compounds in pig and poultry muscle were 0.2–3.8 µg/kg and 1.0–10.9 µg/kg, respectively. The recoveries were all above 80.37% when the muscle samples were spiked with the four target compounds at the LOQ, 0.5 maximum residue limit (MRL), 1.0 MRL, and 2.0 MRL levels. The intraday relative standard deviations (RSDs) were less than 5.11%, and the interday RSDs were less than 6.29%.

Simultaneous Determination of Tetracyclines and Fluoroquinolones in Poultry Eggs by UPLC Integrated with Dual-Channel-Fluorescence Detection Method

An innovative, rapid and stable method for simultaneous determination of three tetracycline (oxytetracycline, tetracycline and doxycycline) and two fluoroquinolone (ciprofloxacin and enrofloxacin) residues in poultry eggs by ultra-high performance liquid chromatography–fluorescence detection (UPLC-FLD) was established and optimized. The samples were homogenized and extracted with acetonitrile/ultrapure water (90:10, v/v) and then purified by solid-phase extraction (SPE). LC separation was achieved on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm), and the mobile phase was composed of acetonitrile and a 0.1 mol/L malonic acid solution containing 50 mmol/L magnesium chloride (the pH was adjusted to 5.5 with ammonia). When the five target drugs were spiked at the limit of quantification, 0.5 times the maximum residue limit (MRL), 1.0 MRL and 2.0 MRL, the recoveries were above 83.5% and the precision ranged from 1.99% to 6.24%. These figures of merit complied with the parameter validation regulations of the EU and U.S. FDA. The limits of detection and quantifications of the targets were 0.1–13.4 µg/kg and 0.3–40.1 µg/kg, respectively. The proposed method was easily extended to quantitative analyses of target drug residues in 85 egg samples, thus demonstrating its reliability and applicability.

A Novel UPLC Method for Simultaneous Estimation of Ertugliflozin and Sitagliptin in Bulk and Tablet Dosage Form

Aim: The proposed study aimed to develop a novel ultra-performance liquid chromatography (UPLC) method for the estimation of Ertugliflozin and Sitagliptin in Bulk and Tablet dosage form and validate the method in accordance with the International Conference on Harmonization guidelines. Methods: The optimized conditions for the developed UPLC method are Acquity UPLC HIBRA C18 (100mm × 2.1mm, 1.8µ) column maintained at 30°C with a mobile phase consisting of Buffer (0.01N sodium hydrogen phosphate) pH adjusted to 4.0 with dil. orthophosphoric acid: Acetonitrile in the ratio of 60:40%v/v on isocratic mode at a flow rate of 0.3mL/min. Results and conclusion: The sample was detected at 220nm. The retention time for Ertugliflozin and Sitagliptin was deemed at 1.873min and 1.260min. The developed method was validated for accuracy, precision, specificity, ruggedness, robustness, and solution stability. The method obeyed Beer’s law in the concentration range of 3.75µg/mL to 22.5µg/mL for Ertugliflozin and 25µg/mL to 150µg/mL for Sitagliptin with a correlation coefficient of 0.999 for Ertugliflozin and Sitagliptin respectively. Forced degradation studies were conducted by exposing the drug solution to numerous stress conditions such as acidic, basic, peroxide, neutral, photolytic, and thermal conditions. The net degradation was considered within the limits, indicating that the drug is stable in stressed conditions. The developed method for the estimation of Ertugliflozin and Sitagliptin can be utilized for the routine analysis of Pharmaceutical dosage form.

Pharmacokinetics of the Couple of Radix Aconiti Lateralis Preparata and Cinnamomum Cassia in Rats with Osteoporosis with Kidney-yang Deficiency by UPLC and Microdialysis Method

Background: To confirm the metabolites of the Aconite and Cinnamon herb couple in rat, and characterize its pharmacokinetics.Methods: Microdialysis probes were inserted into the jugular vein and knee joint of Sprague Dawley rat, and dialysates of different administration groups were collected. Target analytes were separated on a hydrophilic interaction liquid chromatography column (ACQUITY UPLC BEH C8 2.1×100 mm, 1.7 μm) and analyzed with an ultra-performance liquid chromatography (UPLC) under multiple reaction monitoring modes.Results: The experiment shows that the concentrations of quality of the three metabolites in mixing extraction of the herb couple were 0.3701% for trans-cinnamic acid, 0.1249 % for mesaconitine, and 0.0469 % for hypoaconitine, and Cinnamon group was 0.1731 % for trans-cinnamic acid, Aconite group were 0.0017 % for mesaconitine, and 0.0300 % for hypoaconitine. The concentrations of quality of the metabolites in rat plasma of the herb couple group were 0.0028% for trans-cinnamic acid, 0.0947% for mesaconitine, and 0.1124% for hypoaconitine. And the concentrations of quality of Aconite group and Cinnamon group were 0.0019% for trans-cinnamic acid, 0.0307% for mesaconitine, and 0.0220% for hypoaconitine. Pharmacokinetic results showed that the mean half-lives of the microdialysis samples of blood of Cinnamon group was 492.18 min for trans-cinnamic acid, and Aconite group were 102.48 min for mesaconitine and 93.27 min for hypoaconitine, and the herb couple ware 181.36 min for trans-cinnamic acid, 103.9 min for mesaconitine and 116.01 min for hypoaconitine, and the microdialysis samples of joint of Cinnamon group was 190.85 min for trans-cinnamic acid, and Aconite group were 48.51 min for mesaconitine and 46.01 min for hypoaconitine, and the herb couple was 131.19 min for trans-cinnamic acid, 49.36 min for mesaconitine and 146.68 min for hypoaconitine.Conclusions: The mixed extraction of aconite and cinnamon can promote the dissolution of the active ingredients. The herb couple can promote the absorption of the active ingredients, improve the distribution of the active ingredients in the joint and blood, prolong the half-lives of the active ingredients. It shows that the compatibility of aconite and cinnamon can increase the bioavailability of the drug and improve the clinical efficacy.

EXPRESS: A rapid LC-MS/MS assay for the measurement of serum methotrexate in patients who have received high doses for chemotherapy

Background Methotrexate is used in high doses to treat a number of cancers, particularly certain haematological malignancies. Monitoring of serum methotrexate concentration is important due to the potential toxicity of methotrexate and the variation in methotrexate pharmacokinetics in different patients on the same treatment regimen. Objective To develop a rapid liquid chromatography-tandem mass spectrometry method for monitoring serum methotrexate in patients on high-dose chemotherapy. Method Isotopically labelled internal standard was added to sample prior to protein precipitation with methanol. Diluted supernatant was injected into a Waters Acquity UPLC system linked to a TQS-Micro mass spectrometer. Separation by chromatography was achieved with a Waters Phenyl Vanguard with a retention time of approximately 0.5 minutes. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2 respectively. Results Mean recovery was 111% for 3 different concentrations of methotrexate spiked into 7 different patient samples, with ion suppression <1%. Between-batch and within-batch CVs were <5% at three different concentrations of methotrexate in fresh frozen plasma. The lower limit of quantification was 0.02 µmol/L and the assay was shown to be linear to approximately 25 µmol/L. The LC-MS/MS assay showed a mean bias of -8.6% compared to an immunoassay, whilst mean bias compared to weighed in targets in EQA samples was 1.6 %. Discussion A rapid LC-MS/MS assay for methotrexate has been developed and validated. The LC-MS/MS method is likely to offer superior accuracy and specificity to more widely available immunoassays.