Kinetex C18 Column Research Articles

OPTIMIZATION OF ROBUST HPLC APPROACH FOR ASSESSING PROCESS RELATED IMPURITIES OF LEMBOREXANT AND ELUCIDATION OF STRESS-INDUCED DEGRADATION PRODUCTS THROUGH LCMS/MS

This study primarily aimed to establish an uncomplicated yet highly responsive HPLC technique to effectively separate and quantify process-related impurities of Lemborexant. Additionally, it aimed to explore the forced degradation behavior of lemborexant through systematic assessments by utilizing LCMS. The chromatographic separation of drug substance, process related impurities and its degradation products (DPs) was achieved on Kinetex C18 (150×4.6 mm, 5 μm) column at that was maintained at 350C temperature using 10 mM ammonium formate buffer pH 4.2, acetonitrile and methanol in 65:25:10 (v/v) isocratic elution at 0.7 mL/min. Detection wavelength was selected as 265 nm. In the proposed conditions, lemborexant is identified at 4.06 and 6.19, 9.33 min and 1.60 min respectively for impurity 1, 2 and 3 min with acceptable system suitability and specificity. The method produces LOD at 0.009 for impurities with calibration range of 40–280 µg/mL for lemborexant and 0.04 - 0.28 µg/mL for impurities. The remaining validation parameters were observed to fall within acceptable ranges for both lemborexant and its impurities. The compound underwent exposure to various stress conditions (acid, base, peroxide, thermal, and UV light) as outlined in accordance with ICH Q1A (R2) guidelines. The degradation products formed during the stress study were detected and characterized using LCMS/MS in ESI positive mode. This involved a thorough comparison of collision-induced dissociation mass spectrometry data between the degradation products and lemborexant. Consequently, potential structures for five degradation compounds were proposed. The outcomes of supplementary validation investigations were equally satisfactory, confirming their appropriateness for the regular quantification of lemborexant and its related impurities in both bulk drug and pharmaceutical formulations. Furthermore, these findings have the potential to shed light on the mechanism of stress-induced degradation in lemborexant

Determination of furmonertinib in human plasma and cerebrospinal fluid by UPLC-MS/MS: Application in lung cancer patients with and without brain metastasis

Furmonertinib (AST2818) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) being developed for the treatment of patients with EGFR mutation-positive non-small cell lung cancer. Quantification of furmonertinib in plasma and cerebrospinal fluid (CSF) can be used to assess penetration of furmonertinib into the central nervous system (CNS). This paper described ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for quantification of furmonertinib in human plasma and CSF. Sample separation was achieved on a Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm) after simple protein precipitation with acetonitrile. The mobile phase was composed of acetonitrile and 5 mM ammonium acetate with 0.2 % formic acid in water. Quantitative ion pairs were m/z 569.3 → 72.2 for furmonertinib and m/z 526.5 → 72.2 for aumolertinib, which was used as the internal standard (IS). The calibration curves showed good linearity (r2 > 0.99) over concentration range of 0.5–200 ng/mL(plasma sample) and 0.05–30 ng/mL(CSF sample). The precision (RSD) was ≤7.86 %, and the accuracy fell within the range of 96.2 % to 109.3 %, all meeting acceptance criteria. The matrix effect was from 94.3 % to 102.1 %. The recovery of analytes fell within the range of 93.3 % to 98.9 %. The established analytical methods showed great sensitivity, simplicity, accuracy and reliability for the analysis of furmonertinib in human plasma and CSF. This assay would be helpful to predict the effectiveness and toxicities of furmonertinib in the pursuit of precision medicine for lung cancer patients.

Determination and Quantification of Related Impurity Substances in Elvitegravir Drug Substance by Full Factorial Design Evaluated Liquid Chromatography Method

ABSTRACTHerein, our work deals with developing a rapid, accurate, and reliable high‐performance liquid chromatographic technique with the application of quality by design (QbD) experimental tool to quantify Elvitegravir drug (Integrase inhibitors of antiretroviral drug) and its novel related impurities at low levels. Further, this unique method has been validated to prove its consistency. Separation of N‐methyl Elvitegravir (Impurity‐I), 2‐fluoro Elvitegravir (Impurity‐II), and Elvitegravir isopropyl ester (Impurity‐III) in Elvitegravir drug substance was executed by using Kinetex C18 column (150 mm × 2.1 mm × 1.7 µm) with water, acetonitrile, formic acid, and triethylamine (690:300:6:2, v/v/v/v/), mobile phase (MB‐A) and acetonitrile, formic acid and triethylamine (990:6:2, v/v/v), MB‐B. This developed analytical technique gave a well‐separated peak at the retention times of 9.126 min (Elvitegravir), 7.713 min (Impurity‐I), 7.972 min (Impurity‐II), and 8.548 min (Impurity‐III) adequate with the peak properties as per the United States Pharmacopeia guideline. The method's sensitivity and linearity are established by its detection and quantification limits at low levels with a correlation coefficient of 0.998. The method's repeatability, specificity, and accuracy suggest that this developed technique is a reliable determination strategy for Elvitegravir drug substance and its related impurities (Impurity‐I, Impurity‐II, and Impurity‐III) in a simple, feasible, and affordable way.

B-298 Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine by LC-MS/MS

Abstract Background The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine, a clonidine analog is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing. Methods Remnant urine samples were identified when tested positive for xylazine during routine clinical LC-MS/MS testing. All experiments were performed using an Acquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. Urines were diluted with xylazine-D6 internal standard, hydrolyzed using β-glucuronidase and separated on a 100mm x 2.1mm, 2.6μm Kinetex C18 column (Phenomenex) with a 4 minute step gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Xylazine and 4-OH-xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards (Cayman Chemical and/or Sigma Aldrich), resulting in the following transitions: xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137. Xylazine and 4-OH-xylazine were quantified using calibration curves prepared from 1 - 5,000 ng/mL in negative urine. Product ion scanning (PIS) was used to detect four additional xylazine metabolites, based on spectral matching to previously published full scan high resolution product ion spectra. The highest abundance fragment ions and identified retention times from PIS experiments were used to perform targeted detection for sulfone-xylazine (253>181, 253>147), oxo-xylazine (235>122, 235>114), OH-sulfone-xylazine (269>197, 269>163), and OH-oxo-xylazine (251>197, 251>148). Results Xylazine was detected at >=1 ng/mL in 38% (54/138) of fentanyl positive urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested but xylazine was detected in only 1 urine that did not contain fentanyl.Xylazine concentrations quantified in 61 remnant urines ranged from <1 to >5,000 ng/mL. 4-OH-xylazine concentrations ranged from <1 to 509 ng/mL. We observed five previously reported xylazine metabolites: 4-OH-xylazine, oxo-xylazine, sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine. The mean concentration ratio of 4-OH-xylazine-to-xylazine was 0.11. The mean peak area ratios (metabolite-to-xylazine) for sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine, and oxo-xylazine were 3.5, 3.8, 2.1, and <0.1, respectively. The proposed sulfone-xylazine and OH-oxo-xylazine metabolites were observed to have the most abundant signal of all detected metabolites, although there is currently no commercially available standard to facilitate quantification. Conclusions Xylazine and 4-OH-xylazine were quantitated in 61 patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in plasma and association of xylazine detection with clinical presentation and patient outcomes.

Ultra-performance liquid chromatography-mass spectrometry methods for the determination of the residual quantities of ramipril and hydrochlorothiazide for controlling the cleaning of equipment

Monitoring the completeness of equipment cleaning is essential to prevent cross-contamination of medicinal products. Therefore, it is necessary to develop fast and sensitive methods for studying residual quantities of active ingredients on the surfaces of technological equipment. The aim of the work was to develop and validate analytical methods for the determination of ramipril and hydrochlorothiazide in wash waters by ultra-performance liquid chromatography–mass spectrometry method. Materials and methods. In the study, standard samples of ramipril (USP RS) and hydrochlorothiazide (USP RS), as well as class A reagents, were used. Samples were analysed on a liquid chromatograph with an MS detector (Agilent 6420 and Waters Xevo TQD ACQUITY). We used the Kinetex C18 column (2.1 mm × 30 mm × 1.7 μm); mobile phase – 0.1% formic acid solution in deionised water – Acetonitrile (ratio 73:27 for the determination of ramipril and 91.5:8.5 for the determination of hydrochlorothiazide); mobile phase rate of 0.4 mL/min for the determination of ramipril and 0.35 mL/min for the determination of hydrochlorothiazide; column temperature 45 °C for the determination of ramipril and 40 °C for the determination of hydrochlorothiazide, ionisation mode - electric spray in positive mode; The detection parameters are the mode of registration of the daughter ion 417 → 234 m/z for the determination of ramipril and 298 → 281 m/z for the determination of hydrochlorothiazide. Results and discussion. Methods for the determination of ramipril and hydrochlorothiazide in wash waters by ultra-performance liquid chromatography–mass spectrometry have been developed. The developed methods have sufficient linearity, correctness and precision. The sensitivity of the techniques was confirmed at the level of 0.0026 μg/ml. The techniques can be used in the concentration range of 0.0026 – 0.0255 μg/ml Conclusions. Analytical methods for determining ramipril and hydrochlorothiazide in wash waters have been developed and validated.

Simultaneous determination of icotinib, osimertinib, aumolertinib, and anlotinib in human plasma for therapeutic drug monitoring by UPLC-MS/MS

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as icotinib, osimertinib, and aumolertinib have emerged as promising treatment options for EGFR mutated Non-small cell lung cancer (NSCLC) patients. Additionally, anlotinib, an anti-angiogenic agent targeting VEGFR, FGFR, and PDGFR, has been used in combination with EGFR-TKIs in NSCLC cases. A method utilizing ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated for quantifying icotinib, osimertinib, aumolertinib and anlotinib simultaneously in clinical TDM. The chromatographic separation was performed using a Kinetex C18 column (100 mm × 2.1 mm) and an elution gradient of ammonium acetate in water acidified with 0.1 % formic acid and in acetonitrile. The assay was validated over a linear range of 4–2000 ng/mL for icotinib, 2–1000 ng/mL for osimertinib, 1–500 ng/mL for aumolertinib, and 0.8–400 ng/mL for anlotinib, following the guidelines on bioanalytical methods by FDA. The quantification method exhibited satisfactory performance in terms of selectivity, accuracy (from 91.3 % to 107 %), precision (intra- and inter-day coeffficients of variation ranged from 0.944 % to 7.48 %), linearity, recovery (from 86.0 % to 91.9 %), matrix effect (IS-normalized matrix factors were from 96.7 % to 102 %), and stability. Overall, the method proved to be sensitive, reliable, and straightforward, enabling successful simultaneous determination of blood concentrations of icotinib, osimertinib, aumolertinib, and anlotinib in patients. The validity of the method has been confirmed across various instruments.

Identification and characterization of the rat in-vivo and in-vitro metabolites of tazemetostat using LC-QTOF-MS

In drug discovery, metabolite profiling unveils biotransformation pathways and potential toxicant formation, guiding selection of candidates with optimal pharmacokinetics and safety profiles. Tazemetostat (TAZ) is employed in treating locally advanced or metastatic epithelioid sarcoma. Identification of drug metabolites are of significant importance in improving safety, efficacy and reduced toxicity of drugs. The current study aimed to investigate the comprehensive metabolic fate of TAZ using different in vivo (rat) and in vitro (RLM, HLM, HS9) models. For in vivo studies, drug was orally administered to Sprague–Dawley rats with subsequent analysis of plasma, feces and urine samples. A total of 21 new metabolites were detected across various matrices and were separated on Phenomenex kinetex C18 (2.5 μm; 150 × 4.6 mm) column using acetonitrile and 0.1% formic acid in water as mobile phase. LC-QTOF-MS/MS and NMR techniques were employed to identify and characterize the metabolites from extracted samples. The major metabolic routes found in biotransformation of TAZ were hydroxylation, N-dealkylation, N-oxidation, hydrogenation, hydrolysis and N-acetylation. In silico toxicity revealed potential immunotoxicity for TAZ and few of its metabolites. This research article is the first time to discuss the complete metabolite profiling including identification and characterization of TAZ metabolites as well as its biotransformation mechanism.

Study on optimal harvesting period and rational medicinal parts of Zanthoxylum nitidum based on statistics of main effective components

To determine the optimal harvesting period and rational medicinal parts of Zanthoxylum nitidum, the main effective components of cultivated Z. nitidum samples, which originate from various growth years, harvesting months, and different parts were analyzed and compared with the wild samples. HPLC was performed on a Kinetex C18 column(4. 6 mm×100 mm, 2. 6 μm) with the gradient elution of 0. 3% phosphoric acid solution-acetonitrile(80 ∶ 20) containing 0. 2% triethylamine. The flow rate was 1. 0 m L·min-1, and the detection wavelength was 273 nm. The column temperature was 30 ℃. Nitidine chloride and chelerythrine, the main effective components, were determined as the markers. The results showed there was no significant difference in the contents of the main effective components among the roots of wild and cultivated Z. nitidum, as well as the roots and roots + stems of cultivated Z. nitidum. The statistical results of HCA and PCA indicated that the roots and stems could be clearly distinguished, but no distinction could be made between wild and cultivated products, which was consistent with the results of the significance analysis. The total contents of nitidine chloride and chelerythrine in roots and stems of Z. nitidum of 1-6 years old were 0. 114%-0. 256% and 0. 030%-0. 133%, respectively. These results suggested a positive correlation between the content of the main effective components and the growth years. No significant difference was observed between the contents of samples harvested in different seasons, indicating that the harvest season had no effect on the content of the main effective components of the Z. nitidum samples. The total contents of nitidine chloride and chelerythrine of the dried Z. nitidum samples(excluding branches) from three plantation bases were 0. 308%±0. 123% in Yunfu, 0. 192%±0. 025% in Maoming, and 0. 197%±0. 052% in Nanning, respectively, and they were all not less than 0. 15%, or in other words, the roots(including fibrous roots, taproots, and underground stems) and stems(aboveground stems) of Z. nitidum transplanted for more than 2. 5 years can meet the medical requirements. This study demonstrates that the cultivated Z. nitidum could be used as a valid substitute for the wild Z. nitidum, which provides a guarantee for the sustainable development and the application of Z. nitidum resources. The stems and roots could be considered medicinal parts of Z. nitidum. It is recommended to revise the medicinal parts of Z. nitidum to dried roots and stems in the next edition of Chinese Pharmacopoeia, and the medicinal parts can be harvested all year round. In order to ensure the content of effective components and clinical effectiveness, the root and stem should be harvested for medical use after the seedlings of Z. nitidum have been transplanted for more than three years.

Development and validation of a high-performance liquid chromatography with tandem mass spectrometry method for quantification of tofacitinib in human plasma

Introduction. The use of tofacitinib as a pharmacological treatment for rheumatoid arthritis remains relevant in the light of the predicted increase in prevalence of the disease. At the same time, due to the withdrawal of several foreign pharmaceutical companies from the Russian pharmaceutical market, there has been a heightened demand for domestically produced medications, including generic formulations of tofacitinib. Registration of generic drugs necessitates the conduct of bioanalytical studies. Development and validation of a method for quantifying the analyte in biosamples remains to be a crucial part of the bioequivalence studies.Aim. The aim of this research is to develop and validate a method for the quantitative determination of tofacitinib in human plasma using high-performance liquid chromatography as the separation system coupled with a tandem mass spectrometer for detection purposes.Materials and methods. Biosample preparation was based on plasma proteins precipitation using acetonitrile. Baricitinib was selected as an internal standard. The analytical range of the method was 1.00 to 200.00 ng/mL and was further expanded to 0.30 to 200.00 ng/mL during the analytical phase of the study. The mobile phase consisted of water and acetonitrile, both acidified with formic acid (0.1 % v/v). The stationary phase was a Phenomenex Kinetex C18 column [100 × 3.0 mm, with a particle size of 5 µm (Phenomenex, USA)]. Sample separation and detection were carried out using high-performance liquid chromatography coupled with a tandem mass spectrometry (HPLC-MS/MS), operating in positive ion mode. The multiple reaction monitoring (MRM) transitions selected for the analyte and the internal standard were 313.30 to 173.00 m/z and 371.90 to 186.00 m/z, respectively.Results and discussion. The developed assay was validated in accordance with the current requirements of regulatory documentation from the EAEU (Eurasian Economic Union), FDA (US Food and Drug Administration), and EMA (European Medicines Agency) with the following parameters being evaluated: selectivity, specificity, carry-over, matrix effect, recovery, calibration curve, lower limit of quantitation, accuracy, precision, stability. The validated method was applied in the analytical part of a bioequivalence study of domestically produced generic tofacitinib.Conclusion. A method for the quantitative determination of tofacitinib in human blood plasma with an analytical range of 0.30–200.00 ng/mL was developed and validated. Application of the assay during the analytical phase of bioequivalence study of generic tofacitinib confirms the possibility of using the method in similar bioanalytical investigations.

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.

Simultaneous Determination of Fipronil, Permethrin, and Their Key Related Substances in a Topical Drug Product by a Single Stability-Indicating High-Performance Liquid Chromatography Method.

The topical veterinary drug product containing fipronil and permethrin provides an effective repellent protection and high insecticidal efficacy for dogs. The objective of this study was to develop a stability-indicating high-performance liquid chromatography (HPLC) method for simultaneous detection and quantification of fipronil, permethrin, their key degradation products, and butylated hydroxytoluene (BHT) in a topical drug product. The two active ingredients, their degradation products, and the antioxidant (BHT) were separated by a gradient elution on a Phenomenex Kinetex C18 column (150 × 3 mm, 2.6 µm particle size) maintained at 37°C with H2O acetonitrile isopropyl alcohol 85% H3PO4 (65.5 + 32.5 + 4/0.0053, v/v/v/v) as mobile phase A and acetonitrile (100%) as mobile phase B. The flow rate was 0.9 mL/min, and analytes were detected and quantified at 235 nm. The specificity of the method was demonstrated by adequate separation of fipronil, permethrin, their degradation products, and BHT in the forced degraded finished product. The linearity of the method was demonstrated in the range of 0.2% to 150% of target analytical concentration of both active ingredients and 50% to 150% for BHT. Excellent recoveries of fipronil, permethrin, and BHT in placebo spiked active ingredient solutions in the linearity range showed sufficient accuracy of the method. The LOQ and LOD of the method were determined to be 0.2% and 0.07% of the analytical concentration. A robustness study did not identify any critical parameter that adversely affected the separation and quantification. Here, we report the development and validation of a robust, stability-indicating HPLC method for identification and assay of fipronil, permethrin, and BHT, including estimation of fipronil's and permethrin's degradation products in a topical drug product for dogs. The new HPLC method permits the acquisition of data for all analytes of interest for a topical finished drug product containing fipronil, permethrin, and BHT.

Metabolomic Profiles in Patients with Cervical Cancer Undergoing Cisplatin and Radiation Therapy.

This study was aimed to evaluate endogenous metabolic changes before and after cisplatin and radiation therapy in patients with cervical cancer via untargeted metabolomic analysis using plasma samples. A total of 13 cervical cancer patients were enrolled in this study. Plasma samples were collected from each patient on two occasions: approximately one week before therapy (P1) and after completion of cisplatin and radiation therapy (P2). Of the 13 patients, 12 patients received both cisplatin and radiation therapy, whereas one patient received radiation therapy alone. The samples were analyzed using the Ultimate 3000 coupled with Q ExactiveTM Focus Hybrid Quadrupole-OrbitrapTM mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation utilized a Kinetex C18 column 2.1×100 mm (2.6 μm) (Phenomenex, Torrance, CA, USA), and the temperature was maintained at 40°C. Following P2, there were statistically significant increases in the concentrations of indoxyl sulfate, phenylacetylglutamine, Lysophosphatidyethanolamine (LysoPE) (18:1), and indole-3-acetic acid compared with the concentrations observed at P1. Specifically, in the human papillomavirus (HPV) noninfection group, indoxyl sulfate, LysoPE (18:1), and phenylacetylglutamine showed statistically significant increases at P2 compared with P1. No significant changes in metabolite concentrations were observed in the HPV infection group. Indoxyl sulfate, LysoPE (18:1), phenylacetylglutamine, and indole-3-acetic acid were significantly increased following cisplatin and radiation therapy.

Novel method for determination of colistin sulfate in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its clinical applications in critically ill patients

Colistin is a last-resort antibiotic used for treating infections caused by carbapenem-resistant Gram-negative bacteria, particularly in critically patients, nevertheless its therapeutic window is narrow, and requires monitoring. A determination method suitable for clinical detection is conducive to ensure its efficacy and safety of patients with severe infection. We developed and validated a concise and accurate high-performance liquid chromatography-tandem mass spectrometry method for the determination of colistin A and B in human plasma. We used a Kinetex C18 column (50 mm × 2.1 mm, 2.6 μm) with acetonitrile (containing 0.1% formic acid) as the protein precipitant and water (containing 0.2% formic acid and 5 mmol/L ammonium formate) - acetonitrile (containing 0.2% formic acid) as the gradient elution. The calibration curves were linear over concentration ranges of 0.06–4.00 μg/mL (colistin A) and 0.1–7.0 μg/mL (colistin B). The precision, accuracy, matrix effect, extraction recovery, and stability were all validated. This method was applied to the therapeutic drug monitoring for 50 critically ill patients. The trough, peak, and average steady-state concentrations of these patients were 0.8 ± 0.4, 1.4 ± 0.5, and 1.0 ± 0.4 μg/mL, respectively. And the concentrations of colistin in human plasma were closely related to the patient's renal function.

Development and validation of a simple and affordable LC-UV method for identification and assay of selected antimicrobial medicines

Antimicrobials, particularly antibiotics, are among the most common classes of drugs reported as substandard and falsified (SF) in developing countries. Therefore, it is important to develop simple and affordable analytical methods for the quality control of antimicrobial medicines. In this study, a liquid chromatographic method with ultraviolet detection (LC-UV) was developed and validated for the screening and quantification of 13 antimicrobial medicines and one beta-lactamase inhibitor in pharmaceutical formulations. LC separation was carried out on a Kinetex C18 column (150 mm × 4.6 mm, 2.6 µm) with gradient elution. The mobile phase consisted of mixtures of acetonitrile-water-10 mM phosphate buffer pH 3.5 at ratios of 3:92:5, v/v/v for mobile phase A and 50:45:5, v/v/v for mobile phase B with a flow rate of 0.5 mL/min. The screening method was intended for confirmation of the identity of the actives and validated for specificity and robustness, whereas the quantification method (using only a different detection wavelength) was further validated in terms of linearity, accuracy, sensitivity and precision (repeatability, intermediate precision). For all compounds, the method was found to be linear (r2 > 0.999), precise (%RSD < 1%), accurate (% recovery of 98–102%), sensitive, specific and robust. The developed LC method was successfully applied for the identification and assay of 12 antimicrobial samples from Ethiopia. Among the 12 samples analyzed, one (8.3%) product was confirmed to be falsified.

An LC-MS/MS method for determination of the bromodomain inhibitor ZEN-3694 and its metabolite ZEN-3791 in human plasma.

We have developed and validated a novel LC-MS/MS method for the simultaneous quantification of ZEN-3694 and its active metabolite ZEN-3791 in human plasma afterprotein precipitation. Stable isotope-labeled versions were used as internal standards. Chromatographic separation was achieved on a Kinetex C18 column using 0.1% formic acid in H2O and 0.1% formic acid in MeOH as mobile phases. Detection was performed via positive electrospray ionization mode with multiple reaction monitoring. The assay exhibited linearity in the concentration range of 5-5000ng/ml for both analytes. Intra- and inter-assay precision and accuracy were within ±11%. ZEN-3694 and ZEN-3791 recoveries were between 93 and 105%. This LC-MS/MS assay is an essential tool to study ZEN-3694 in an ongoing clinical trial (NCT04840589).

A validated UPLC-MS/MS method for quantification of pyrotinib and population pharmacokinetic study of pyrotinib in HER2-positive breast cancer patients.

Pyrotinb has been approved for the treatment of HER2-positive advanced or metastatic breast cancer in China. However, the plasma concentration of pyrotinb in different patients varies greatly, and in the course of treatment, if patients have intolerable adverse reactions, the drug dosage will be reduced or even stopped. This study set out to establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the determination of pyrotinb in human plasma, analyze the population pharmacokinetics (PPK) of pyrotinib and assess the influence of patient variables on PK of pyrotinib in patients with HER2 positive breast cancer. An UPLC-MS/MS method was developed to measure pyrotinib in human plasma. Utilizing a gradient elution procedure and a Kinetex C18 column (2.1mm × 100mm, 1.7μm), sample separation was accomplished in 5.5min. Pyrotinb extraction via protein precipitation was used as a sample pre-treatment technique. In total, 50 patients provided 158 plasma samples, which were identified and used in the PPK investigation. The non-linear mixed-effects modeling (NONMEM) approach was used to assess the plasma concentrations and covariates information. For the final PPK model evaluation, external evaluation, non-parametric bootstrap, visual predictive check (VPC), and goodness-of-fit (GOF) were used. The UPLC-MS/MS method for determining plasma concentration of pyrotinib in patients had good selectivity and linearity in the range of 1-1,000ng/mL. Pyrotinib concentration profile in HER2-positive breast cancer patients was well described by a single-compartment PPK model with first-order absorption and elimination. The formulas for the final estimated values of overall parameters of CL/F and Vd/F and Ka are respectively: , , . No dosage adjustment was advised, despite the possibility that the total protein levels could have a substantial impact on the apparent distribution volume of pyrotinib with limited magnitude. In this study, an UPLC-MS/MS method was established to determine the concentration of pyrotinib in human plasma. A population pharmacokinetic model of pyrotinib in HER2 positive breast cancer patients suggested that low serum total protein reduced the clearance rate of pyrotinib in patients. Clinical medical staff should pay attention to the liver function of patients with abnormal serum total protein and be alert to the occurrence of adverse drug reactions.

Determination of Tyrosine Kinase Inhibitor Icotinib in Rat Plasma using UPLC-MS/MS and its Application In vivo Pharmacokinetic

Objective: The purpose of this study was to develop a UPLC-MS/MS method for the determination of icotinib concentrations in blood plasma. Methods: For plasma sample preparation, protein precipitation with acetonitrile was utilized. Analytes were separated on a Kinetex C18 column using 10 mM ammonium acetate containing 0.2% formic acid and methanol (30:70) as the mobile phase, with a gradient flow rate ranging from 0.2 ml·min-1 to 0.4 ml·min-1. The total chromatographic analysis duration was 4.5 minutes. The UPLC system was connected to a mass spectrometer via an electrospray ionization (ESI) interface operated in positive ion mode. Mass monitoring was conducted in multiple reaction monitoring (MRM) modes, with precursor-to-product transitions being m/z 392.06→304.07 for icotinib and m/z 248.00→120.09 for the internal standard, tinidazole. This method has been used for a pharmacokinetic study in rats that were orally administered a single dose of 30 mg/kg icotinib. Results: The assay showed good linearity over concentration ranges of 1-1000 ng/ml for icotinib, with the correlation coefficient exceeding 0.99. The lower limit of quantitation (LLOQ) was established at 1 ng/ml. Both intra- and inter-day precisions (RSD, %) were below 8.23%. The results demonstrated that stability, matrix effect, extraction recovery, carryover effect and dilution stability were all within the acceptable conditions. The primary pharmacokinetic parameters in SD rats after oral administration of icotinib (30 mg·kg-1 ) were as follows: t1/2 = (2.92 ± 0.87)h, Cmax = (2168.65 ± 268.72)ng/ml, Tmax = (0.70 ± 0.27)h, AUC=(9.69 ± 1.95)ug/mL•h, Vd = (14.51 ± 5.60)L, and CL = (3.19 ± 0.59)L/h. Conclusion: A simple and sensitive UPLC-MS/MS method was developed and validated for the determination of icotinib in pharmacokinetic studies.

A Rapid and Simple HPLC-MS/MS Method for the Quantitative Determination of Colistin for Therapeutic Drug Monitoring in Clinical Practice.

Colistin is the last-line option for the treatment of multidrug-resistant gram-negative bacterial infections with narrow therapeutic window. It is essential to ensure its efficacy and safety by therapeutic drug monitoring (TDM). Quantitative determination of colistin is difficult due to its complex ingredients. Previous determination methods demand intricate sample pre-treatment which are not only time-consuming but also costly, and is difficult to apply in clinical practice. Therefore, in order to carry out quantitative determination of colistin accurately and quickly, we establish a rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with simple sample pre-treatment process. The sample was purified by acetonitrile to remove the plasma protein. Then purified colistin was effectively separated from terfenadine, an internal standard (IS) using Phenomenex Kinetex C18 column (50.0×2.1mm, 5µm) with acetonitrile and water mobile phase at a flow rate of 0.5 mL/min and 40°C column temperature. Colistin and IS were monitored in positive ion mode. Our method expressed good linearity in 50.0~6000 ng/mL of colistin B and 28.31~3397.51 ng/mL of colistin A in plasma. Methodology validations, including selectivity, precision, accuracy, recovery, stability, matrix effect, and dilution integrity met acceptance criteria of Bioanalytical Method Validation (M10) of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).

Determination of 145 pharmaceuticals and personal care products in eleven categories in water by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry

Pharmaceuticals and personal care products (PPCPs) are emerging contaminants frequently detected in aquatic environments at trace levels. These chemicals have diverse structures and physicochemical properties and includes pharmaceuticals like antibiotics, antihypertensive drugs, antiviral drugs, and psychotropic drugs that are widely used in large quantities worldwide. Considering the large number of pharmaceuticals currently in usage, it is crucial to establish a priority list of PPCPs that should be monitored and/or treated first. An accurate understanding of the occurrence and levels of PPCPs in aquatic environments is essential for providing objective materials for monitoring these emerging contaminants. Therefore, accurate, efficient, sensitive, and high-throughput screening techniques need to be established for determining and quantifying PPCPs. This study developed a method for the determination of 145 PPCPs (grouped into eleven categories: antibiotics, antihypertensive drugs, antidiabetic drugs, antiviral drugs, β-receptor agonists, nitroimidazoles, H2 receptor antagonists, psychotropic drugs, hypolipidemic drugs, non-steroidal anti-inflammatory drugs, and others) in water. The method was based on large volume direct injection without sample enrichment and cleanup and used ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS). Water samples were collected and filtered through a 0.22-μm regenerated cellulose (RC) filter membrane. Subsequently, Na2EDTA was added to the samples to adjust their pH to 6.0-8.0. Internal standards were mixed with the solutions, and because of the addition of Na2EDTA, the interference of metal ions could be eliminated in the determination of compounds, especially for tetracycline and quinolone antibiotics. Among the six filter membranes tested in this study (PES, PFTE-Q, PFTE, MCE, GHP, and RC), RC filter membranes were screened for water sample filtration. The UHPLC-MS/MS parameters were optimized by comparing the results of various mobile phases, as well as by establishing the best instrumental conditions. The 145 PPCPs were separated using an Phenomenex Kinetex C18 column (50 mm×3 mm, 2.6 μm) via gradient elution. The mobile phases were 0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium formate and acetonitrile for positive ion modes, 5 mmol/L aqueous solutions of ammonium formate and acetonitrile for negative ion modes. The samples were quantified using the scheduled multiple reaction monitoring (scheduled-MRM) mode with electrospray ionization in positive and negative ion modes. A standard internal calibration procedure was used to calculate contents of sample. The established method was systematically verified, and it demonstrated a good linear relationship. The average recoveries of the 145 PPCPs at the three spiked levels were in the range of 80.4%-128% with relative standard deviations (RSDs, n=6) of 0.6%-15.6%. The method detection limits (MDLs) ranged from 0.015 to 5.515 ng/L. Finally, the optimization method was applied to analyze the 145 PPCPs in 11 surface water samples and 6 drinking water samples. Overall, 93 (64%) out of the 145 analytes were detected. The total contents of the PPCPs in surface water samples ranged from 276.9 to 2705.7 ng/L. The detection frequencies of antidiabetic, antiviral, and psychotropic drugs were 100%. The total contents of the PPCPs in drinking water samples ranged from 140.5 to 211.5 ng/L, and antibiotics, antidiabetic drugs, and antiviral drugs comprised the largest proportion of analytes (by mass concentration) in drinking water samples. Our method exhibited high analytical speed and high sensitivity. It is thus suitable for the trace analysis and determination of the 145 PPCPs in environmental water and showed improved detection efficiency for PPCPs in water, indicating that it has a high potential for practical applications. This study can extend technical support for further pollution-level analysis of PPCPs in water and provide an objective basis for environmental management.

Identification, characterisation and in silico ADMET prediction of ozenoxacin and its degradation products using high‐performance liquid chromatography‐photodiode array and liquid chromatography‐quadrupole time‐of‐flight‐tandem mass spectrometry

RationaleOzenoxacin (OXC) is an antibiotic used topically to treat impetigo. This study aimed to evaluate the degradation products (DP) of OXC drug substance under different stress conditions, including hydrolysis, oxidation, thermal and photolysis, in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q1A(R2) and Q1B. The analytical technique was validated in compliance with ICH Q2(R1) requirements.MethodsThe drug substance underwent degradation under various forced degradation conditions, including thermal, oxidative, photolytic and hydrolytic (neutral, acidic and basic) degradation. Overall, four DPs were formed under oxidative stress conditions with AIBN. The formed DPs were identified and separated using a Shimadzu LC system with a reversed‐phase Phenomenex Kinetex C18 column (4.6 × 250 mm, 5 μm), using 10 mM NH4CH3COOH buffer (pH −5.0) as mobile phase A and acetonitrile as mobile phase B at a detection wavelength of 254 nm.Results and ConclusionThe drug was found to be stable in neutral, acidic, basic and oxidative degradation conditions with hydrogen peroxide. Liquid chromatography‐electrospray ionisation‐quadrupole time‐of‐flight‐tandem mass spectrometry‐ was employed in positive ionisation mode to analyse both the drug and the mass of the identified DP. The mechanism and the pathway of mass fragmentation have been proposed. The developed method was accurate, repeatable, linear and selective for further research. The ADMET Predictor software was applied to predict the in silico toxicity of the drugs and its DPs as well as their physicochemical characteristics.