Ammonium Formate Aqueous Solution Research Articles

Impurity profile analysis of amphotericin B using on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Amphotericin B (AmB) is a polyene-macrolide antimicrobial drug with a broad antibacterial spectrum and remarkable efficacy against deep fungal infections. It binds to ergosterol on the fungal cell membrane and alters its permeability, thereby destroying the membrane. AmB is a multicomponent antimicrobial medication that contains a wide range of impurities, rendering quality analysis extremely difficult. In the current Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3), high performance liquid chromatography (HPLC) is applied to examine related substances in AmB. However, this technique presents a number of issues. For instance, the mobile phases used in the HPLC method described in both references contain nonvolatile inorganic salts, which cannot be coupled with a mass spectrometry (MS) detector. In addition, because the mobile phases used have a low pH, the component/impurities of AmB drug can easily be degraded or interconverted during the analytical process, leading to reduced analytical accuracy. Therefore, the accuracy and sensitivity of this method must be improved. In this study, a method based on on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (2D HPLC-Q TOF/MS) was developed to analyze the impurity profile of AmB in accordance with the Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3). The method combines on-line dilution and a multiple-capture HPLC system to achieve the efficient separation of AmB component/impurities. It also resolves the issue of poor solvent compatibility in 2D HPLC, increases the analytical flux, enhances the automation capability, reduces the mutual conversion of AmB and its impurities during the analytical process, and increases the detection sensitivity of the method. MS was also used to determine the structural inference of unstable components and impurities. An XBridge Shield C18 column (250 mm×4.6 mm, 3 μm) was used for first-dimensional-liquid chromatography with gradient elution using methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (10∶30∶60, v/v/v, pH 4.7) as mobile phase A and methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (12∶68∶20, v/v/v, pH 3.9) as mobile phase B. An Xtimate C8 column (10 mm×2.1 mm, 5 μm) was used as the trap column, and trapping and desalting were performed using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v). An Xtimate C8 column (250 mm×2.1 mm, 5 μm) was used for second-dimensional-liquid chromatography with gradient elution using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v) and 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (5∶95, v/v) as mobile phases. The data were collected in positive-ion mode. In this study, the structures of six impurities in amphotericin B were inferred, according to the fragmentation, the MS and MS2 spectra of each impurity. The developed method can be used to quickly and sensitively analyze the impurity profile of AmB. Furthermore, the research results on impurity profiles can be applied to guide improvements in AmB production.

Ultra-performance liquid chromatography-tandem mass spectrometry for quantification of pyrethroid pesticide residues in freshwater fish and shrimp

A sophisticated Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) approach was devised for the concurrent quantification of pyrethroid pesticide residues in freshwater fish and shrimp. This analytical method involved ultrasonic extraction using acetonitrile, succeeded by sample purification employing Primary Secondary Amine (PSA) and Zirconium Dioxide Silica (Z-Sep+) following low-temperature centrifugation. Chromatographic separation was achieved with an ACQUITY UPLC BEH C18 column utilizing a mobile phase of a 2 mmol/L ammonium formate aqueous solution and methanol, with detection conducted through ESI + ionization in Multiple Reaction Monitoring (MRM) mode. The assay demonstrated exceptional linearity for seven pyrethroids across a concentration range of 0.5–10 μg/L, evidenced by correlation coefficients surpassing 0.999. Detection limits were established between 0.08 and 0.52 μg/kg, and the lower limits of quantification ranged from 0.28 to 1.72 μg/kg. Furthermore, recovery rates for blank samples were outstanding, ranging from 91.40% to 108.83% at concentrations of 1.0, 4.0, and 20.0 μg/kg, with Relative Standard Deviations (RSDs) between 0.94% and 8.13%. This methodology proved to be rapid, convenient, sensitive, and robust, offering stable recovery rates. Notably, it facilitated the detection of seven pyrethroid pesticide residues in freshwater fish and shrimp within an expedited timeframe of 15 min. This pioneering technique constitutes a significant advancement in the development of new methods for ensuring food safety.

Degradation kinetics of β-nicotinamide mononucleotide based on reliable HPLC quantitative method

To explore the stability characteristics of β-nicotinamide mononucleotide(NMN) and provide data support for NMN production, preparation, and related product development, this study established a simple HPLC content determination method for NMN in simple substrate and investigated the degradation behavior, degradation products, and degradation kinetics of NMN under various chemical, physical, and biological conditions. The HPLC method employed a Welch Xtimate AQ-C_(18) column(4.6 mm×250 mm, 5 μm), a detection wavelength of 266 nm, a column temperature of 30 ℃, a flow rate of 1.0 mL·min~(-1), an injection volume of 5 μL, and a mobile phase consisting of methanol(A) and a 10 mmol·L~(-1) ammonium formate aqueous solution(B) with a gradient elution(0-6.7 min, 0-4% A; 6.7-13 min, 4%-18% A; 13-14.2 min, 18% A; 14.2-15 min, 18%-0 A; 15-22 min, 0 A). This method provided good separation between NMN and potential impurities and degradation products, and had a wide linear range, short analysis time, good durability, high accuracy, an average sample recovery rate of 98.71%, and an RSD of 1.2%. The instrument precision had an RSD of 0.26%, and the linearity within the examined range was excellent(R~2≥0.999 9). This method can be applied for NMN content determination in simple substrate. The degradation process of NMN in aqueous solution followed apparent first-order kinetics, with the degradation rate primarily influenced by high temperature and pH. NMN was more stable in low-temperature, neutral, or weakly acidic/alkaline environments. Strong acids or strong alkalis could accelerate its degradation, and its degradation rate was less affected by pepsin and trypsin. In an aqueous solution at room temperature, it followed the kinetic equation lg C_t=0.005 7t + 4.817 2, with t_(0.9) and t_(1/2) values of 95.58, 860.26 h, respectively. The results suggest that pH and temperature are the main factors affecting the stability of NMN in aqueous solution, and low temperature, moisture protection, and a weakly acidic environment are more conducive to the storage and application of NMN and its products.

Liquid chromatography-high resolution mass spectrometry analysis of 300 illegally added drugs and their analogues in functional milk powder for the elderly

Various types of milk powder purportedly providing diverse health functions have emerged with the growth of the country's elderly population. Some manufacturers illegally add chemical drugs to their products to achieve their reported benefits, which poses a threat to consumer health. The existing standard methods are inapplicable to such complex sample matrices and require testing based on functional claims and classification. These limitations not only consume manpower and resources but also seriously impede daily regulatory efforts to detect unknown risk substances. In this study, a high-throughput method for the screening and quantitative analysis of 300 illegally added chemical drugs in functional milk powder and an identification strategy for unknown structural analogues were established using Zeno SWATH® data-independent acquisition (DIA) ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) technology combined with a QuEChERS sample purification method. The QuEChERS purification process was developed according to the characteristics of milk powder matrix. The supernatant was separated on a Kinetex F5 column (100 mm×3.0 mm, 2.6 μm) by gradient elution using 5 mmol/L ammonium formate aqueous solution (0.1% (v/v) formic acid, ) and methanol-acetonitrile (1∶1, v/v) as mobile phases. The method was validated in terms of selectivity, linearity, limits of detection and quantification (LODs and LOQs, respectively), matrix effect, accuracy, and precision. Based on a screening database for the 300 target substances, electron-activated dissociation (EAD) fragmentation was applied to obtain rich secondary MS fragmentation information, and unknown structural analogues were identified and confirmed through fragment attribution analysis. The results indicated that all compounds had good linear relationships in certain ranges with correlation coefficients >0.99. The LODs and LOQs were 0.04-2.7 and 0.2-8.0 μg/kg, respectively. The average recoveries at three spiked levels were in the range of 73.1%-125.2%, and the relative standard deviations were ≤14.8% (n=6). When the developed method was applied to detect illegally added chemicals in 60 functional milk powder samples, it detected benzoguanidine and sildenafil and successfully identified ethylphenidate, which is the structural analogue of an amphetamine. The proposed method is simple, sensitive, and accurate; thus, it may have practical application value for the daily supervision and law enforcement of milk powders with reported health functions.

Development of A Liquid-Phase Microextraction Method for Simultaneous Determination of Parabens in Lipstick Samples at Trace Levels by High-Performance Liquid Chromatography.

The endocrine-disrupting potential of parabens, as well as their relation to cancer, has sparked significant discussions over their impact. Consequently, analyses of cosmetic products are an essential necessity, particularly in terms of human health and safety. In this study, a highly accurate and sensitive liquid-phase-based microextraction method was developed to determine the five parabens at trace levels by high-performance liquid chromatography. All prominent parameters of the method such as extraction solvent type and amount (1,2-dichloroethane/250μL), and dispersive solvent type and amount (isopropyl alcohol/2.0mL) were optimized to enhance the extraction efficiency of the analytes. The mobile phase consisting of 50mM ammonium formate aqueous solution (pH4.0) and acetonitrile (60:40, v/v) was used to elute the analytes at a flow rate of 1.2mLmin-1 in the isocratic mode. Analytical performance of the optimum method for methyl, ethyl, propyl, butyl and benzyl parabens were determined and the analytes recorded detection limit values of 0.78, 0.75, 0.34, 0.33 and 0.75μgkg-1, respectively. Four different lipstick samples were analyzed under optimum conditions of the developed method, and the amount of parabens quantified in the samples using matrix matched calibration standards was in the range of 0.11-1.03%.

Hydrophilic interaction liquid chromatography–electrospray ionization mass spectrometry combined with fabric phase sorptive extraction for therapeutic drug monitoring of pioglitazone, repaglinide, and nateglinide in human plasma

Polypharmacy in type 2 diabetes is an issue of major concern as the prescription of multiple medi-cations for the management of diabetes-associated comorbidities can lead to drug-to-drug interactions, which can pose serious risks to patients’ health. Within this context, the development of bioanalytical methods for monitoring the therapeutic levels of antidiabetic drugs is notably useful to ensure patients’ safety. In the present work, a liquid chromatography–mass spectrometry method for the quantitation of pioglitazone, repaglinide, and nateglinide in human plasma is described. Sample preparation was performed by fabric phase sorptive extraction (FPSE), and hydrophilic interaction liquid chromatography (HILIC) was implemented for the chromatographic separation of the analytes, using a ZIC®-cHILIC analytical column (150 × 2.1 mm, 3 µm) under isocratic elution. The mobile phase consisted of 10 mM ammonium formate aqueous solution (pH = 6.5)/ acetonitrile, 10/90 v/v, and was pumped at a flow rate of 0.2 mL min−1. Design of Experiments was used during the development of the sample preparation method to gain deeper insight into the effect of various experimental parameters on extraction efficiency, their potential interactions and to optimize the recovery rates of the analytes. The linearity of the assay was assessed over the ranges of 25 to 2000, 6.25 to 500, and 125 to 10000 ng mL−1 for pioglitazone, repaglinide, and nateglinide, respectively. The presented method was fully validated and can be used for the therapeutic monitoring of the targeted analytes in human plasma samples.

A study of impurities in the repurposed COVID-19 drug hydroxychloroquine sulfate using ultra-high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry and liquid chromatography-solid-phase extraction-nuclear magnetic resonance.

RationaleHydroxychloroquine sulfate is effective in the treatment of malaria and autoimmune diseases and as an antiviral drug. However, unreported impurities are often detected in this drug, which pose a health risk. In this study, the structures of hydroxychloroquine and six unknown impurities were analyzed using ultra‐high‐performance liquid chromatography‐quadrupole/time‐of‐flight‐tandem mass spectrometry (UHPLC­Q/TOF/MS/MS), and the structures were characterized using liquid chromatography‐solid‐phase extraction‐nuclear magnetic resonance (LC‐SPE‐NMR) spectroscopy.MethodsAn Agilent InfinityLad Poroshell HPH‐C18 column (100 × 4.6 mm, 2.7 μm) was used. For the analysis of hydroxychloroquine and six unknown impurities, the mobile phase was 20 mM ammonium formate aqueous solution and methanol/acetonitrile (80:20, v/v) using gradient elution. Full‐scan MS and MS2 were performed to obtain as much structural information as possible. In addition, six unknown impurities were separated by semi­preparative liquid chromatography and characterized using LC‐SPE‐NMR.ResultsThe MS2 fragmentation patterns of the impurities were investigated, leading to more structural information and an understanding of the fragmentation pathways of the impurities. The structures of the unknown impurities were confirmed using NMR. In addition, some possible pathways of the formation of the impurities in the drugs were outlined, and these impurities were found to be process impurities.ConclusionsBased on the identification and characterization of these impurities, this study also describes the cause of the production of the impurities and provides insights for companies to improve their production processes and a scientific basis for the improvement of the related pharmacopoeias.

Chiral separation of new chiral insecticide pyraquinil isomers and establishment of analytical methods in vegetables

以全新手性杀虫剂唑虫酯为研究对象,通过筛选手性色谱柱和优化流动相比例,建立了唑虫酯及其氧化代谢物异构体的拆分方法,在此基础上开发利用高效液相色谱-串联质谱(HPLC-MS/MS)同时测定小白菜和蕹菜中唑虫酯及其氧化产物手性异构体的分析方法。以纤维素-三(3,5-二氯苯基氨基甲酸酯)共价键合手性柱(Chiral INC)(250 mm×4.6 mm, 5 μm)为分析柱,乙腈和2 mmol/L甲酸铵水溶液作为流动相进行梯度洗脱分离,在多反应监测负离子模式下进行检测,唑虫酯4个异构体分离度分别为1.63、2.83和1.74,唑虫酯氧化产物异构体分离度为5.82。通过衍生化的方法进一步确定出峰顺序为RS-唑虫酯、SS-唑虫酯、RR-唑虫酯、SR-唑虫酯、S-唑虫酯氧化产物和R-唑虫酯氧化产物。唑虫酯和其氧化产物的手性异构体分别在1.25~1250 μg/L和2.5~2500 μg/L范围内具有良好的线性关系,相关系数(R2)大于0.99。在蕹菜和小白菜样品中同时添加唑虫酯和唑虫酯氧化产物消旋体进行添加回收试验,添加水平为1、20、400 μg/kg(即唑虫酯异构体为0.25、5、100 μg/kg;唑虫酯氧化代谢产物异构体为0.5、10、200 μg/kg),回收率为72.6%~110.6%,相对标准偏差(RSD)均在9.4%以下,其中日内重复性的RSD在0.5%~9.4%之间;日间重复性的RSD在1.0%~8.6%之间,表明该方法具有良好的回收率和精密度。该研究可为唑虫酯这一新型手性农药的环境行为研究及后续质量控制、药效评价等提供相应的分析技术,为新农药开发应用提供有力的技术支撑。

Multi-residue analytical method for detecting pesticides, veterinary drugs, and mycotoxins in feed using liquid- and gas chromatography coupled with mass spectrometry

Modified QuEChERS and triple quadrupole mass spectrometry (LC and GC-MS/MS) technology were used to sequentially analyze pesticides, veterinary drugs, and mycotoxins in feed. In order to analyze the harmful substances that may remain or occur in the feed, we performed optimization experiments for sample preparation and LC-MS/MS and GC-MS/MS conditions. Optimized sample preparation involves extracting 5 g of sample with 15 mL of 0.25 M EDTA and 10 mL of acetonitrile. And some extracts were diluted 10-fold with 100 mM ammonium formate aqueous solution and analyzed by LC-MS/MS, and some extracts were purified through 25 mg PSA and analyzed by GC-MS/MS by adding an analyte protectant. We confirmed the matrix effect of feed ingredients and compound feeds, and added a dilution process after extraction to increase on-site efficiency. Matrix-matched calibration was applied for quantification. Method validation was performed for 197 pesticides, 56 components for veterinary drugs, and 5 components for toxins. All the components showed good linearity (r2 ≥ 0.98) in the developed analytical method. For most compounds, the limit of quantitation was 0.05 mg/kg. The recovery rate experiment was repeated three times at three concentrations including LOQ in feed ingredient, compound feed for livestock, and compound feed for pets. The recovery rate was 70.09–119.76% and relative standard deviations were ≤ 18.91%. And the accuracy and precision were further verified through cross-validation between laboratories. The developed analytical method was used to monitor 414 domestically distributed and imported feeds.

LC-MS/MS monitoring of the colorectal carcinoma cellular uptake and entrapment of sorafenib and its N-oxide active metabolite

Sorafenib (SOR) is a multikinase inhibitor with a mild activity against colorectal cancer cells due to multi-drug resistance mechanisms. Potentiated SOR activity was expected upon combination with some ginger derived compounds due to their interference with intracellular drug metabolism. Studying such combination necessitates the development of a sensitive validated LC-MS/MS method for the determination of intra and extracellular concentration of SOR and its N-oxide metabolite (SNX) in colorectal cancer cells. SOR, SNX and the internal standard (diclofenac sodium) were efficiently separated on Eclipse plus C18 column (3.0 ×150 mm, 5 µm) using isocratic elution with acetonitrile and 0.01 M ammonium formate aqueous solution containing 0.1% formic acid (69:31, v/v). Sample pretreatment using solid phase extraction was optimized and the mean percent recoveries were more than 97.01% for both analytes. Detection was conducted at positive ion multiple reaction monitoring (MRM) mode and the monitored mass transitions were 465.2 → 252.2 for SOR and 481.1 → 286.0 for SNX. The method was linear over the range 0.25 – 200.00 ng/mL (r2 ≥ 0.9992) for SOR and 0.10 – 125.00 ng/mL (r2 ≥ 0.9990) for SNX in both intra and extracellular matrices. The lower limits of quantification (LLOQ) were 0.25 and 0.10 ng/mL for SOR and SNX, respectively. Accuracies were within 94.25 – 109.45% and precision CV values did not exceed 7.63%. The method was able to monitor the cellular uptake and entrapment of both analytes and to prove the positive effect of the ginger derived compounds on SOR activity.

Simultaneous determination of fourteen β2-agonist enantiomers in food animal muscles by liquid chromatography coupled with tandem mass spectrometry

Illegal drug residues in animal derived foods are closely related to human's life and health. Studies on illegal drug residues and the metabolism, such as β2-agonists in animals have attracted more and more attention. In most cases, β2-agonists are suppliedand used astheracemate. The metabolic process and distribution of the two enantiomers in animal tissues are different. Therefore, it is very necessary to develop a simple and fast method for chiral resolution of these drugs in animal tissues. In this paper, a reliable resolution and determination method was presented using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for fourteen enantiomers of seven β2-agonist racemates, clenbuterol (CLE), salbutamol (SAL), cimaterol (CIM), terbutaline (TER), clorprenaline (CLO), tulobuterol (TUL), penbuterol (PEN) in pork, beef, and lamb muscle samples. The samples were added the internal standard solution (IS) and extracted in the alkaline medium with acetonitrile. The further sample purification was accomplished through MCX solid phase extraction cartridge. Chromatographic chiral separation was carried out on a VancoShell chiral column (100 mm × 4.6 mm, 2.7 μm) with an isocratic mobile phase consisting of methanol and 10 mmol mL−1 ammonium formate aqueous solution (85:15, v/v). Under the optimized conditions, the resolution (R) of CIM was 2.0, CLE and PEN were 1.5, the others were all greater than 1.0. Enantiomeric determination was performed in the positive electrospray ionization mode using multiple reaction monitoring (MRM). The correlation coefficient (r) in the range of 0.2–25.0 μg L−1 was above 0.993. The average recoveries at the three spiking levels ranged from 95.3% to 117.7% with the relative standard deviation (RSD) lower than 15%. The limit of detection (LOD) and the limit of quantification (LOQ) of β2-agonist enantiomers was 0.2 μg kg−1 and 0.5 μg kg−1 respectively. The method was successfully applied in the analysis and evaluation of β2-agonist enantiomers in positive food animal muscle samples, CLE, SAL, TEB and CIM enantiomers were detected. The concentrations of the corresponding enantiomers were in the range of 1.06–17.3 μg kg−1, the lowest enantiomer fraction (EF) value was 0.42, and the highest value was 0.69. The work is expected to provide a method for chiral separation and enantiomeric determination of the further study of pharmacology, toxicity and residue elimination of β2-agonist enantiomers.

Development of an eco-friendly and fast HPLC method for quantitative analysis of four nucleosides in Cordyceps and related products.

An eco-friendly and fast HPLC method was developed for the determination of adenosine, inosine, guanosine and uridine in Cordyceps and related products (fermented mycelia of Hirsutella sinensis andPaecilomyces hepiali). The sample was ultrasonically extracted using 0.5% phosphoric acid solutions for 2.5 min. Sample separation was performed on a Poroshell SB-Aq column (50 mm × 4.6 mm, 2.7 μm) using eco-friendly mobile phase consisting of formic acid and ammonium formate aqueous solution at a flow rate of 1.0 mL·min-1. The detection wavelength was 260 nm. The developed HPLC method showed good linearity with correlation coefficients of 1.0000 in the test range. Good precision, repeatability and stability of this method were also observed (RSD ≤ 2.81%). The recovery ranged from 91.84%-105.19% (RSD ≤ 2.59%). Compared with reported methods, the current method did not use harmful organic solvent and took only 10.5 min. It obtained a high eco-score of 91 by the "Analytical Eco-Scale" tool. The developed method is eco-friendly and fast, which is suitable for the quality evaluation of Cordyceps and related products.

Simultaneous determination of nicotinamide and N1 -methylnicotinamide in human serum by LC-MS/MS to associate their serum concentrations with obesity.

A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of nicotinamide and its metabolite N1 -methylnicotinamide in human serum. Serum samples were prepared by protein precipitation with acetonitrile. The chromatographic separation was performed on a Waters Spherisorb S5 CN microbore column (2.0 × 100 mm, 5μm) with gradient elution within 7min. Acetonitrile and 5mm ammonium formate aqueous solution (containing 0.1% formic acid) were used as mobile phases. Nicotinamide, N1 -methylnicotinamide and N'-methylnicotinamide (internal standard) were detected with a triple-quadrupole tandem mass spectrometer in the positive ion mode. Multiple reaction monitoring was used to monitor precursor to product ion transitions of m/z 123.1 → 80.1 for nicotinamide, m/z 137.1 → 94.1 for N1 -methylnicotinamide and m/z 137.1 → 80.1 for the internal standard. The linear ranges of nicotinamide and N1 -methylnicotinamide were 5.000-160.0and 2.500-80.00 ng/ml, respectively. The intra- and inter-day precisions (RSD) of both analytes were within 6.90%. The recoveries were >88%. The analytes were proven to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to determine the concentrations of nicotinamide and N1 -methylnicotinamide in human serum to investigate the association between their concentrations and obesity in 1160 Chinese subjects.

A Validated HPLC-MS/MS Method for Quantification of Methotrexate and Application for Therapeutic Drug Monitoring in Children and Adults with Non-Hodgkin Lymphoma.

PurposeA simple, rapid and reliable method to quantify methotrexate (MTX) in human plasma by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) was established and validated in two laboratories.Patients and MethodsSample separation was achieved on a Synergi Hydro-RP column (50 mm×2.0 mm, 2.5 μm) with a gradient elution program in 3.5 min after a simple protein precipitation with methanol (MeOH) and acetonitrile (ACN) (1:1). About 5 mM ammonium formate aqueous solution with 0.2% formic acid and ACN were used as mobile phase with a flow rate of 0.5 mL/min at 40 °C. Mass spectrometry detection using AB Sciex Triple Quad 4500 mass spectrometer (4500 QQQ) and Qtrap 5500 mass spectrometer (5500 Q-trap) were both characterized by electrospray ionization (ESI) for positive ions in multiple reaction-monitoring (MRM) mode. Quantitative ion pairs were m/z 455.1→m/z 308.0 for MTX and m/z 248.1→m/z 121.0 for tinidazole (TNZ) used as internal standard (IS).ResultsLinear calibration curves were generated over the range of 5–1000 ng/mL (r2> 0.99) on both the 4500 QQQ and 5500 Q-trap, both of the intra- and inter-batch precision were less than 7.67% and accuracy ranged from 96.33% to 108.94%. The recovery and matrix effect were 82.20–93.98% and 102.69–105.28%, respectively.ConclusionAn analytical method transfer was achieved by re-verification in two laboratories to ensure stability and reproducibility and this method has been applied for therapeutic drug monitoring (TDM) successfully in children and adults with NHL, and during routine TDM, two delayed elimination of MTX cases were observed and analyzed.

Pharmacokinetic comparison between Tanreqing Capsules Substitute and Tanreqing Capsules in rats by LC-MS/MS

Due to the limited resource of bear bile powder, the major raw material of Tanreqing Capsules(TRQ), cultured bear bile powder is used as a replacement to develop the Tanreqing Capsules Substitute(TRQS). An LC-MS/MS method was established in this study for simultaneous quantitation of 8 compounds from TRQS in rat plasma: tauroursodeoxycholic acid(TUDCA), taurocheno-deoxycholic acid(TCDCA), ursodeoxycholic acid(UDCA), chenodeoxycholic acid(CDCA), ferulic acid, wogonoside, baicalin, and forsythoside A. Thereby, the pharmacokinetic behaviors of TRQ and TRQS were evaluated. Concentration of endogenous compounds TUDCA, TCDCA, UDCA, and CDCA was determined with the stable isotope surrogate analytes: D4-TUDCA, D4-TCDCA, D4-UDCA, and D4-CDCA. Plasma samples were extracted by acetonitrile-induced protein precipitation. The LC conditions are as follows: Waters BEH C_(18) column(2.1 mm×100 mm, 1.7 μm), mobile phase of 10 mmol·L~(-1) ammonium formate aqueous solution(containing 0.01% formic acid) and acetonitrile-methanol mixture(1∶5). MS conditions are as below: multiple reaction monitoring(MRM), ESI~(+/-). Concentration of UDCA, CDCA, TUDCA, and TCDCA was corrected with a response factor, which is the ratio between the responses recorded for the surrogate and the authentic analyte at the equal concentration. Each of the plasma components showed good linearity(r > 0.995 1). Accuracy and precision met the criteria(inter-day RSD<7.0%, RE 89.98%-112.0%; intra-day RSD<12%, RE 90.41%-111.2%). The recovery was 64.83%-119.9% and matrix effect was 87.15%-113.8%. The validated method was applied for pharmacokinetic study of TRQS and TRQ(po, 0.94 g·kg~(-1)). There was no significant difference in C_(max) and AUC_(0-24 h) of baicalin, UDCA, TUDCA, and TCDCA between the two groups, indicating similar pharmacokinetic behaviors between TRQS and TRQ in rats.

Determination of methamphetamine and methylphenidate in urine by liquid chromatography/time-of-flight mass spectrometry coupled with solid-phase dispersive extraction, and its pharmacokinetic application

A simple and rapid analytical method for the determination of methamphetamine (MA) and methylphenidate (MPD) in urine was developed, which involves liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) and solid-phase dispersive extraction (SPDE). The developed method could be used in pharmacokinetic studies. Oasis® WCX gel was used in SPDE. LC separation was carried out on a Poroshell 120 EC-C18 column (100 mm × 4.6 mm I.D., 2.7 µm) using a mixture (30:70) of methanol/10 mM ammonium formate aqueous solution (pH 3.0). The calibration curves showed good linearity in the range of 2 to 100 ng/mL for MA and 0.5 to 200 ng/mL for MPD. The limits of detection were 0.5 ng/mL and 0.1 ng/mL, and the limits of quantification were 2 ng/mL and 0.5 ng/mL for MA and MPD, respectively. The average recoveries of MA and MPD from urine samples spiked with MA at 5 and 50 ng/mL and MPD at 1 and 100 ng/mL were 93.9–94.8% (RSD: 1.6–2.1%) and 91.5–106.5% (RSD: 1.3–3.0%), respectively. Periodic analyses of urine samples collected from a volunteer receiving clinical doses of MPD revealed the presence of ritalinic acid (RA), an inactive major metabolite of MPD. Furthermore, when the RA/MPD ratios corrected by the urinary creatinine level were plotted against time, the regression curve showed linearity after 4 h. Pharmacokinetic study revealed that the extrapolated line of the regression line reached the origin in the graph, suggesting that the regression line could be used to calculate the dosage and timing of administration.

Determination of Intact Parabens in the Human Plasma of Cancer and Non-Cancer Patients Using a Validated Fabric Phase Sorptive Extraction Reversed-Phase Liquid Chromatography Method with UV Detection.

Parabens have been widely employed as preservatives since the 1920s for extending the shelf life of foodstuffs, medicines, and daily care products. Given the fact that there are some legitimate concerns related to their potential multiple endocrine-disrupting properties, the development of novel bioanalytical methods for their biomonitoring is crucial. In this study, a fabric phase sorptive extraction reversed-phase liquid chromatography method coupled with UV detection (FPSE-HPLC-UV) was developed and validated for the quantitation of seven parabens in human plasma samples. Chromatographic separation of the seven parabens and p-hydroxybenzoic acid was achieved on a semi-micro Spherisorb ODS1 analytical column under isocratic elution using a mobile phase containing 0.1% (v/v) formic acid and 66% 49 mM ammonium formate aqueous solution in acetonitrile at flow rate 0.25 mL min−1 with a 24-min run time for each sample. The method was linear at a concentration range of 20 to 500 ng mL−1 for the seven parabens under study in human plasma samples. The efficiency of the method was proven with the analysis of 20 human plasma samples collected from women subjected to breast cancer surgery and to reconstructive and aesthetic breast surgery. The highest quantitation rates in human plasma samples from cancerous cases were found for methylparaben and isobutylparaben with average plasma concentrations at 77 and 112.5 ng mL−1. The high concentration levels detected agree with previous findings for some of the parabens and emphasize the need for further epidemiological research on the possible health effects of the use of these compounds.

Simultaneous determination of twelve antiepileptic drugs in serum by ultra high performance liquid chromatography-tandem mass spectrometry

A sensitive, high-throughput method was established for the simultaneous determination of 12 antiepileptics in serum by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The antiepileptics were gabapentin, lamotrigine, pregabalin, lacosamide, levetiracetam, topiramate, oxcarbazepine, clonazepam, sodium valproate, carbamazepine, phenobarbital and phenytoin sodium. Phenacetin and chlorzoxazone were used as internal standards. The antiepileptics and internal standards were extracted from serum by protein precipitation using acetonitrile as the precipitant. Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column with a gradient mobile phase comprising 10 mmol/L ammonium formate aqueous solution and methanol (containing 10 mmol/L ammonium formate) at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode with ion mode switching. The results showed good linear trends in their respective concentration ranges and the correlation coefficients (r2) were greater than 0.992. The spiked recoveries of the 12 antiepileptics in serum were 90.80%-114.0% at the three spiked levels. The intra-assay (n=6) and inter-assay (n=3) precisions were less than 13.2% and 14.8%, respectively. The method has high specificity and sensitivity, and it can be used for clinical blood concentration monitoring and pharmacokinetic studies of the 12 antiepileptics.

Intrinsic stability study of L-α-glycerylphosphorylcholine with HPLC method development and validation

L-α-Glycerylphosphorylcholine (L-α-GPC) is effective to control the symptoms of cognitive decline for the patients of Alzheimer’s disease. In this study, an HPLC method coupled with a refractive index detector was developed to evaluate the intrinsic stability of L-α-GPC. The separation of L-α-GPC and its major potential degradation products was achieved on a normal-phase silica gel column (4.6 mm × 250 mm) with the mobile phase consisting of methanol–20 mM ammonium formate aqueous solution (pH 3.2) (65:35, v/v) in isocratic mode. The HPLC method was validated satisfactorily with respect to precision, accuracy and robustness. It is found that L-α-GPC is stable under the photolytic, thermal, oxidative and acidic conditions, while relatively sensitive to alkaline condition due to the specific breakage of phosphate ester bond in the moiety of L-α-GPC. A preliminary kinetics study for the alkaline degradation was conducted with the corresponding kinetics parameters obtained. It can be concluded that the developed HPLC method is capable of distinguishing the stability difference between the two phosphate ester bonds characterized on the L-α-GPC chemical structure.

LC-MS/MS method for quantitation of the CK2 inhibitor silmitasertib (CX-4945) in human plasma, CSF, and brain tissue, and application to a clinical pharmacokinetic study in children with brain tumors

Silmitasertib (CX-4945) as a potent and selective inhibitor of CK2 exhibited promising in vitro and in vivo anti-cancer activity. An assay employing cation-exchange solid phase extraction (SPE) followed by LC-MS/MS analysis was successfully developed and validated for the quantitation of silmitasertib in human plasma, brain tissue, and human cerebrospinal fluid (CSF). Reverse phase chromatographic separation was achieved using Synergi™ hydro-RP column (4 μm, 75 × 2.0 mm) and gradient elution with 5 mM ammonium formate aqueous solution (pH 6.5) as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. Multiple reaction monitoring (MRM) transition of m/z 350.2 → 223.2 and m/z 316.2 → 223.2 were chosen for detection of silmitasertib and internal standard (CX-4786) respectively. Since silmitasertib concentration in patient plasma is expected to be in a wide range due to the study design, two calibration curves with range 0.2–125 ng/ml and 32–20,000 ng/ml were established. A different curve ranging from 2 to 40 ng/g was used for measurement of silmitasertib in brain tissue, while another calibration curve ranging from 0.2 to 20 ng/ml was established for CSF. All these calibration curves corresponding to different matrices showed good linearity (R2 > 0.99) over the concentration range. This assay demonstrated excellent precision below 15% and accuracies between 85% and 115% within–day and between–day for all the concentration levels in each matrix. This assay was also validated for each matrix for selectivity, sensitivity, matrix effects, recovery, and stability. We applied the validated method to the analysis of plasma silmitasertib for a clinical study.