Ammonium Formate Solution Research Articles

Development and validation of HPLC-UV and LC-MS/MS methods for the quantitative determination of a novel aminothiazole in preclinical samples

Aminothiazoles are the important class of chemical groups which have proven their broad range of biological activities. A novel aminothiazole (21MAT) was quantified in analytical solutions using a high-performance liquid chromatography (HPLC) approach that was developed and partially validated for the analysis of in vitro experimental samples. An isocratic elution on reverse phase Phenomenex® Luna C18 (50 mm × 4.6 mm, 5 μm) column with 55% 0.1% v/v orthophosphoric acid in water and 45% of orthophosphoric acid in acetonitrile at a flow rate of 1 mL/min was used. The analyte was detected at 272 nm. Similar to this, a robust bioanalytical technique, LC-mass spectrometry (LC-MS/MS) was created and verified to measure 21MAT in rat plasma for use in in vitro screening study samples and early-stage pharmacokinetic research. The protein precipitation method was used to extract 21MAT from plasma. The mixture of 95: 5% v/v methanol: acetonitrile and 0.1% v/v formic acid, along with 15% of 5 mM ammonium formate solution, was used to separate the mixture on a reverse phase Waters Xterra RP® C18 (150 mm × 4.6 mm, 5 μm) column at a flow rate of 1 mL/min. Using electro spray ionisation mode in multiple reaction monitoring mode, the analyte and internal standard (a structural analogue) were both identified. According to current criteria, all validation parameters (specificity, selectivity, accuracy, precision, recovery, matrix factor, hemolysis effect, and stability) were evaluated in rat plasma. The area response of 21MAT was found to be linear over the concentration range of 1.25–1250 ng/mL in rat plasma. Both techniques are suitable for use in any format of preclinical research and were sufficiently reliable to measure 21MAT precisely in various matrices. In silico prediction helped in understanding absorption, distribution, metabolism, excretion, and toxicity (ADMET) behaviour of the molecule. Both developed LC-MS/MS and HPLC-UV methods were successfully used to quantify the analyte in in vitro screening study samples.

Determination of three methylimidazole compounds in cosmetics by high performance liquid chromatography-tandem mass spectrometry

Methylimidazole compounds are byproducts formed during the caramel-coloring process and are used in various cosmetics. In addition metronidazole is an antibacterial and anti-inflammatory drug commonly used in modern medicine and is used in cosmetics to treat acne in the short-term. The illegal addition of metronidazole during cosmetics production can result in residual 2-methylimidazole (2-MEI), which, along with 4-methylimidazole (4-MEI), is a class 2B carcinogen. Therefore, establishing efficient, accurate, and sensitive analytical techniques for analyzing methylimidazole compounds in cosmetics is an urgent objective. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining 1-methylimidazole (1-MEI), 2-MEI, and 4-MEI in cosmetics was developed. Cosmetics samples were extracted via ultrasonication in acetonitrile and purified using a mixed cation-exchange (MCX) solid-phase extraction (SPE) column, with subsequent drying under a stream of nitrogen and redissolution in acetonitrile. The resulting solution was then filtered through a 0.22 μm organic filter membrane for further testing. The analytes were separated using an XBridge® shield RP18 chromatographic column (150 mm×4.6 mm, 3.5 μm) and isocratically eluted with 20 mmol/L ammonium formate solution (containing 0.1% formic acid)-acetonitrile (98∶2, v/v). The target compounds were ionized by electrospray ionization (ESI) source, analyzed in multi-reaction monitoring (MRM) mode, and quantified using the external standard method, with the peak area of the quantitative ion and the mass concentration of the compound taken as the longitudinal and transverse coordinates, respectively. Matrix-matching working curves were also constructed. 1-MEI exhibited good linear relationships in the range of 5-200 μg/L, with correlation coefficients (r2)≥0.9994, while 2-MEI and 4-MEI showed good linearities in the range of 2-100 μg/L with r2≥0.9984. The three methylimidazole compounds exhibited limits of detection (LODs) and quantification (LOQs) of 10-30 μg/kg and 25-100 μg/kg, respectively. Under three spiked levels (LOQ, 2LOQ, 10LOQ), the recoveries of three methylimidazole compounds were 80.9%-107.9%, with relative standard deviations (RSDs, n=6) of 1.2%-12.8%. The practicability of the method was examined using 48 cosmetic samples; 4-MEI was detected in nine samples at contents of 26-1000 μg/kg, while two samples contained 240 and 267 μg/kg of 2-MEI, respectively. 1-MEI was not detected in any of the 48 samples tested. The developed method is simple, fast, and highly sensitive, and provides methodological support for assessing risks and monitoring the three methylimidazole compounds in cosmetics through screening.

Advanced HPLC Method with Diode Array Detection Using a Phenyl-Bonded Column for Simultaneous Quantitation of Three Sunscreen Filters in a Moisturizing Sunscreen Cream for Acne-Prone Skin

This study introduces a novel, robust, and efficient method for the simultaneous quantitative determination of three sunscreen filters, namely, 4-methylbenzylidene camphor, octyl methoxycinnamate, and avobenzone, in a moisturizing sunscreen cream specifically designed for acne-prone skin. The method employs high-performance liquid chromatography with photodiode-array detection, providing a reliable separation of the analytes. Chromatographic separation was achieved using a Fortis Phenyl analytical column (150.0 × 2.1 mm, 5 μm), with isocratic elution at a flow rate of 0.4 mL/min. The mobile phase was composed of a 57/43 (v/v) mixture of acetonitrile/45 mM aqueous ammonium formate solution, ensuring sufficient resolution and peak symmetry for the target compounds. The method was validated comprehensively for critical performance parameters, including linearity, precision, accuracy, and robustness. Linearity was established across a suitable range for all three analytes, with high correlation coefficients. Precision was confirmed with intra-run and total precision coefficients of variation of ≤4.6%, while accuracy assessments yielded a percent recovery between 98.6 and 100.4, for all quality control levels. Additionally, the method was able to effectively separate the sunscreen filters from other cosmetic ingredients, such as [β-(1.3), (1.6)-D-glucan], low molecular weight (LMW) hyaluronic acid and plant extracts ensuring specificity in complex formulations. This straightforward and time efficient sample preparation process, involving methanol extraction followed by serial dilution, makes the method suitable for routine quality control in cosmetic laboratories. The method was successfully applied to the analysis of two different lots of a commercial sunscreen cream, achieving excellent recovery for all filters, ranging between 94.6% and 99.8%, thus demonstrating its reliability and applicability for the quality control of cosmetics.

Development of a UHPLC-MS/MS Method for the Determination of Moxidectin in Rat Plasma and Its Application in Pharmacokinetics.

The aim of the present study was to establish a simple and reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method and apply it for the determination of pharmacokinetics of moxidectin-loaded microspheres (MOX-MS) in rats. Plasma samples were processed using a simplified liquid-liquid extraction method and were separated using an Agilent Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of a 10 mM ammonium formate solution with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min for 5 min. Avermectin B1a was used as an internal standard (IS). The sample was injected at a volume of 10 μL with a column temperature of 35 °C and detected in a positive ion mode. A good linear response across the concentration range of 1.00-200 ng/mL (r2 > 0.99) and a lower limit of quantification (LLOQ) of 1.00 ng/mL were achieved. The extraction recovery of moxidectin exceeded 94.1%, the matrix effect was between 91.2% and 96.2%, the accuracy ranged from 100.1 to 103.6%, and the relative standard deviation (RSD) did not exceed 15% for the intra- and inter-day accuracy and precision. The pharmacokinetic results showed that MOX-MS significantly decreased Cmax, prolonged T1/2, and improved bioavailability. The developed method significantly reduced the assay volume, shortened detection time, simplified sample processing methods and saved assay costs, which may contribute to the development of the new antiparasitic drug.

The retention features of nitrogen-containing heterocyclic compounds in reversed-phase and hydrophilic HPLC-MS modes

Aromatic five- and six-membered nitrogen-containing heterocyclic compounds are biologically active substances and are widely used in biochemistry and medicine. In addition, such compounds are known as ecotoxicants formed during oxidation processes in industrial wastewater. High-performance liquid chromatography-mass spectrometry is widely used to determine nitrogen-containing heterocycles in various complex mixtures. Octadecyl silica gel is the main sorbent for chromatographic separation. However, octadecyl silica gel does not always enable satisfactory separation in the presence of highly polar isomeric molecules, and more selective chromatographic approaches are required. One of these is hydrophilic chromatography, which facilitates the separation of polar compounds. The aim of the study was to comparatively characterize the retention of a number of five- and six-membered nitrogen-containing heterocyclic compounds during reversed-phase and hydrophilic liquid chromatography with mass spectrometric detection. In addition, spectrophotometric detection at a wavelength of 210 nm was carried out. In the study, we used sorbents based on octadecyl silica gel and unmodified silica gel. High-resolution mass spectrometry was used in the electrospray ionization mode. We studied 19 nitrogen-containing heterocyclic compounds. We used aqueous acetonitrile mobile phases with added aqueous solutions of formic acid and diethylamine as acid-base modifiers. An ammonium formate solution was used as a modifier during hydrophilic chromatography. Reversed-phase chromatography showed that the introduction of 0.1% aqueous formic acid solution increased the ionization and the mass spectrometer signal intensity, but did not always result in a satisfactory separation of isomers upon elution from the octadecyl silica gel surface. The introduction of 0.01% diethylamine solution provided no significant improvement in the separation compared to the mobile phase without modifiers. The use of unmodified silica gel in hydrophilic chromatography mode allowed us to separate isomeric five-membered heterocycles, which was demonstrated by narrow symmetric peaks, but did not result in selective separation of isomeric diazines. Thus, we studied the retention of 19 nitrogen-containing heterocyclic compounds using reversed-phase and hydrophilic liquid chromatography with electrospray ionisation. It was determined that hydrophilic chromatography on unmodified silica gel could be used for satisfactory separation of isomeric five-membered heterocyclic compounds of different classes. For six-membered heterocycles, satisfactory separation was achieved by elution from the surface of octadecyl silica gel using aqueous acetonitrile mobile phase without modifiers.

Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry

The pool of free, genetically encoded AA in plasma plays an essential role not only as substrate for every protein synthesized in the body but also as signaling molecules that regulate a wide range of physiological processes. Here we present a method for the analysis of 19 of the 20 encoded AA (except Cys) in dairy cow plasma. Isolated plasma or standards for the 19 AA were gravimetrically mixed with an internal standard mix consisting of 13C isotopes of each AA. Plasma proteins were precipitated on acetonitrile and supernatants transferred to glass vials. For pre-column derivatization, plasma supernatants were buffered with sodium borate (pH 9.5 −10), and AA were derivatized with 9-fluorenylmethoxycarbonyl (Fmoc) chloride. Analytes were isolated by solid-phase extraction using a strong-anionic ion exchange column and dry eluates were reconstituted in mobile phase consisting of 70% water solution of ammonium formate and 30% acetonitrile. Amino acid derivatives were separated by reverse phase liquid chromatography over 17.5 min with a C18 column in which acetonitrile increased to 80% over the first 11 min of the method, before returning to initial levels. Electrospray ionization on negative mode was used for most AA, except Arg and Pro, for which positive mode yielded superior results. Single or double (only Lys) derivatives were measured by single quadrupole mass spectrometry. We hypothesized that pre-column Fmoc derivatization would yield optimal resolution for quantitative analysis of the 19 targeted AA and their respective 13C internal standards, with limits of quantitation beyond physiological ranges. All the 19 AA were detected with minimal background noise. An 11-point standard curve was developed for each AA. Limits of quantitation were beyond concentrations observed in plasma samples of lactating dairy cows, except for Gly that upper curve points had to be removed to maintain linearity, limiting quantitation to the upper range of physiological concentration. After removing the 4 highest concentrations from the Gly standard curve, coefficients of determination were greater than 0.999 for all the AA. Recovery of spiked AA from plasma samples ranged from 89.9% for Phe to 100.3% for Trp. Instrument repeatability averaged 0.91 and ranged from 0.33 for Val to 2.29 for Arg. Meanwhile, sample preparation method repeatability averaged 2.02 and ranged from 1.14 for Tyr to 3.34 for Arg. While robust methods have been developed, they depend on either availability of sophisticated instruments, mostly limited to core facilities (i.e., tandem MS methods), long and expensive chromatography without specific internal standards for each AA (i.e., HPLC-UV and HPLC-FLD), or unstable derivatization (GC-MS). Here we describe a method with high throughput, stable derivatization, high precision and recovery, and potentially more affordable than most existing methods. This method could help dairy nutritionists to consider plasma AA information for diet formulation strategies, potentially reducing feeding costs and N emissions.

Determination of 11 nutrients in liquid milk by ultra-performance liquid chromatography-tandem mass spectrometry

To establish an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of 11 nutritional components(thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, biotin, choline, L-carnitine) in liquid milk. Milk samples were shaken with 20 mmol/L ammonium formate solution and heated in a water bath at 100 ℃ for 30 min, then incubated with papain and acid phosphatase at 45 ℃ for 16 h, the lower liquid was collected after centrifugation for analysis. UPLC separation was performed on an ACQUITY~(TM) HSS T3(3.0 mm×150 mm, 1.8 μm) column, 2 mmol/L ammonium formate(containing 0.1% formic acid) solution and acetonitrile(containing 0.1% formic acid) were used as mobile phase. Quantitative detection was performed by internal standard method. 11 nutritional components can be effectively separated and detected in 12 min, and the linear correlation coefficients(R~2) were all above 0.995. The limits of detection(LODs) were between 0.05 and 0.50 μg/L, and the limits of quantification(LOQs) were between 0.20 and 1.25 μg/L. The recovery rates of three-level addition were 85.6%-119.3%, and the precision RSDs were between 3.68% and 7.82%(n=6). Based on the detection of 60 liquid milk samples from 5 different animals, it was found that the contents of 11 nutrients in liquid milk from different milk sources were significantly different, but pyridoxine could not be detected. The method can quantitatively detect 11 water-soluble nutrients, including free and bound forms, by effective enzymolysis. It is sensitive, reproducible and can meet the needs of quantitative detection.

Silver-decorated palladium on carbon catalyst for enhanced ammonium formate dehydrogenation

Palladium (Pd)-based catalysts efficiently convert ammonium formate solution to hydrogen at low temperatures (<100 °C), but they tend to deactivate quickly during stability testing.

Separation and characterization of related substances of Lurasidone hydrochloride by LC-QTOF-MS techniques

In the present study, a reliable LC-QTOF-MS method was developed and employed for the separation and characterization of process-related substances and forced degradation products of Lurasidone hydrochloride. The chromatographic separation was carried out using an Agilent Poroshell 120 Bonus-RP C18 column (100 mm × 4.6 mm, 2.7 µm) and a mobile phase consisting of a gradient elution of 10 mM ammonium formate solution and methanol. The degradation studies followed the guidelines outlined in ICH Q1A (R2). It was observed that Lurasidone hydrochloride exhibited instability under photolytic, alkaline, and oxidative stress conditions, while remaining relatively stable under acidic and thermal stresses. Through positive ESI-QTOF mass spectrometric analysis, fourteen related compounds in total, including both process-related and stress degradation products, were identified based on the accurate masses of parent and product ions and calculated elemental compositions. Amongst these substances, nine had not been previously reported, and their formation mechanisms were speculated. The process-related substances were further confirmed by NMR spectra determination, and suggestions were proposed to eliminate them. This study highlights the potential for monitoring and controlling related substances during the manufacturing processes, providing valuable insights for process optimization and quality control of Lurasidone hydrochloride.

Determination of Mycotoxins in Wheat Grain by LC-MS/MS using Modified QuEChERS Sample Preparation

The procedure of the mycotoxins determination in wheat grain was developed by LC-MS/MS using modified QuEChERS sample preparation. The method of simultaneous determination of six mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, T-2 toxin and zearalenone) in wheat grain was validated according to the following criteria: specificity, linearity, limits of detection (LOD), limits of quantification (LOQ), accuracy, and precision. The chromatographic and mass spectrometric conditions for separation and determination of the mycotoxins were optimized during the study. It was established that the most optimal mobile phase for the separation of six mycotoxins is 5 mМ ammonium formate solution in methanol : water : formic acid (5 : 94.9 : 0.1 by volume, eluent A and 95:4.9:0.1 by volume, eluent B). It was found that the correlation coefficients for six mycotoxins range from 0.9990 to 0.9998 (R2 > 0.995). Limits of detection (LOD) and limits of quantification (LOQ) of the mycotoxins are below the maximum permitted levels set by the European Union (EU). Percent recovery in the range from 88 to 103 % indicates the acceptability of the mycotoxin extraction procedure. The relative standard deviation (RSD, %) of the measurement results under conditions of repeatability ranged from 1.94 to 8.76 %. The obtained data suitability evaluation of method (validation) corresponds to criteria European Commission Regulation (EC) No. 401/2006. The validation results showed that LC-MS/MS method with using of modified QuEChERS sample preparation is effective and suitable for the simultaneous quantitation of mycotoxins in wheat grain.

Development of a double monitoring system for the determination of Cr(VI) in different water matrices by HPLC-UV and digital image-based colorimetric detection method with the help of a metal sieve-linked double syringe system in complexation.

This study reports a cheap, efficient, sensitive, and simple double monitoring analytical method for trace determination of Cr(VI), which is toxic and harmful even at very low concentrations. A metal sieve-linked double syringe (MSLDS) system was used to help the formation of chromium complex (Cr-diphenyl carbazide, DPC) subsequently determined by high-performance liquid chromatography-ultraviolet (HPLC-UV) and digital image-based colorimetry (DIC) systems. The metal complex was eluted through a Phenomenex-Aqua C18 with a mobile phase comprising of 50mM ammonium formate solution (pH 4.0):acetonitrile (78:22, v/v) and detected by the UV detector at the wavelength of 581nm. Under their optimum conditions, the HPLC-UV and DIC systems exhibited good linearity in ranges of 10-500µg L-1 and 100-1000µg L-1, respectively. The percent relative standard deviations (RSD%s) calculated for the lowest concentrations of both systems fell below 10%, and this confirmed good repeatability for replicate measurements. The accuracy of the proposed methods was evaluated by performing spike recovery experiments on wastewater, river water, and tap water samples. The calculated recovery results were in the range of 81.5-105.5% for HPLC-UV system and 93.8-111.1% for the DIC system. These results indicate that the proposed methods are suitable for routine Cr(VI) determination in terms of their rapidness, simplicity, good repeatability, and low cost.

Retention mechanisms of imidazoline and piperazine-related compounds in non-aqueous hydrophilic interaction and supercritical fluid chromatography based on chemometric design and analysis

The experimental design methodology based on central composite design of experiments was applied to compare the retention mechanisms in supercritical fluid chromatography (SFC) and non-aqueous hydrophilic interaction liquid chromatography (NA-HILIC). The selected set consists of 26 compounds that belong to imidazoline and serotonin receptor ligands. The different chemometric tools (multiple linear regression, principal component analysis, parallel factor analysis) were used to examine the retention, as well as to identify the most significant retention mechanisms. The retention mechanism was investigated on two different stationary phases (diol, and mixed-mode diol). In NA-HILIC, the mobile phase contains acetonitrile as a main component, and methanolic solution of ammonium formate (+ 0.1% of formic acid) as a modifier. The same mobile phase modifier was used in SFC, with a difference in the main component of the mobile phase which was CO2.The retention behaviour differs significantly between HILIC and SFC conditions. The retention pattern in HILIC mode was more partition-like, while in SFC the solute-sorbent interactions allowed retention. The retention mechanism between mixed-mode diol and the diol phases varies depending on the applied chromatographic mode, e.g., in HILIC the type of stationary phase significantly affects the elution order, while in SFC this was not the case. The HILIC retention behaviour was influenced by the number of tertiary amines-aliphatic, and N atom-centred fragments in tested compounds. On the other hand, the number of pyrrole and pyridine rings in the structure of the compound showed correlation with their SFC retention, simultaneously increasing the molecular weight and rapid elution of larger compounds. It was found that temperature surprisingly plays a major role in SFC mode. The increase in temperature reduces the relative contribution of enthalpy factors to total retention, so the separation in SFC was more entropy-controlled. For further pharmaceutical research and optimization, the SFC would be considered more beneficial compared to HILIC since it gives good selectivity in separation of chosen impurities.

Establishment to measure oxycodone in plasma with liquid chromatography-tandem mass spectrometry.

Oxycodone (OXY) is classified as a “strong opioid” in the World Health Organization system of cancer pain treatment. However, OXY also causes severe adverse reactions, such as respiratory depression. Thus, in order to adjust the dosage of OXY for well‐managed pain relief with less toxicity, we tried establishing and validating a system for measuring plasma concentrations of OXY using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Human pooled plasma samples containing OXY diluted with 0.1% formic acid solution and internal standard (papaverine) were used for solid‐phase extraction. The eluents were injected into LC–MS/MS with Unison UK‐Silica column (100 × 2 mm, 3 μm, Imtakt). Mobile phase was a mixture of 1 mM ammonium formate solution and acetonitrile containing 0.1% formic acid (50:50). OXY in plasma could be measurable with good linearity in a concentration range of 2–100 ng/ml by using 100 μl of plasma within 4 min. Relative standard deviations of all validation results were within ±15%. These results suggest that our established method using LC–MS/MS to measure OXY in plasma would be useful to adjust the dosage of OXY in order to obtain its efficacy and to avoid its adverse reactions.

Determination of 19 illegally added chemical ingredients in hair loss prevention cosmetics by ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry

建立了超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)法,结合高分辨质谱数据库,用于快速筛查及定量分析防脱发化妆品中19种非法添加化学成分。实验比较了提取溶剂的影响,优化了色谱条件和质谱条件。采用ACQUITY UPLC BEH C18色谱柱(100 mm×2.1 mm, 1.7 μm)进行色谱分离,以乙腈和2 mmol/L甲酸铵水溶液(含0.05%甲酸)为流动相进行梯度洗脱,运行时间为20 min。在电喷雾电离源、正离子扫描模式下,采用MSE模式采集19种化合物的质谱信息。根据标准品的色谱保留时间、母离子和碎片离子精确质量数及丰度比,在UNIFI软件中构建了非法添加化学成分的筛查数据库。结果表明,19种化合物线性关系良好,检出限为0.025~0.05 μg/g,定量限为0.075~0.15 μg/g。以溶液型防脱育发剂和膏霜型防脱洗发水两种常见防脱发化妆品为代表性基质进行加标回收试验,在不同的添加水平下,19种化合物的平均回收率为68.6%~118.8%,相对标准偏差(RSD)为0.3%~10.3%。通过对比保留时间以及母离子和碎片离子的精确质量数,该方法可用于77批样品的筛查。最终两批育发剂产品中均确证检出米诺地尔和非那雄胺两种非法添加化学成分,并对米诺地尔的裂解途径进行了推测;采用基质匹配外标法定量,结果表明其中米诺地尔含量高达60 mg/g,非那雄胺含量高达0.31 mg/g,说明防脱发类化妆品中存在多种化学组分同时添加的情况,且添加的药物含量非常高,具有很高的安全风险。该方法操作简便、灵敏度高、重现性好,能够同时用于防脱发化妆品中多种类型非法添加化学药物的筛查和定量分析,为防脱发化妆品的安全监管提供了强有力的技术手段。

The use of atmospheric-pressure chemical ionization for pesticide analysis using liquid chromatography mass spectrometry.

Based on the regulations of the Ministry of Health and Welfare (MOHW) of Taiwan in 2017, an analysis of 373 pesticides in food was conducted using the MOHW official method. The analyses involved the use of either liquid chromatography mass spectrometry (LC-MS) with electrospray ionization (ESI) or gas chromatography mass spectrometry (GC-MS) with electron ionization (EI). In this study, the applicability of detecting pesticides using atmospheric pressure chemical ionization (APCI) was investigated and evaluated. The pesticides were separated using an aqueous solution of ammonium formate with methanol as the mobile phase, and ionization efficiency was compared between APCI, ESI, and EI coupled with triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) acquisition. Among the 196 pesticides that were originally analyzed by ESI, 164 could be successfully detected by APCI with 6 showing a higher sensitivity when APCI was used. Among the 177 pesticides that were analyzed by EI, 43 could be successfully detected by APCI. The results also showed that APCI gave superior ionization efficiency for pesticides containing triazine, imidazole, triazole, and pyrazole groups.

Simultaneous determination of beauvericin and four enniatins in eggs by ultra-performance liquid chromatography-tandem mass spectrometry coupled with cold-induced liquid-liquid extraction and dispersive solid phase extraction

新型生物毒素白僵菌素(BEA)和恩镰孢菌素(ENNs)是由镰刀菌种产生的有毒代谢产物,主要污染谷物及其制品,会威胁人类健康,因此受到人们越来越多的关注。该工作建立了冷诱导液液萃取-分散固相萃取净化-超高效液相色谱-串联质谱法(CI-LLE-DSPE-UPLC-MS/MS)同时测定鸡蛋中白僵菌素和4种恩镰孢菌素残留的分析方法。以乙腈-水-乙酸(79:20:1, v/v/v)为提取溶剂,采用冷诱导液液萃取与分散固相萃取净化相结合的方法进行样品处理,同时,对影响待测物提取与净化效率的提取溶剂、冷冻萃取温度与时间、净化剂用量等因素和色谱条件进行了优化。样品经20 mL提取液涡旋提取20 min,放入-40 ℃冰箱静置30 min后,取2 mL上层溶液经70 mg C18粉末净化,离心,上清液于40 ℃浓缩至近干,残留物用1 mL 80%(v/v)乙腈水溶液溶解,进样分析。以乙腈与5 mmol/L甲酸铵溶液作为流动相进行梯度洗脱,经ACQUITY UPLC BEH C18色谱柱(100 mm×2.1 mm, 1.7 μm)分离,采用ESI+电离,在多反应监测模式下采集,白僵菌素采用稳定同位素内标法定量,4种恩镰孢菌素采用基质匹配曲线外标法定量。结果表明,5种待测物在0.1~50.0 μg/L范围内线性关系良好,相关系数(r2)为0.9983~0.9997,该方法的检出限(LOD)为0.05~0.15 μg/kg,定量限(LOQ)为0.20~0.50 μg/kg。以阴性鸡蛋样品为基质,在低、中、高3个浓度水平(0.5、5.0、25.0 μg/kg)下进行加标试验考察方法的准确度与精密度,各待测物的平均回收率为81.1%~106%,相对标准偏差(RSD)为0.27%~9.79%。采用所建立的方法对农村散养鸡蛋与市售鸡蛋进行检测,结果表明,BEA在散养鸡蛋的检出率为30.4%, 4种ENNs均未被检出。该方法灵敏度高,稳定性好,回收率高,定量准确,简单易操作,适用于禽蛋食品中白僵菌素与恩镰孢菌素的同时快速测定。

Separation and structural elucidation of cefsulodin and its impurities in both positive and negative ion mode in cefsulodin sodium bulk material using liquid chromatography/tandem mass spectrometry

The structure identification of impurities in cephalosporins was reported. However, to the best of our knowledge, there was no report on impurities of cefsulodin sodium, which is necessary for the quality control. Thus, the aim of this study was to separate and characterize the impurities in cefsulodin sodium raw material using LC-MS/MS. The analytes were separated on a Kromasil 100-5C18 column (4.6 mm ×250 mm, 5 μm) by a gradient elution with a mobile phase consisting of 1% ammonium sulphate aqueous solution and acetonitrile in the first dimension. The separation in the second dimension was carried on a Shimadzu Shim-pack GISS C18 column (50 mm × 2.1 mm, 1.9 μm) with a mobile phase consisting of 10 mM ammonium formate solution and methanol. The fragmentation behaviors of cefsulodin and its impurities were studied and the structures of impurities were deduced based on the MSn data. The structures of ten unknown impurities were proposed based on the work carried out in this study. The degradation behaviors of cefsulodin sodium were also studied. It revealed that the storage environment of cefsulodin sodium should be preserved in a dry, cool and dark closed container. Based on the characterization of impurities, this study not only revealed the mechanism of impurity production, thus providing guidance to pharmaceutical companies for manufacturing process improvement and impurity control, but also provided a scientific basis for further improvement of official monographs in pharmacopoeias.

Effect of intestinal flora changes on pharmacokinetics of astragaloside Ⅳ

The effect of intestinal flora changes on the pharmacokinetics of astragaloside Ⅳ in rats with type 2 diabetes mellitus was explored in this study. The rat model in preliminary experiment was established by high-sugar and high-fat diet combined with the intraperitoneal injection of low-dose streptozotocin(STZ). Rats were divided into model group, astragaloside Ⅳ group, berberine group and combination group(five rats in each group). After two weeks of gavage, the rats' feces was taken for 16 S rRNA sequencing of intestinal flora. Pharmacokinetic experiments were performed on astragaloside Ⅳ in the four groups one day after the preliminary experiment. Plasma samples were precipitated in methanol with ginsenoside Rb_1 as an internal standard, and the plasma concentrations of astragaloside Ⅳ at different time points were determined by UPLC-MS/MS. The chromatographic separation was performed on a Waters Acquity UPLC BEH-C_(18) column(2.1 mm×100 mm, 1.7 μm) via gradient elution. The mobile phase was acetonitrile(A) and 5 mmol·L~(-1) ammonium formate solution with 0.2% formic acid(B). The flow rate was 0.4 mL·min~(-1), the injection volume 5 μL and the column temperature 40 ℃. The mass spectrometry was carried out with electrospray ionization source(ESI) in multiple reaction monitoring and positive ion modes. The specificity, linearity range, accuracy, precision, stability and dilution effect of the method all met the requirements for the determination of astragaloside Ⅳ in plasma. Plasma concentration-time curves were plotted and relevant pharmacokinetic parameters were calculated by DAS 3.2.8. The results showed that the concentration of absorbed astragaloside Ⅳ increased within 0-3.95 h and began to decline since 3.95 h. After 36 h, the metabolism was complete. The area under the plasma concentration-time curve(AUC_(0-t)) and the peak concentration(C_(max)) of astragaloside Ⅳ were increased in the three administration groups compared with the model group, but without significant difference, which suggested that the pharmacokinetic characteristics of saponin components would not necessarily change after the drug-induced alteration of intestinal flora.

Revealing Changes in Curcumin Bioavailability using Vitamin C as an Enhancer by HPLC-MS/MS

Background: Curcumin (CUR), a natural isolated compound from turmeric, helps in fighting many diseases, but the broad application of curcumin has been limited ascribed to low bioavailability. Objective: The aim of this study is to pursue the enhancement of curcumin bioavailability through coadministration of vitamin C. Methods: Such purpose was achieved through the analysis of curcumin pharmacokinetics by highperformance liquid chromatography coupled with electrospray ionization - tandem mass spectrometry (HPLC - ESI - MS/MS). The plasma was separated on a C18 reverse-phase column using acetonitrile and ammonium formate solution (pH 6.5; 2.0 mM) at 0.8 mL/min. MS/MS detection was carried out in negative mode using mass patterns of m/z 367.0 > 216.7 for curcumin and m/z 265.2 > 223.9 for internal standard (honokiol). Results: Successful application of the proposed method in the pharmacokinetic study presented clear changes in key pharmacokinetic parameters, including the growth of AUC (0-t) up to 2.4 times, a 2.2- fold increase of Cmax, 2.2-fold loss of CL, and 1.5-fold diminishment of t1/2. Conclusion: An HPLC-ESI-MS/MS method for the determination of curcumin in rat plasma and validated the improvement of bioavailability of curcumin through co-administration of vitamin C was determined. These changes were reasoned to the inhibition of lipid peroxidation induced by the use of vitamin C. Such a simple strategy is possible to become an alternative for enhancing curcumin efficiency in practice.

Separation and characterization of impurities and isomers in cefpirome sulfate by liquid chromatography/tandem mass spectrometry and a summary of the fragmentation pathways of oxime-type cephalosporins.

Although the identification of degradation products of cefpirome sulfate has been reported, there has been no report concerning the impurities in bulk samples of this compound. To meet the requirements of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use, the structures of impurities whose content are over 0.1% need to be confirmed. Thus, characterization of the impurities in cefpirome sulfate bulk samples is critical for controlling the production of this drug. The structures of cefpirome sulfate impurities were investigated using two-dimensional liquid chromatography (LC) coupled to electrospray ionization tandem mass spectrometry. In the first LC dimension, a Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 μm) was used, and the mobile phases were 0.03 M ammonium dihydrogen phosphate solution and acetonitrile. In the second dimension, the column was a Shimadzu Shim-pack GISS C18 column (50 mm × 2.1 mm, 1.9 μm), and the mobile phases were 10 mM ammonium formate solution and methanol. An ion trap time-of-flight mass spectrometer operated in both positive and negative ion mode was employed in this study. Nine impurities and isomers in cefpirome sulfate, eight of which were previously unknown, were separated and characterized. Structures were proposed for the eight unknown compounds based on the MSn fragmentation data. The degradation behavior of cefpirome sulfate was also studied. Based on the characterization of impurities and isomers, this study could be used to improve the quality control of the cefpirome sulfate drug recommended in pharmacopoeias. The degradation behavior of cefpirome sulfate provides a basis for the selection of storage conditions.