Characterization of N-acetyltryptophan degradation products in concentrated human serum albumin solutions and development of an automated high performance liquid chromatography-mass spectrometry method for their quantitation

N-acetyltryptophan (NAT) has long been used as a stabilizer in some protein solutions, such as human serum albumin, to prevent oxidative protein degradation. However, the fate of NAT has not been discussed in literature. Two NAT degradation products have been observed in concentrated albumin solutions (20% and 25%) and identified as 1-acetyl-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid and 1-acetyl-3a,8a-dihydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid. To monitor the levels of these two previously unidentified NAT degradation products in concentrated albumin solutions, a fully automated method, incorporating online size exclusion chromatography (SEC) trapping and reversed-phase high performance liquid chromatography-mass spectrometry (HPLC-MS) with multiple reaction monitoring (MRM) analysis, has been developed and validated for their quantitative analysis. The method does not require an internal standard. The only sample manipulation is to obtain an albumin concentration of 4% in all standards and test HPLC samples. A limit of quantitation (LOQ) as low as 20 ng/mL has been achieved for both compounds. This method can readily be adopted for the quantitative determination of other small molecules in concentrated protein solutions.     

Simultaneous determination of estrogenic and androgenic hormones in water by isotope dilution gas chromatography-tandem mass spectrometry

A rapid gas chromatography-tandem mass spectrometry (GC-MS/MS) analytical method was developed for the simultaneous analysis of 7 estrogenic hormones (17α-estradiol, 17β-estradiol, estrone, mestranol, 17α-ethynylestradiol, levonorgestrel, estriol) and 5 androgenic hormones (testosterone, androsterone, etiocholanolone, dihydrotestosterone, androstenedione) in aqueous matrices. This method is unique in its inclusion of all 12 of these estrogens and androgens and is of particular value due to its very short chromatographic run time of 15 min. The use of isotope dilution for all analytes ensures the accurate quantification, accounting for analytical variabilities that may be introduced during sample processing and instrumental analysis. Direct isotopically labelled analogues were used for 8 of the 12 hormones and satisfactory isotope standards were identified for the remaining 4 hormones. Method detection levels (MDLs) were determined to describe analyte concentrations sufficient to provide a signal with 99% certainty of detection. The established MDLs for most analytes were 1-5 ngL(-1) in a variety of aqueous matrices. However, slightly higher MDLs were observed for etiocholanolone, androstenedione, testosterone, levonorgestrel and dihydrotestosterone in some aqueous matrices. Sample matrices were observed to have only a minor impact on MDLs and the method validation confirmed satisfactory method stability over intra-day and inter-day analyses of surface water and tertiary treated effluent samples.     

Peptide Charge Derivatization as a Tool for Early Detection of Preeclampsia by Mass Spectrometry—A Comparison with the ELISA Test

Early detection of any preeclampsia biomarkers may lower the risk of mortality, both for a mother and a child. Our study focuses on techniques for preeclampsia biomarker identification by comparing the results of a method using liquid chromatography mass spectrometry in multiple reaction monitoring mode (LC-MS/MS) with those by the enzyme-linked immunosorbent assay (ELISA) test, as well as by comparing the obtained results with clinical data. In the proposed LC-MS/MS method a tryptic digest peptide charge derivatization strategy was used as a tool for sensitive detection of podocin, i.e., a previously discovered preeclampsia biomarker present in urine samples from pregnant women. Urine samples from pregnant women with diagnosed preeclampsia were collected at different stages of pregnancy and from healthy subjects, and then were analyzed by ELISA test and the proposed method with LC-MS/MS. Charge derivatization of the ε amino group of C-terminal lysine residues in tryptic digests by 2,4,6-triphenylpyrylium salt was performed to increase the ionization efficiency in the LC-MS/MS mode. Podocin was identified at the early stage of pregnancy, while its detection using an ELISA test was not possible. The protocol for urine sample preparation was optimized. Our results show that the proposed method by LC-MS/MS in combination with peptide charge derivatization, provides an ultrasensitive tool for diagnosis of preeclampsia, and provides earlier detection than a clinical diagnosis or ELISA test. The proposed solution may revolutionize medical diagnostics.     

Rapid quantification of iridoid glycosides analogues in the formulated Chinese medicine Longdan Xiegan Decoction using high-performance liquid chromatography coupled with mass spectrometry [corrected

Longdan Xiegan Decoction (LXD) is a formulated preparation composed of 10 ingredient herbs, with iridoids as the main bioactive components. In this study, a rapid, simple and reliable method of simultaneous determination of four iridoid glycosides in LXD using high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (MS) was first developed and validated. The four iridoid glycosides references were isolated from LXD extract and purified using a preparative HPLC chromatography. The sample preparation for quantification comprised of a simple ultrasonic extraction and the satisfactory chromatographic separation of the four structurally similar iridoid glycosides was effected in less than three minutes on a CAPCELL PAK C(18) MGII column (3 microm, 100 mm x 2.0 mm), using an elution system of 10% methanol and their concentrations in different batches of LXD and ingredient herbs were simultaneously determined by HPLC-MS/MS using a multiple reaction monitoring (MRM) mode. The method was validated with respect to the overall intra- and inter-day variation (RSD less than 8%) and the limits of quantification for the four iridoid glycosides were 35, 20, 37 and 33 ng/mL, respectively.     

Determination and quantification of astragalosides in Radix Astragali and its medicinal products using LC–MS

In the present study, a liquid chromatography–tandem mass spectrometry method was developed for the separation and simultaneous quantification of astragalosides I–IV in samples of Radix Astragali and a medicinal product thereof (Jinqi Jiangtang tablets). Chromatographic separation was achieved on an Agilent Eclipse XDB (ODS)‐C18 column with a mobile phase consisting of acetonitrile and 0.05% formic acid aqueous solution by use of an efficient 17‐min program. A triple quadrupole mass spectrometer was operated in positive ionization mode with multiple reaction monitoring for the detection of four astragalosides. The saponin ginsenoside Rg1 (similar structure to astralagosides) was used as an internal standard. All calibration curves showed excellent linear regressions (r² ? 0.9912) within the range of tested concentrations. The intra‐ and inter‐day variations were below 4.57% in terms of RSD. The recoveries were 94.38–103.53% with RSD of 1.39–3.58% for spiked Radix Astragali samples. The method was successfully used for the analysis of samples of Radix Astragali and Jinqi Jiangtang tablets. In conclusion, we have developed a rapid, efficient, and accurate LC–MS/MS method for the detection of astragalosides, which can be applied for quality control of Radix Astragali and related medicinal products.     

液相色谱-串联质谱法测定海参和海胆中的单唾液酸神经节苷脂

建立了液相色谱-四极杆串联质谱法定量检测海参和海胆中单唾液酸神经节苷脂的分析方法。采用Svennerholm法从海胆或海参样品中提取神经节苷脂,经C8固相萃取柱净化,采用APS-2 NH₂柱(150 mm×2.1 mm, 3 μm),以乙腈和50 mmol/L乙酸铵溶液(pH 5.6)为流动相,梯度洗脱。样品中每种成分的定量在多反应监测模式下进行。该方法具有极高的灵敏度,定量限可低至纳克级。非硫酸酯化单唾液酸神经节苷脂(NMG)和硫酸酯化单唾液酸神经节苷脂(SMG)在1～40 ng进样量范围内呈现良好的线性关系;定量结果显示所测海参样品中美国红参的NMG含量最高,海胆样品中紫海胆的SMG含量最高;海胆中总的单唾液酸神经节苷脂含量(4.30～6.40 mg/g)明显高于各海参样品(8～131 μg/g)。该方法稳定可靠,适合海胆和海参中微量单唾液酸神经节苷脂的定量分析。     

Simultaneous Determination of Preservatives,Artificial Sweeteners,and Synthetic Dyes in Kimchi by Ultra-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (UPLC-ESI-MS/MS)

Abstract

A simple and rapid method was developed for the simultaneous determination of five preservatives, six artificial sweeteners, and nine synthetic dyes in kimchi using ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) with multiple reaction monitoring (MRM). The chromatographic separation was performed in 5.5?min using an Acquity UPLC BEH C18 column (100?mm × 2.1?mm, 1.7?µm) with a mobile phase composed of 0.002% trifluoroacetic acid and 10?mM aqueous ammonium acetate or 9:1 (v/v) methanol:acetonitrile. Linear calibration curves were obtained with correlation coefficients above 0.98. The limits of quantification ranged between 0.227 and 8.569?ng/mL, while the recovery values in kimchi samples were from 83.1 to 113.5%. Forty kimchi samples were analyzed for the food additives, with sweeteners detected in more than half of these samples. The most commonly detected sweetener was saccharin, and six of the kimchi samples contained a combination of sweeteners; dulcin, sucralose, acesulfame K, preservatives, and synthetic dyes were not detected. Based on these results, the developed method can be used for the rapid quality control of food additives in kimchi.     

Simultaneous Determination of Phenolics and Polymethoxylated Flavones in Citrus Fruits by Ultra-High Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry (UHPLC-QqQ-MS)

Phenolic and polymethoxylated flavones are important bioactive components in citrus fruit. Here, a rapid and sensitive method based on ultra-high performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ-MS) was developed for the simultaneous determination of phenolic and polymethoxylated flavones in the peels and pulp of mandarins, tangelos, and oranges. Three phenolic acids and eight flavonoids, including polymethoxylated flavones, were separated and determined using positive and negative ion modes in a single chromatographic run of only 11?min using the multiple reaction monitoring detection mode. The method was validated with high recoveries from 96.1% to 103.5%, good precision with interday relative standard deviations less than or equal to 7.3%, intraday relative standard deviations ≤2.64%, low limits of detection from 1.0 to 18?µg L^–1, and low limits of quantitation in the range from 3.0 to 61?µg L^–1. The application of this UHPLC-QqQ-MS/MS method to the citrus extracts of three cultivars showed that mandarin fruits contained the highest total amounts of the 11 analytes, followed by tangelos and oranges. This study provides a reliable and quantitative method that can be used for the development of functional products and quality evaluation of citrus fruits.