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Chinese Journal of Chromatography
2021, Vol. 39, No. 5
Online: 08 May 2021

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Research progress of solid phase extraction materials in the application of metal ion pretreatment XING Shige, HE Muyi, LIU Tong, YONG Wei, ZHANG Feng 2021, 39 (5):  455-462.  DOI: 10.3724/SP.J.1123.2020.07004 Abstract ( 240 )   HTML ( 206 )   PDF (2640KB) ( 159 )   Monitoring of trace heavy metal pollutants released during industrial and agricultural processes is essential because of their widespread distribution in the environment and health hazards. Several techniques, including inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), electrothermal atomic absorption (ETAAS), and flame atomic absorption spectrometry (FAAS), have been proposed for the determination of heavy metals in serum, plasma, whole blood, and food. All these techniques have earned robust recognition in the field of trace heavy metals and have many advantages such as multi-elemental analysis capability, large dynamic linear range, low detection limits, and high productivity. Nevertheless, most of the recommended techniques require digestion of the sample and extraction with an organic solvent for isolation of the metal ion from the sample solution prior to analysis. Despite improvements in the performance of modern analytical instruments, the direct determination of heavy metal ions in real samples is difficult because of their low concentration levels and matrix interference. Thus, extraction and clean-up steps are required for pre-concentration of the analyte, so that detection and elimination of the interfering matrix component are possible. Solid-phase extraction (SPE) is one of the popular metal ion pretreatment methods. The advantages of SPE include easy cartridge/column regeneration, high analytical frequency, and high preconcentration factors for sorbents with high adsorption capacities. On the other hand, when the analytes are extracted from a complex matrix such as serum and meat samples, large amounts of proteins from the samples can be retained on the sorbent surface, obstructing the binding sites on the sorbent and leading to poor precision and accuracy. The key to metal ion detection is the development of new SPE materials with high efficiency and enrichment factors as well as an effective pretreatment technology. Nanomaterials such as restricted-access carbon nanotubes, nanoadsorbents, nanoparticle carriers, and magnetic nanoparticles have shown great promise in advancing biomedical and environmental analysis because of the unique properties originating from their ultrafine dimensions. Nanomaterials can provide large specific surface areas and tunable functional groups to facilitate metal ion absorption. They could also possess superior optical properties and allow for high sensitivity in simple fluorescent or colorimetric detection methods. Owing to their excellent mechanical and chemical stability, polymer materials have been of great interest as adsorbents for the SPE of metal ions from solution. Moreover, a designed polymeric material can show triple functionality such as physical adsorption, chelate formation, and ion exchange for the target metal ions. A dual-functional nanomaterial-DNAzyme platform can simultaneously allow for the sensitive detection and effective removal of heavy metal ions in water. Thus, this platform can serve as a simple, cost-effective tool for rapid and accurate metal quantification in the determination of human metal exposure and inspection of environmental contamination. Furthermore, the new photocaged chelator can uncage and release the combined metal ions into an aqueous solution that is free of the other components of the matrix. In this manner, we can develop diagnostic tests for metal ions that are often difficult to detect using other methods. In this paper, the characteristics of new SPE materials, including nanomaterials, polymer materials, and functional materials as well as advances in their applications to the preparation of complex samples are summarized, and the direction for future development is proposed.

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Effect of sample preparation on analysis of human milk endogenous peptides using liquid chromatography-tandem mass spectrometry YU Wenhao, YU Yang, WANG Wendan, LI Yitong, SZETO Ignatius M., JIN Yan 2021, 39 (5):  463-471.  DOI: 10.3724/SP.J.1123.2020.08019 Abstract ( 126 )   HTML ( 37 )   PDF (2577KB) ( 77 )   Hundreds of endogenous peptides were released from milk proteins within the human mammary gland and some of them possess a variety of bioactive functions. Thus, it is important to investigate human milk endogenous peptides for infant health. Peptidomics based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to investigate human milk endogenous peptides. Extraction of endogenous peptides from human milk is an essential and key procedure for analyzing human milk peptides using LC-MS/MS. This study aimed to compare methods for extracting endogenous peptides from human milk using LC-MS/MS. Ultrafiltration methods including that not involving denaturation (UF 1), that involving heat denaturation (UF 2), and that involving chemical denaturation (UF 3), precipitation methods using trichloroacetic acid (PCPN 1) and alcohol (PCPN 2), and an enrichment method using highly ordered mesoporous carbon (OMC) were used to extract endogenous peptides from human milk. Extracted endogenous peptides were then analyzed using LC-MS/MS. The samples extracted using UF 1 and UF 2 comprised 1161±8 and 1017±91 endogenous peptides, respectively. More than 70% peptide sequences in each sample extracted using UF 1 and UF 2 overlapped. The results revealed that endogenous peptides extracted using UF 1 and UF 2 showed similar characteristics. UF 1 yielded the highest number of peptides, whereas UF 3 extracted the least number of peptides at 366±18. The number of endogenous peptides extracted using PCPN 1 and PCPN 2 were 779±69 and 876±55, respectively. However, their characteristics were quite different, and only about 50% peptide sequences overlapped. The number of peptides extracted using OMC (549±151) was not remarkable compared with that using other methods. However, the isoelectric point (pI) and grand average of hydropathicity (GRAVY) of the peptides extracted using OMC were different from those extracted using other methods. This method presented no selectivity for the endogenous peptides with different pI and GRAVY and may be used to extract unique peptides from human milk. A total of 205 peptides were commonly identified in the samples using each of the six methods. The percent of shared peptides across the six samples ranged from 13% to 23%. The number of unique peptides in the samples extracted using UF 1 and UF 2 (226 and 228, respectively) were the highest among those extracted using the six methods. The results showed that all six methods could be used to extract endogenous peptides from these high-abundance precursor proteins. A total of 21, 38, and 19 peptides were extracted from lactotransferrin using UF 2, UF 3, and OMC, respectively, and the coverage rates of these peptides in lactotransferrin were 14%, 16%, and 19%, respectively. These three methods could extract the endogenous peptides from lactotransferrin in human milk, but PCPN 1 that has been commonly used in previous studies could not. The peptides from β-casein, polymeric immunoglobulin receptor, osteopontia, αS1-casein, κ-casein, and bile salt-activated lipase were identified in all samples extracted using the six methods. Moreover, these precursor proteins contributed 88% peptides in the samples extracted using the six methods. In conclusion, UF 1 and UF 2 were efficient procedures for extracting endogenous peptides from human milk. In addition, UF 2 could extract peptides from lactotransferrin, which is the optimum choice for extracting endogenous peptides from human milk. Additionally, the OMC enrichment method can be used to enrich and extract specific endogenous peptides from human milk. This study systematically compared the sample preparation methods commonly used in human milk endogenous peptidomics in recent years. The results provide strong support for uniform and standardized sample preparation methods. An ultrafiltration method without denaturation, which is more advantageous than the currently commonly used trichloroacetic acid precipitation method, was also established to prepare human milk endogenous peptide samples. In combination with OMC, this method can help in a more comprehensive and in-depth understanding of the endogenous peptidome of human milk.

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Determination of 18 amino acids in three different kinds of milk powder by ultra performance liquid chromatography coupled with pre-column derivatization QU Li, GU Shuqing, ZHANG Jiaqi, ZHAO Chaomin, DENG Xiaojun 2021, 39 (5):  472-477.  DOI: 10.3724/SP.J.1123.2020.07008 Abstract ( 206 )   HTML ( 47 )   PDF (794KB) ( 182 )   In recent years, goat milk powder and camel milk powder have gained popularity among consumers. Due to their potential low allergenicity, these milk powders have become a substitute for breast milk, especially for infants, and for people with lactose intolerance. In this paper, a method was developed for the simultaneous determination of 18 amino acids (AAs), histidine (His), serine (Ser), arginine (Arg), glycine (Gly), aspartic acid (Asp) combined with asparagine (Asn), glutamic (Glu), glutamine (Gln), threonine (Thr), alanine (Ala), proline (Pro), lysine (Lys), tyrosine (Tyr), methionine (Met), valine (Val), isoleucine (Iso), leucine (Leu), and dimer of cysteine (Cys) combined with cysteine (L-Cys-Cys), phenylalanine (Phe), taurine (Tau) in milk, goat milk, and camel milk power. The aim of the research was to compare the three kinds of milk powder from the perspective of the constituent amino acids. Therefore, the amino acid compositions and contents were compared. Thus, 2.0 g of the sample was accurately weighed, added to 16 mL H2O, and mixed thoroughly. Then, 200 mg of the sample was weighed in a glass tube with a stream of nitrogen to displace oxygen. The samples were hydrolyzed in HCl for 24 h at 110 ℃. Then, the amino acids were pre-column derivatized by 6-aminoquinoline-n-hydroxysuccinimide carbamate (AQC). In precolumn derivatization combined with reverse-phase chromatography, both 2,4-dinitrofluorobenzene (DNFB) and phenylisothiocyanate (PITC) can react with primary amines and secondary amines. However, the derivatization time is approximately 1 h. In contrast, the derivatization time of AQC was greatly shortened. Derivatization led to the conversion of free amino acids into highly stable derivatives, which were separated by ultra performance liquid chromatography (UPLC) with UV detection at 260 nm and quantified by the external standard method. The samples were separated on a BEH C18 column (150 mm×2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min. The calibration curves showed good linearity, with correlation coefficients greater than 0.999. The limits of detection (LODs) and limits of quantification (LOQs) of the 18 amino acids were 1.3-2.5 (mg/100 g) and 3.9-7.5 (mg/100 g), respectively. Quality control samples of SRM 1849a were used as the reference material. The results were in accordance with the content range. The RSDs ranged from 2.04% to 3.65%. Furthermore, the developed method was successfully applied to determine the types and concentrations of amino acids in 11 samples purchased from local markets in Shanghai and online shops. Abundant amino acids were detected in the three types of milk powder. While all the milk powder samples contained 18 types of amino acids, Tau was not detected in some of the goat and camel milk powder samples. Total essential amino acids (TEAA) in total amino acids (TAA) of milk powder was the highest of all. The TEAA values of TAA in the goat and camel milk powders were similar. The developed method requires only 22 min for the separation of 18 amino acids. This method is suitable for the large-scale analysis of milk powder samples, and it demonstrates high sensitivity and accuracy for the determination and confirmation of the 18 amino acids in different types of milk powders.

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Chemome profiling of Pien-Tze-Huang by online pressurized liquid extraction-ultra-high performance liquid chromatography-ion trap-time-of-flight mass spectrometry LI Wei, JIANG Zhenzhen, LI Han, TU Pengfei, SONG Qingqing, YU Juan, SONG Yuelin 2021, 39 (5):  478-487.  DOI: 10.3724/SP.J.1123.2020.10011 Abstract ( 121 )   HTML ( 25 )   PDF (4098KB) ( 86 )   Pien-Tze-Huang is one of the most famous traditional Chinese medicine prescriptions and consists of several precious medicinal materials, such as Notoginseng Radix et Rhizoma, Bovis Calculus, Snake Gall, and Moschus. However, its formula has not been completely revealed. It is mainly applied for the treatment of acute and chronic viral hepatitis, carbuncle, and boils caused by blood stasis, unknown swelling, bruises, and various inflammation disorders. The chemical composition of Pien-Tze-Huang is extremely complicated. Thus far, extensive attention has been paid to the principal chemical families in Pien-Tze-Huang, such as ginsenosides, bile acids, and muscone derivatives. Comprehensive chemical profiling, although of immense importance for systematic quality control, has not been achieved. Therefore, we configured a platform, namely online pressurized liquid extraction-ultra-high-performance liquid chromatography-ion trap-time-of-flight mass spectrometry (online PLE-UHPLC-IT-TOF-MS), to characterize the chemical profile of Pien-Tze-Huang in detail as well as to conduct source attribution, aiming to clarify the chemome of Pien-Tze-Huang and to provide a reliable method for quality assessment. A sub-microgram amount of Pien-Tze-Huang powder (0.3 mg) was placed in a hollow guard column, which was subsequently filled with clear silica gel. Filter membranes were used to seal the extraction vessel. The vessel was then placed in an adapted guard column holder and maintained in a thermal column oven (70 ℃). Metal tubing was used to connect the outlet of the guard column holder to the mass spectrometer. The extraction phase was maintained for 3 min by employing 0.1%(v/v) formic acid aqueous solution as the extraction solvent with a flow rate of 0.2 mL/min. Moreover, a six-port two-position electronic valve was introduced to automatically switch the system from extraction to elution phases. Within the elution phase, 0.1%(v/v) formic acid aqueous solution and acetonitrile composed the mobile phase, and the extracts were eluted with a gradient program. Because of the elevated temperature and pressure, the physical and chemical properties of water, especially polarity and solubility, were modified. Therefore, warm water could be an eligible green solvent to achieve wide polarity-spanned extraction. In addition, IT-TOF-MS was employed to acquire tandem mass spectrometry information. The mass fragmentation pathways of saponins and bile acids were carefully studied. Finally, according to authentic compounds, mass fragmentation pathways, reference information in the literature, and accessible databanks, a total of 73 signals were observed from Pien-Tze-Huang, of which 71 components were tentatively identified and assigned. Among them, 36 were from Notoginseng Radix et Rhizoma, 15 from Snake Gall, and 9 from Bovis Calculus, while the occurrences of the other 11 components were synergistically contributed by both Bovis Calculus and Snake Gall, through retrieving the in-house chemical database that was built by considering all accessible chemical information from Notoginseng Radix et Rhizoma, Bovis Calculus, Snake Gall, and Moschus. The other two compounds were assigned as unknown compounds. However, none of the components were assigned to Moschus because they mainly contained hydrophobic compounds, such as cycloketones, cholesterol, and sterols, among others, and it was difficult to detect them with the current measurement program. The extraction efficiency of online PLE was assessed by comparing it with the efficiency obtained from ultrasonication at the same time. According to base peak ion current chromatograms (BPCs) and mass spectrometry information, the efficiency of online PLE was greater than that of ultrasonic extraction, even through direct analysis. Online PLE-UHPLC-IT-TOF-MS is not only a tool fit for the concept of green analytical chemistry, but also a reliable analytical pipeline for the direct characterisation of other complicated matrixes. Above all, this study clarified the chemome of Pien-Tze-Huang and provided meaningful information for the quality control of this famous TCM prescription.

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Simultaneous determination of eight additives in polyethylene food contact materials by ultrahigh-performance liquid chromatography LING Yun, BI Jingbo, YONG Wei, YAO Meiyi, ZHANG Yujia, ZHANG Feng 2021, 39 (5):  488-493.  DOI: 10.3724/SP.J.1123.2020.12002 Abstract ( 205 )   HTML ( 36 )   PDF (1019KB) ( 123 )   Measurement of additive residues in food contact materials is important for safety monitoring at the initial stage. Most of the current studies focus on the determination of the migration amounts of chemical hazards from food contact materials into food simulants. Studies on chemical hazard residues in food contact materials are limited to monomers, oligomers, heavy metals, phthalic acid esters, and biphenols, which are known environmental pollutants. Only a few studies have investigated analysis methods for additive residues in food contact materials. In this study, the main factors (monitoring wavelength, chromatographic column, mobile phase, extraction solvent, etc.) that affect the accuracy and sensitivity of eight compounds, including three antioxidants, three light stabilizers, and two plasticizers, were investigated during sample preparation and instrument analysis. A method based on ultrahigh-performance liquid chromatography (UPLC) was developed for the simultaneous determination of these eight additives in polyethylene (PE). The PE food contact material sample was ground to homogenize the particle sizes under freeze-grinding. After comparing the extraction efficiencies of methylbenzene, chloroform, acetone, and acetonitrile, 2.0 g of the sample was extracted with methylbenzene at 80 ℃ and 10.34-11.72 MPa (1500-1700 psi) by accelerated solvent extraction (ASE) for 10 min once. The exaction solvent (10 mL) was transferred and concentrated to near dryness under a gentle stream of nitrogen gas and then re-dissolved in 10 mL of the initial mobile phase (70% (v/v) methanol in water). Finally, the eight compounds were analyzed by UPLC. After optimization of the analytical column and mobile phases, the eight analytes were separated on an ACQUITY UPLC BEH C8 chromatographic column (100 mm×2.1 mm, 1.7 μm) by gradient elution using water and acetonitrile as the mobile phases. The column oven temperature, flow rate of the mobile phase, and injection volume were 30 ℃, 0.3 mL/min, and 5 μL, respectively. The analytes were detected by a diode assay detector (DAD) in the scanning range of 210 nm to 400 nm. The monitoring wavelength was set at 230 nm, 250 nm, 280 nm, and 330 nm. External standard calibration curves were used for quantification. Under the optimized conditions, the calibration curves for the eight compounds showed good linearity in the range of 0.2 μg/mL to 10 μg/mL, and the correlation coefficients were >0.999. The recoveries in spiked blank polyethylene samples at the level of 0.05% were in the range of 83.8% to 103.4%, with relative standard deviations (RSDs) ranging from 0.14% to 7.86%. To validate the method, PE reference materials containing these eight compounds were manufactured at the content level of 0.2% to 0.9%. The recoveries using the prepared reference materials ranged from 63.5% to 118.5%, and the RSDs were in the range of 4.61% to 15.6%. The limits of detection (LODs, S/N=3) of all the eight compounds were 0.005% and the limits of quantification (LOQs, S/N=10) were 0.02%, in compliance with the current legislation. To assess the feasibility and potential of the proposed approach for routine analyses of these eight compounds, the developed method was applied to the analysis of these compounds in ten PE food packages and PE gloves. In six samples, tris(2,4-di-tert-butylphenyl)phosphite (Irganox 168) was detected at a level of 0.02%-0.07%, which was lower than the maximum level of this compound in PE food contact material products regulated in GB 9685-2016 at 0.2%. The method is compliant with the current legislation, and it can be used for the monitoring and supervision of these eight additives in PE food contact materials.

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Screening and confirmation of 244 pesticide residues in chilli by gas chromatography-quadrupole time-of-flight mass spectrometry CAO Qi, ZHANG Yazhen, ZHU Zhengwei, WU Wanqin, JIANG Feng, YU Tingting 2021, 39 (5):  494-509.  DOI: 10.3724/SP.J.1123.2020.11019 Abstract ( 155 )   HTML ( 49 )   PDF (5670KB) ( 125 )   QuEChERS pretreatment combined with gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF/MS) has been investigated for application in screening 244 pesticide residues in chilli. Fresh chilli samples were extracted with acetonitrile, and dried chilli samples were extracted using an acetonitrile/acetic acid (99∶1, v/v) mixture. The two extraction solvents were stored at -20 ℃. After salting out and cleaning by dispersive solid phase extraction (dSPE), heptachlor epoxide B was added as an internal standard, and the resulting residues were dissolved in 1.00 mL acetone. The dissolved sample solution was loaded onto an HP-5MS UI column (30 m×0.25 mm, 0.25 μm) and eluted by GC-Q-TOF/MS with a programmable temperature vaporizer and splitless injection in the full-scan mode. The compensation effects of the analytical protectant (AP) and matrix-matched calibration method on the matrix effect were established. AP could be used in the fresh chilli matrix to compensate for matrix effects, but it was not effective in the dried chilli matrix. The matrix-matched calibration method was effective in both matrices, which was selected for the quantification of pesticide residues in the samples. Because of the existence of the isomers of one compound and the same characteristic ions of different compounds, analyte detection was based on a flexible retention time deviation of ±0.25 min and accurate mass deviation of ±20×10-6. Screening was performed by the software in the automatic matching mode. Compound identification and quantitation were based on a database and calibration curve established with reference materials. Suspicious samples were subjected to manual analysis. Quantitative analysis of 244 pesticide residues in fresh chilli and 222 pesticide residues in dried chilli was performed. The results showed that the developed database and method can provide a reference for the high-throughput screening and quantitation of fresh and dried chilli. Different levels of pesticides were added to the blank chilli samples, and the addition level corresponding to a signal-to-noise ratio (S/N) of 10 was used as the limit of quantification (LOQ). The LOQs of 44 pesticides with a maximum residue limit (MRL) ≤0.05 mg/kg in fresh chilli did not exceed 0.010 mg/kg. The linear ranges of these 44 pesticides were 0.01-1.00 mg/L. At spiked levels of the LOQ and 2.5 times the LOQ, the ratios of the 44 pesticides with recoveries of 60% to 120% were 88.64% and 100%, respectively. The LOQs of 200 pesticides with MRLs ≥0.05 mg/kg or without MRLs in fresh chilli did not exceed 0.025 mg/kg. The linear ranges of these 200 pesticides were 0.05-1.00. At spiked levels of the LOQ, twice the LOQ, and 10 times the LOQ, the ratios of the 200 pesticides with recoveries of 60% to 120% were 49.50%, 87.00%, and 89.50%, respectively. The linear correlation coefficients (r 2) of the 244 pesticides in fresh chilli were greater than 0.99. The LOQs of 222 pesticides in dried chilli were less than 0.15 mg/kg, and the linear ranges were 0.04-1.00 mg/L. The ratios of these 222 pesticides with r2 greater than 0.99 was 95.46%. At spiked levels of the LOQ, twice the LOQ and 10 times the LOQ in dried chilli, the ratio of the 222 pesticides with recoveries of 60% to 120% were 72.52%, 73.42%, and 81.53%, respectively. The established screening and confirmation method was used to analyze 12 fresh chilli samples and 14 dried chilli samples. Eight pesticides were found in nine fresh chilli samples and three dried chilli samples, all of which were confirmed to be positive after manual identification. The concentrations of these pesticides were lower than the MRLs required by GB 2763-2019: National Food Safety Standard-Maximum Residue Limits for Pesticides in Food. The results demonstrate that the established method is rapid, easy to execute, efficient, and reliable. It can be used for the high-throughput screening and quantitation of pesticide residues in fresh and dried chilli.

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Simultaneous determination of 46 semi-volatile organic compounds in water by liquid-liquid extraction-gas chromatography-mass spectrometry LIU Lingling, ZHANG Lijun, DONG Xiliang, CHEN Xiaomei, ZHAO Chuanming 2021, 39 (5):  510-517.  DOI: 10.3724/SP.J.1123.2020.07006 Abstract ( 216 )   HTML ( 36 )   PDF (1501KB) ( 142 )   Semi-volatile organic compounds (SVOCs) include polycyclic aromatic hydrocarbons (PAHs), phthalic acid esters (PAEs), organochlorine pesticides (OCPs) and nitrobenzenes (NBs). Most of them have carcinogenic, teratogenic, mutagenic and endocrine disrupting effects. Therefore, rapid and accurate determination of SVOCs in water is very important. As the most traditional pretreatment method, liquid-liquid extraction (LLE) has the advantages of wide extraction range, high extraction efficiency, simple operation and lower cost, which is very suitable for the simultaneous extraction of multiclass SVOCs. Dichloromethane has good solubility for most SVOCs, and is slightly soluble in water with low boiling point. It is a good broad-spectrum extractive solvent of organic compounds. But at present, there is no detection standard of SVOCs in water in China. In this study, three factors including nitrogen blowing temperature, pH of water sample and extraction time were optimized. It was aimed to establish a liquid-liquid extraction-gas chromatography-mass spectrometry (GC-MS) method for the simultaneous determination of the 46 SVOCs in water. At first, the effect of nitrogen blowing temperature (30, 35, 40 ℃) was investigated. The results showed that under different nitrogen blowing temperature, the recoveries of the 46 SVOCs were slightly different, but the differences were not significant. Considering the recovery and concentration efficiency, the nitrogen blowing temperature was finally set at 35 ℃. Dichloromethane was selected as the LLE solvent of the SVOCs and its extraction efficiency was investigated. The recoveries of the 46 SVOCs were satisfactory for the determination. Then sample pH (neutral and alkaline condition) was investigated. Most of the SVOCs in this paper have no obvious acid-base property. The extraction effect of water sample under neutral conditions was the best and the most stable, and under alkaline condition, the recovery of each substance was generally low. Finally, extraction time (7, 10, 15 min) was studied. In a certain range, with the increase of extraction time, the recovery also increased, but when the time increased to 15 min, the recovery of some compounds increased or decreased, and the time-consuming was longer, the recovery of most substances could meet the requirements when the extraction time was set to 10 min. The optimized experimental conditions were determined as follows: under neutral conditions, the water sample was extracted by dichloromethane for three times, each extraction time was 10 min, and concentrated at the nitrogen blowing temperature of 35 ℃. GC-MS was used for detection, and internal standard method was used for quantitative analysis. The results showed that the linearity of the method was good in the range of 20-2000 μg/L, the correlation coefficients (r 2) were no less than 0.9916, the limits of detection (LODs, S/N=3) ranged from 0.28 to 16.55 ng/L, and the limits of quantification (LOQs, S/N=10) ranged from 0.92 to 55.16 ng/L. The average recovery was 63.6%-125% at three spiked levels of 0.02, 0.2, 0.4 μg/L, with the relative standard deviations (n=6) ranging from 1.03% to 17.0%. This method was applied for the determination of 27 surface water samples in Jinan section of the Yellow River. The pollutants were mainly PAEs and PAHs, while NBs were not detected, only two kinds of OCPs were detected at some sites. The method is simple, universal, accurate and precise, and has low detection limit. It is suitable for the simultaneous determination of the 46 SVOCs in surface water and groundwater.

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Quantitative analysis of tryptophan and its metabolites in urine by ultra performance liquid chromatography-tandem mass spectrometry LI Hui, CUI Lanchong, ZHANG Guolei, ZHANG Mengmeng, JIAO Lili, WU Wei 2021, 39 (5):  518-525.  DOI: 10.3724/SP.J.1123.2020.06022 Abstract ( 359 )   HTML ( 40 )   PDF (1249KB) ( 147 )   Tryptophan (Trp), also known as α-amino β-indolepropionic acid, is an essential amino acid, which is involved in various physiological processes. Studies have shown that tumors, infectious diseases, and neurological diseases are accompanied by Trp-related metabolic disorders. Understanding the excretion of Trp and its metabolites in normal individuals is of great significance for treating Trp-related diseases and monitoring the health. A rapid quantitative method was developed based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Further, this method was applied to the simultaneous determination of Trp and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA). The excretion and amount of target compounds in random urine samples collected from healthy participants were studied using this method. Urine samples were collected from healthy male volunteers (between 20-22 years old) without any diet and exercise restrictions. Urine samples were collected between 11∶00-13∶00 daily for 10 d. Thereafter, the urine samples were diluted, centrifuged, and subjected to pre-column derivatization with dansyl chloride (DNS-Cl). Caffeic acid (CA) was used as the internal control. Later, the derivatives were detected using triple quadrupole mass spectrometry with electron pray ionization (ESI) in positive and multi reaction monitoring (MRM) modes. The samples were separated using a Thermo C18 column (50 mm×3 mm, 2.7 μm) with 0.1% aqueous formic acid aqueous solution and methanol as mobile phases at a flow rate of 0.2 mL/min. The three most abundant ions for each derivative were selected for downstream analysis, and the internal control was used for quantification. The polarity and molecular weight of the compounds were found to be altered effectively after DNS-Cl derivatization treatment. The dansyl group effectively altered the polarities of the derivatives, such that their retention behaviors in the reverse elution system were similar and they were well separated. The interference due to impurities was effectively eliminated using the MRM mode. The results showed significant linear correlation, since the correlation coefficients were greater than 0.9740. The recoveries were between 93.24%-107.65%, and the LODs were 0.005-0.5 ng/mL for the eight compounds. Trp prototype and the seven target metabolites, including 3-OH-Kyn, 3-OH-AA, XA, Kyn, KA, 5-HIAA, and 5-HT generated through Trp-5-HT and Trp-Kyn pathways were detected in the urine samples. These results indicated that Trp was excreted in a prototypic form or after being metabolized. The level of the target compounds in random urine samples of individuals were 0.99-3.72 (3-OH-Kyn), 2.51-21.11 (3-OH-AA), 0.25-1.12 (XA), 0.15-1.53 (Kyn), 0.24-2.58 (KA), 0-0.31 (5-HT), and 2.2-17.94 (5-HIAA) μg/mL. For the same individual, in the state of physical health, the fluctuations of Trp and its metabolites in urine were large. Due to these large fluctuations in the absolute content, the difference between individuals was not significant. The data generated using 70 urine samples revealed that the amount of excreted Trp being metabolized was 124%-268% of prototype, which further indicated that the excretion after metabolism was the major underlying mechanism. Upon comparing the levels of metabolites in the Trp-5-HT and Trp-Kyn pathways, the results indicated that the levels of 3-OH-AA and 3-OH-Kyn generated upon Trp degradation through the Kyn pathway was higher than those of the other products. Trp was degraded via Kyn pathway to produce 3-OH-AA, which was the main metabolite of Trp found to be present in the body. This manuscript detected the levels of Trp and its metabolites, as well as summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.

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Determination of nine ginsenosides in health foods by solid extraction phase-ultra performance liquid chromatography-tandem mass spectrometry CHEN Shudong, FENG Rui, LIN Xiaojia, LIANG Tujin, HE Qiuting 2021, 39 (5):  526-533.  DOI: 10.3724/SP.J.1123.2020.04028 Abstract ( 180 )   HTML ( 35 )   PDF (1445KB) ( 212 )   Ginsenosides are the main active compounds of ginseng, American ginseng and Panax notoginseng. They have certain pharmacological effects on the cardiovascular, immune and central nervous systems. Most ginsenosides are naturally classified as protopanaxatriol (PPT), protopanaxadiol (PPD), and oleanolic acid (OA) according to their aglycone skeletons. The nine main ginsenosides are Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1 and Rg2. Accurate quantification of ginsenosides is imperative because they are the characteristic components and quality evaluation indicators of health foods. A new method based on solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) was developed for the determination of the nine ginsenosides in health foods. First, the pretreatment conditions were optimized. With the aim of purifying the samples and removing impurities, SPE cartridges with different packing materials, such as Alumina-N/XAD-2 SPE Cartridge, C18 and HLB were investigated. Based on the purification efficiencies, recoveries and other factors, the Alumina-N/XAD-2 SPE cartridge composite SPE column was selected as the pretreatment purification column. The eluents were then optimized. When water was used as the eluent, the ginsenosides could remain adsorbed on the SPE column, and could not be eluted down with other water-soluble substances. By increasing the proportion of ethanol in the eluent, the ginsenoside adsorbed on the filler of the SPE column could be gradually eluted. When the proportion of ethanol in the eluent reached 70%, the ginsenosides could be completely eluted. The effects of different volumes of 70% ethanol elution solvent (5-30 mL) on the extraction efficiencies of ginsenosides were also investigated. The results showed that when the volume of the elution solvent reached 20 mL, the ginsenosides were completely eluted. Then, the chromatographic conditions and MS parameters were optimized. By examining the ionization cracking of ginsenosides, the quasi-molecular ions and corresponding fragment ions in ginsenoside primary MS were determined. After optimizing the chromatographic conditions and MS parameters, not only the sensitivity of the method was improved, but also the isomers Rb2, Rb3 and Rc with the same quasi-molecular ions and the corresponding fragment ions were completely separated. Good separation was achieved for the nine ginsenosides, thus meeting the requirements for accurate quantification. Finally, chromatographic separation was achieved on a Hypersil Gold C18 column (100 mm×2.1 mm, 1.9 μm) under linear gradient elution using a 5 mmol/L ammonium acetate solution (with 0.1% formic acid) and acetonitrile as the mobile phases. The nine ginsenosides were detected using a triple quadrupole MS detector under ESI - and multiple reaction monitoring (MRM) modes, and quantified by the external standard method. The nine ginsenosides showed a strong positive linear correlation (r 2>0.9950) in the range of 0.005-0.5 μg/mL. The sample recoveries and the corresponding relative standard deviations (RSDs) were 81.1%-114.2% and 0.4%-8.0% (n=6), respectively. Eleven batches of health foods on the market, among which six batches contained ginseng, American ginseng or Panax notoginseng ingredients, were analyzed by the developed method, and the ginsenosides were detected. The total ginsenosides contents were close to those mentioned on the label. However, the nine ginsenosides were detected in one batch of health food, whose label did not indicated ginseng, American ginseng or Panax notoginseng. The nine ginsenosides were not detected in the remaining batches of health foods.The health food extract was directly loaded and purified without any complex pretreatment. The UPLC⁃MS/MS method, not only helped shorten the analysis time, but also accurate quantification of low ginsenoside contents in complex matrix samples. The developed method is simple and rapid, with high throughput, thus being suitable for the quantitative analysis of the nine ginsenosides in health foods.

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Design and application of special solid phase extraction column for three cannabinol compounds in hemp SHEN Shuchang, LI Shaohua, GUO Li, LÜ Weichao, LI Qiushi 2021, 39 (5):  534-540.  DOI: 10.3724/SP.J.1123.2020.09025 Abstract ( 147 )   HTML ( 22 )   PDF (1148KB) ( 85 )   Cannabidiol (CBD), cannabinol (CBN), and Δ9-tetrahydrocannabinol (THC) are the most important components of hemp, whose concentrations determine the properties and applications of hemp. Hemp contains a large number of impurities, which must be removed from the extracting solution before determining the cannabinol contents by ultra-high performance liquid chromatography (UHPLC). Neutral alumina, magnesium silicate, and graphitized carbon black have different surface characteristics when used as adsorbents. The removal rates of pigments, total sugar, total fatty acid glyceride, and metal ions as well as the recoveries of the three cannabinols in the extraction solution were evaluated. The amounts of neutral alumina, magnesium silicate, and graphitized carbon black were 1.80 g, 0.15 g, and 0.05 g, respectively. The three adsorbents were mixed well and packed into a polypropylene pipe to prepare a special 2 g/6 mL solid phase extraction (SPE) column for determining the three cannabinol compounds in hemp. The chemical components of the hemp flowers and leaves were extracted with an ethyl acetate/methanol (9∶1, v/v) mixture. After the extracting solution was allowed to pass through the SPE column, the recoveries of CBD, CBN, and Δ9-THC were 98.9%, 95.7%, and 99.2%, respectively. The removal rates of xanthophyll, chlorophyll a, and chlorophyll were 96.3%, 99.2%, and 95.5%, respectively. The removal rates of total sugar, total fatty glyceride, and metal ions were 98.5%, 96.9%, and 85.4%, respectively. In this study, the chromatographic conditions for analyzing the three cannabinol compounds were optimized. The cannabinol compounds were separated within 10 min on an Eclipse Plus C18 column (50 mm×2.1 mm, 1.8 μm) using a mobile phase consisting of 1% (v/v) acetic acid and acetonitrile (30∶70, v/v) at a flow rate of 0.5 mL/min. The detection wavelength was set at 210 nm with a diode array detector, and the sample injection volume was 1 μL. Good linear relationships were observed between the mass peak areas and mass concentrations of CBD, CBN, and Δ9-THC in the range of 0.5-50 mg/L. The corresponding correlation coefficients (R2) were 0.9983, 0.9995, and 0.9981, while the detection limits were 0.45 μg/L, 0.53 μg/L, and 0.38 μg/L. The recoveries of CBD, CBN, and Δ9-THC were 90.3%-96.9%, 93.7%-95.6%, and 90.8%-96.1%, with relative standard deviations (RSDs) of 2.2%-6.1%, 4.1%-8.0%, and 2.4%-4.8%, respectively. The results were satisfactory, demonstrating that the special SPE column made of neutral alumina, magnesium silicate, and graphitized carbon black was well suited for the determination of the three cannabinol compounds in hemp.

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Determination of atmospheric organochlorine pesticides using isotope dilution high-resolution gas chromatography/high-resolution mass spectrometry ZHANG Jingxing, ZHENG Xiaoyan, TAN Li, LIU Jinbin, YU Haibin 2021, 39 (5):  541-551.  DOI: 10.3724/SP.J.1123.2021.01001 Abstract ( 123 )   HTML ( 22 )   PDF (890KB) ( 84 )   Supporting Information A method for the determination of 25 organochlorine pesticides (OCPs) in the atmosphere using isotope dilution high-resolution gas chromatography/high-resolution mass spectrometry (ID-HRGC/HRMS) was developed. Sample extraction was performed using an accelerated solvent extractor (ASE). The extraction parameters were as follows: the extraction solvent was 50% (v/v) hexane in dichloromethane, the extraction temperature was 100 ℃, the static time was 8 min, the cell was rinsed with 60% cell volume using the aforementioned extraction solvent, the purging time was 180 s with N2 gas, and the extraction proceeded through three cycles. The eluting solutions of common cartridges such as florisil, graphitized carbon black, alumina, and silica were determined via cartridge elution tests. Use of the aforementioned cartridges alone cannot remove the pigments in the air sample solution. Subsequently, all possible pairwise combinations of the four cartridges were used for sample cleaning, and only the combination of florisil and graphitized carbon black was found to completely remove the pigments. Thus, the combination of florisil and graphitized carbon black cartridges using 10 mL toluene for elution was determined as the final cleaning method in this study. A high-resolution mass spectrometer equipped with a gas chromatograph was used for quantification. A fused-silica capillary column (Rtx-CL Pesticides2, 30 m×0.25 mm×0.2 μm) was used to separate the target compounds. Injection was performed in the splitless mode at 250 ℃. The flow rate of nitrogen gas was maintained constant at 1 mL/min. The oven temperature was 110 ℃ (1 min), 20 ℃/min up to 210 ℃, 1.5 ℃/min up to 218 ℃ (1 min), and 2 ℃/min up to 260 ℃ (1 min). HRMS was conducted at >8000 resolution, the source temperature was 280 ℃ in the electron impact mode using ionization energy of 35 eV, and measurements were performed in the selective ion monitoring (SIM) mode. Twenty-five OCPs were identified by comparing their GC retention times with those of the corresponding labeled compounds, and the actual ion abundance ratios of two exact m/z values with the corresponding theoretical values. The 25 OCPs were quantified by average relative response factors (RRFs), and the relative standard deviations (RSDs) of the RRFs with six calibration solutions were no more than 20%. The linear range of this method was 0.4 to 800 μg/L, and the correlation coefficients (R2) were higher than 0.992. To validate the method, clean materials (one quartz fiber filter (QFF) and two polyurethane foam (PUF) plugs) were spiked with 100 pg, 400 pg, and 15 ng native OCP standards, respectively; the RSDs of the 25 OCPs for each spiked level ranged from 0.64% to 16%. The spiking recoveries of the native OCPs ranged from 67.2% to 135%. Penetration experiments were conducted by sampling various volumes of air (15-1000 m3) using a filter-PUF/PUF high-volume active sampler. The breakthrough volume was sampled when the amount of OCPs collected in the PUF of the non-sampling end reached 5% of the total amount collected by both PUFs. When a high-volume active sampler with filter-PUF/PUF was used as an adsorbent for sampling atmospheric OCPs, a serious breakthrough of pentachlorobenzene (PeCB) occurred. The effective sampling volume of hexachlorobenzene (HCB) was very low, and was no more than 30 m3 under the standard conditions (101.325 kPa, 273 K). The effective sampling volumes of other OCP compounds should be no more than 1200 m3. This will necessitate the use of high-adsorption-capacity adsorbents such as the PUF-XAD (a styrene-divinylbenzene copolymer) sandwich used for sampling air PeCB and HCB. Calculation with the effective sampling volumes from the penetration experiment revealed that the limits of detection of the 25 OCPs were in the range of 0.002 to 0.7 pg/m3. Thus, the detection levels of OCPs in this study were reduced to at least 2% of the current monitoring standards. Analysis of air samples in Beijing showed that all the target compounds except for trans-heptachlor epoxide, endrin, cis-nonachlor and 4,4'-DDD were 100% detected in the air samples. The concentrations of HCB (in volumes of 15-30 m3) ranged from 514 to 563 pg/m3, while those of the other OCPs (in a volume of 600 m3) ranged from 0.01 to 18.9 pg/m3. The recoveries of surrogate standards in this sample analysis were in the range of 33.9% to 155%, which satisfied the requirements of EPA Method 1699. Because of the very high detection limits, the current related monitoring standards cannot meet the requirements of atmospheric OCP analysis, especially at the ultra-trace level. In addition, highly sensitive monitoring standard methods are urgently needed. This method is suitable for analyzing most atmospheric OCPs, even at the ultra-trace level. It also lays the foundation for a new standard method formulation and provides strong support for the implementation of relevant international conventions.

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Determination of 13 sunscreen agents in cosmetics by gas chromatography-mass spectrometry LÜ Wen, LI Hongying, LIU Jie, HAN Wei, HUANG Wei 2021, 39 (5):  552-557.  DOI: 10.3724/SP.J.1123.2020.11003 Abstract ( 325 )   HTML ( 34 )   PDF (781KB) ( 190 )   Sunscreens can be categorized as physical and chemical types. Chemical sunscreens are widely used in cosmetics, and hence, their concentration in the desired products should be strictly monitored. Gas chromatography-mass spectrometry (GC-MS) is widely used for the analysis of cosmetics as it does not require organic mobile phases and allows for accurate qualitative and quantitative analyses. In this study, a method based on GC-MS was established for the determination of 13 sunscreen agents in cosmetics: ethylhexyl salicylate, homosalate, 4-methylbenzylidene camphor, ethylhexyl dimethyl para-aminobenzoic acid, ethylhexyl methoxycinnamate, octocrylene, butyl methoxydibenzoylmethane, diethylamino hydroxybenzoyl hexyl benzoate, 3-benzylidene camphor, benzophenone-3, camphor benzalkonium methosulfate, drometrizole trisiloxane, and isopentyl-4-methoxycinnamate. Accordingly, 0.5 g of the cosmetic product was dissolved in dichloromethane in a 50 mL volumetric flask and extracted ultrasonically for 15 min. Then, 1.0 mL of the extracting solution was withdrawn and diluted to 50.0 mL with dichloromethane. The mixture was filtered through a 0.22-μm membrane. A 1 μL aliquot of the dichloromethane solution was introduced onto the HP-5ms chromatographic column (30 m×250 μm×0.25 μm). The 13 components were separated under programed temperature elevation in the interval from 150 ℃ to 290 ℃. These components could be analyzed within 30 min after being ionized by the EI source, and their determination was achieved in selected ion monitoring (SIM) mode. The external standard method was employed for quantitative determination. Linear equations, linear correlation coefficients, and linear ranges were obtained by analyzing a series of mixed standard working solutions. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) of the 13 sunscreen agents were determined. The matrix effect and average recoveries of the 13 agents with six extraction solvents (dichloromethane, tetrahydrofuran, methanol, acetonitrile, n-hexane, and acetone) were compared. Among these, dichloromethane showed a weak matrix effect and high average recovery. The matrix effect of dichloromethane was 90.1%-100.5%, and the average recovery was 96.3%. All the 13 sunscreen agents showed good linearity in their corresponding ranges. The correlation coefficients (r 2) were higher than 0.998. The LODs and LOQs were in the ranges of 0.04 to 0.63 mg/g and 0.12 to 2.10 mg/g, respectively. Two types of cosmetics were selected to verify the accuracy and precision of the method at three levels. The average spiked recoveries of the 13 sunscreen agents in cream and lotion were 88.7%-103.6%, and 88.4%-102.3%, respectively; the corresponding relative standard deviations (RSDs, n=6) were 1.7%-4.9% and 1.2%-3.9%. Whitening cosmetics are frequently added with sunscreen agents, which is a regulatory blind spot. Five batches of skin whitening products containing sunscreen agents were detected using this method. The contents of five sunscreen agents in skin whitening cosmetics ranged from 0.8% to 5.2%, which were lower than the relevant limits in China. Owing to its advantages of simple operation, high sensitivity, and good recovery, the proposed method is suitable for the qualitative and quantitative determination of 13 sunscreen agents in cosmetics. This method provides technical support for market supervision and laboratory testing.