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  • Title: [Rapid extraction and detection of five alkaloids in dried khat by solvent extraction-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].
    Author: Shi HF, Xu BP, Xu CX, Zhou XQ, Xu HF.
    Journal: Se Pu; 2023 Sep; 41(9):771-780. PubMed ID: 37712541.
    Abstract:
    Khat is a common plant that grows primarily in Eastern Africa and the Arabian Peninsula. Cathinone, norpseudoephedrine, and norephedrine are the main psychoactive components of khat. Experimental studies have shown that red and green khat have similar cathinone contents, but green khat contains more norpseudoephedrine and norephedrine than red khat. Research indicates that Ethiopians believe that red khat has stronger psychoactive effects than green khat. Therefore, we speculated that other substances in red khat may enhance its psychoactive effects. Using the sampling method, we identified two other psychoactive components in khat: methcathinone and ethcathinone. At present, only a few studies on the extraction and detection of alkaloids from khat have been published in China, and no reports on the extraction and detection of methcathinone and ethcathinone from khat are available. In this study, we established an extraction and detection method for five alkaloids in dried khat using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS). To establish the extraction method, we optimized the extraction solvent and process. The amounts of dichloromethane and sodium hydroxide added during the purification step were also optimized. To establish the detection method, we optimized the chromatographic and MS conditions. The final extraction and detection method was as follows: Dried khat powder (0.1 g) was loaded into a polypropylene centrifuge tube, added with 1 mL of 0.05 mol/L hydrochloride aqueous solution, and vortex-oscillated for 3 min for extraction. The sample was centrifuged at 10000 r/min for 3 min. Next, 600 μL of the supernatant was placed in a centrifuge tube, added with 1 mL of dichloromethane, shaken for 1 min, and centrifuged at 10000 r/min for 3 min. Subsequently, 300 μL of the supernatant was placed in a centrifuge tube, added with 80 μL of 1 mol/L sodium hydroxide aqueous solution, shaken for 1 min, and added with 1 mL of acetonitrile. Vortex oscillation was performed for 2 min to extract the sample, after which solid sodium chloride (0.4 g) was added to the mixture, followed by shaking for 1 min to separate the acetonitrile and aqueous phases. The mixture was then centrifuged at 10000 r/min for 3 min. Finally, the supernatant was collected and diluted for further testing. The five target analytes were separated on a ZORBAX Eclipse Plus Phenyl-Hexyl column (100 mm×3.0 mm, 1.8 μm) via gradient elution using 0.1% acetic acid aqueous solution and acetonitrile as mobile phases with a flow rate of 0.3 mL/min and column temperature of 30 ℃. The analytes were identified using the targeted MS/MS method under positive electrospray ionization mode and quantified using the external standard method. The five alkaloids showed good correlations (all correlation coefficients (r2)≥0.9976) with their respective linear ranges. The limits of detection were between 0.08 and 0.75 μg/L, and the limits of quantification were between 0.25 and 2.50 μg/L. The average recoveries of the five alkaloids from two plants with different alkaloid contents were between 90.7% and 105.2%. The intra-sample precision ranged from 0.5% to 2.3%, the intra-day precision was between 1.0% and 2.5%, and the inter-day precision was between 1.3% and 3.3%. Using the developed method, we extracted and analyzed 15 khat samples, and detected five alkaloids. This method enables rapid sample pretreatment and has high sensitivity, good stability, and suitable accuracy. Based on the above results, we conclude that the proposed method meets the inspection and identification requirements for khat. Thus, it can provide a valuable reference for the physical and chemical identification of khat and support for further studies on its psychoactive components. 恰特草是一种常见的毒品原植物,卡西酮、去甲伪麻黄碱、去甲麻黄碱是其主要的精神活性成分。筛查发现恰特草中还存在具有较强精神活性作用的甲卡西酮和乙卡西酮成分。国内关于恰特草中生物碱成分的提取检测研究较少,国外也未见恰特草中甲卡西酮和乙卡西酮成分的研究。本研究通过优选提取溶剂种类、优化净化条件和色谱-质谱条件,利用0.05 mol/L的盐酸水溶液提取,二氯甲烷和1 mol/L的NaOH水溶液净化,再利用乙腈萃取,采用ZORBAX Eclipse Plus Phenyl-Hexyl色谱柱(100 mm×3.0 mm, 1.8 μm)分离,以0.1%甲酸水溶液-乙腈为流动相梯度洗脱,电喷雾双喷离子源(Dual AJS ESI)正离子模式下电离,四极杆飞行时间质谱仪目标离子采集模式(Target MS/MS)检测,外标法定量,建立了溶剂萃取-高效液相色谱-四极杆飞行时间质谱法提取检测干燥恰特草中5种生物碱成分的方法。结果表明,5种生物碱成分在各自的线性范围内线性关系良好,相关系数(r2)≥0.9976,检出限为0.08~0.75 μg/L,定量限为0.25~2.50 μg/L。2份生物碱含量差异较大的恰特草中5种生物碱成分的加标回收率为90.7%~105.2%,实验仪器精密度为0.5%~2.3%,日内方法精密度为1.0%~2.5%,日间方法精密度为1.3%~3.3%。该方法净化效果好,定量准确,灵敏度高,重复性好。利用该方法对国家林业局森林公安司法鉴定中心受理的15份恰特草相关案件中的检材进行检验,均检出5种生物碱成分,能够满足恰特草的理化检验鉴定要求,为恰特草中未知精神活性成分的发现和研究提供参考和指引。
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