衍生化-气相色谱-三重四极杆质谱法测定泼尼松龙中联氨

Determination of hydrazine in prednisolone by derivatization-gas chromatography-triple quadrupole mass spectrometry

泼尼松龙是一种广泛用于临床治疗的肾上腺糖皮质激素药物,其中联氨的残留会直接影响用药安全,但目前国内外还没有出台相应的法律法规和标准来管控药物中联氨的残留限值。联氨具有强极性和强还原性,理化性质很不稳定,易被氧化,又因缺少发色团,相对分子质量太小,检测起来难度很大,需引入一种衍生化试剂,降低其极性,生成相对分子质量较大且理化性质稳定的衍生产物。该研究通过优化衍生化试剂、色谱-质谱条件、溶剂体系和衍生化条件,建立了衍生化-气相色谱-三重四极杆质谱法(GC-MS/MS)测定泼尼松龙中联氨残留的方法,并进行了方法学验证,结果满意。称取1 g泼尼松龙样品置于10 mL具塞离心管中,加入稀释溶剂(甲醇-二氯甲烷(14∶23,v/v))至刻度线,涡旋振荡至样品完全溶解后,吸取100μL置于进样小瓶中,再加入丙酮900μL,涡旋振荡混匀,样品在丙酮-稀释溶剂(9∶1,v/v)中同时完成稀释和衍生化反应后,再经GC-MS/MS检测分析。该研究的衍生化反应无需在添加冰乙酸和超声条件下进行,也无需再添加其他试剂进行萃取操作,联氨与丙酮可瞬间发生衍生化反应,直接实现泼尼松龙中联氨的快速测定。结果表明,联氨在1~12μg/L范围内线性关系良好,线性相关系数(r2)为0.9999;检出限、定量限分别为0.03和0.10 mg/kg;进样精密度(relative standard deviation,RSD)为1.10%。加标回收率和重复性良好,加标水平分别为1、6、12μg/L时的回收率为96.15%~96.46%,对应的RSD值为1.77%~2.12%。中间精密度良好,不同时间、不同人员在同一台仪器上测定结果的RSD值为1.77%。方法耐用性良好,通过改变色谱条件来研究检测结果受影响程度大小,在原条件、初始柱温±5℃、升温速率±2℃/min、柱流量±0.1 mL/min的条件下分别对加标6μg/L的样品溶液中的联氨含量进行检测,检测结果的RSD值为2.58%。应用建立的方法测定泼尼松龙市售标准品和某药企提供的9个不同批次的泼尼松龙样品,均未检出联氨。该方法操作简便、准确可靠、灵敏度高、选择性好,可用于泼尼松龙中联氨的检测。

Prednisolone is an adrenal glucocorticoid drug with immunosuppressive,anti-inflammatory,anti-allergic,and antiviral effects that are widely exploited in clinical treatment.The hydrazine residue to prednisolone directly affects medication safety and threatens the patient’s health.At present,there are no relevant laws,regulations,and standards to control the residual limit of hydrazine in drugs at home or abroad.Therefore,a simple,rapid,accurate,reliable,sensitive,and selective method is urgently needed for the determination of trace hydrazine in prednisolone.Hydrazine has strong polarity and reductivity,with unstable physical and chemical properties,thus being easily oxidized.In addition,because of the lack of chromophores and low molecular weight,the detection of hydrazine is very difficult.Therefore,a derivative reagent should be introduced to reduce its polarity and generate a derivative product with a high molecular weight as well as stable physical and chemical properties.Acetone,as a common laboratory reagent,is inexpensive and can rapidly react with hydrazine;therefore,it is an ideal derivative reagent for the determination of hydrazine.In this study,a method based on precolumn derivatization with gas chromatography-triple quadrupole mass spectrometry(GC-MS/MS)was developed for the determination of hydrazine in prednisolone by optimizing the derivatization reagent,GC and MS conditions,solvent system,and derivatization conditions.Method validation was then carried out using the established method,and the results were satisfactory.In this study,1 g of prednisolone sample was weighed and placed in a 10 mL centrifuge tube with a plug;then,a methanol-dichloromethane dilution solvent(14∶23,v/v)was added to the scale line,and the sample was vortexed until completely dissolved.About 100μL of the test solution prepared above was pipetted into the sample vial,followed by the addition of 900μL acetone.The resulting solution was vortexed and mixed well.The sample was diluted and derivatized simultaneously in acetone solution,acetone/methanol-dichloromethane dilution solvent(9∶1,v/v),and then detected and analyzed by GC-MS/MS.In this study,the derivatization reaction between hydrazine and acetone did not require the addition of acetic acid and ultrasound conditions,or the use of other reagents for the extraction operation.The reaction was instantaneous,and rapid determination of hydrazine in prednisolone could be achieved.The standard curve was obtained with a good correlation coefficient(r2=0.9999)in the range 1-12μg/L.The limits of detection and quantitation were 0.03 mg/kg and 0.10 mg/kg,respectively.The relative standard deviation(RSD)of injection precision was 1.10%.The recoveries and repeatability were good;the recoveries of low-,medium-,and high-concentration spiked samples were 96.15%-96.46%at spiked concentrations of 1,6,and 12μg/L,respectively,and the corresponding RSDs were 1.77%-2.12%.The intermediate precision was good,and the RSD of the determination results obtained on the same instrument by different laboratory technicians at different times was 1.77%.The durability was good,and the degree of influence of the detection results was studied by changing the chromatographic conditions.Under the original condition or conditions with initial column temperature±5℃,heating rate±2℃/min,or column flow rate±0.1 mL/min,the hydrazine content in the sample solution at a spiked concentration of 6μg/L was detected,and the RSD of the detection results was 2.58%.The established method was applied to detect hydrazine in a prednisolone standard substance procured from the market and nine batches of prednisolone samples provided by a pharmaceutical company.No hydrazine was detected in any of these samples.The established method is simple,reliable,highly sensitive,and highly selective,and it can be applied for the detection of hydrazine in prednisolone.