高效液相色谱法快速同时检测全血中两种免疫抑制剂

doi:10.3724/SP.J.1123.2022.03033

摘要/Abstract

摘要：

环孢素A和西罗莫司是许多器官移植手术中广泛使用的免疫抑制剂,且一起使用时会产生协同效应,但这两种免疫抑制剂的治疗窗口都非常窄,仅在特定的血药浓度范围内有预期的治疗效果。因此,快速同时检测全血中这两种免疫抑制剂的浓度,可为患者器官移植手术后的给药方案提供有价值的信息。该工作首先考察了环孢素A和西罗莫司在生物液相色谱柱和传统液相色谱柱上的色谱行为,然后基于生物液相色谱柱,建立了可快速分离和检测全血中环孢素A和西罗莫司的高效液相色谱分析方法。全血样品经样品前处理后进样分析,采用ZORBAX 300SB C8柱(250 mm×4.6 mm, 5.0 μm)进行分离,以乙腈-水(70:30, v/v)为流动相进行等度洗脱,柱温为60 ℃,流速为1.0 mL/min,检测波长为205 nm和278 nm,进样量为20 μL。结果表明,环孢素A和西罗莫司在6 min内可实现较好的分离;环孢素A和西罗莫司在各自的浓度范围内具有良好的线性关系(r>0.997),检出限(S/N=3)分别为10 ng/mL和1 ng/mL,定量限(S/N=10)分别为30 ng/mL和2 ng/mL, 3个水平的平均加标回收率分别为83.5%~89.7%和95.8%~97.8%,相对标准偏差(RSD)分别为3.2%~9.0%和3.4%~6.7%(n=5)。该方法操作简便,流动相简单,分析时间短,线性范围宽,灵敏度高,可用于全血中环孢素A和西罗莫司的含量检测。

关键词: 高效液相色谱, 免疫抑制剂, 环孢素A, 西罗莫司, 生物液相色谱柱

Abstract:

Cyclosporine A and sirolimus are immunosuppressants that are widely used in many organ transplantation procedures. They exhibit some complementary mechanisms of action and interact synergistically when used together. However, they are critical-dose drugs and have a narrow therapeutic index. They provide the desired therapeutic effect with acceptable tolerability only within a specific range of blood concentrations. Therefore, the rapid and simultaneous detection of the concentrations of cyclosporine A and sirolimus in whole blood could provide valuable information on planning medicine administration after organ transplantations. In this study, firstly, the chromatographic behaviors of cyclosporine A and sirolimus on a biological liquid chromatography (BioLC) column and traditional liquid chromatography (TraLC) columns were investigated systematically under the same chromatographic conditions. The results suggested that the peak height and peak width of cyclosporine A and sirolimus on the BioLC column, ZORBAX 300SB C8 (250 mm×4.6 mm, 5.0 μm), were the highest and narrowest, respectively. The number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column increased significantly when the volume ratio of acetonitrile in the mobile phases was greater than 70%. Their retention time on the BioLC and TraLC columns was negligibly affected by the use of formic acid and trifluoroacetic acid as the mobile phases. In the range of the experimental column temperature, the number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column was significantly higher than that on the two TraLC columns. Furthermore, the relationship between the retention factor and column temperature of cyclosporine A on the ZORBAX 300SB C8 column was different from that on the two TraLC columns. Subsequently, a high performance liquid chromatography method based on the ZORBAX 300SB C8 column was established for the rapid separation and determination of cyclosporin A and sirolimus in whole blood. A sample of whole blood with a volume of 50 μL was prepared by protein precipitation with 1 mol/L sodium hydroxide and then extracted into 500 μL of ether-methanol (95:5, v/v). After centrifugation at 14000 r/min for 10 min, the organic layer was removed and evaporated under a stream of nitrogen at 50 ℃. The residue was then reconstituted in 200 μL of methanol for use. Cyclosporin A and sirolimus were separated through isocratic elution on the ZORBAX 300SB C8 column. The column temperature was set at 60 ℃. The mobile phase was acetonitrile-water (70:30, v/v) and the flow rate was 1.0 mL/min. The detection wavelengths were 205 nm for cyclosporine A and 278 nm for sirolimus. The injection volume was 20 μL. The external standard method was used to quantify cyclosporine A and sirolimus. Under the optimized conditions, cyclosporine A and sirolimus were well-separated within 6 min with a resolution of 3.7 at 205 nm. In addition, the endogenous substances in whole blood negligibly interfered in the detection of sirolimus, while two endogenous substances slightly affected the detection of cyclosporine A. Cyclosporine A and sirolimus both showed good linear relationships in their respective concentration (r>0.997). The limits of detection (LODs) of cyclosporine A and sirolimus were respectively calculated to be 10 ng/mL and 1 ng/mL based on a signal-to-noise ratio of 3, and the limits of quantification (LOQs) were 30 ng/mL and 2 ng/mL based on a signal-to-noise ratio of 10. In the whole blood samples, the recoveries of cyclosporine A and sirolimus at three spiked levels were in the ranges of 83.5%-89.7% and 95.8%-97.8% with relative standard deviations (RSDs) of 3.2%-9.0% and 3.4%-6.7% (n=5), respectively. The established method is simple in operation, can be performed with a simple mobile phase, has a short analysis time, and provides a wide linear range and high sensitivity; hence, it can be applied to the determination of cyclosporine A and sirolimus in whole blood.

Key words: high performance liquid chromatography (HPLC), immunosuppressant, cyclosporine A, sirolimus, biological liquid chromatography column

中图分类号:

O658

黄永鹏, 唐慧, 孟祥燕, 钟辉, 宋云扬, 陈博, 邹志云. 高效液相色谱法快速同时检测全血中两种免疫抑制剂[J]. 色谱, 2023, 41(2): 152-159.

HUANG Yongpeng, TANG Hui, MENG Xiangyan, ZHONG Hui, SONG Yunyang, CHEN Bo, ZOU Zhiyun. Rapid and simultaneous determination of two immunosuppressants in whole blood by high performance liquid chromatography[J]. Chinese Journal of Chromatography, 2023, 41(2): 152-159.

图/表 11

图1 免疫抑制剂的化学结构式

Fig. 1 Chemical structures of immunosuppressants

表1 使用的色谱柱基本信息

Table 1 Basic information of the employed chromatography columns

No.	Column style	Model	Specification	Maximum operating temperature/ ℃	Status
1	traditional	ZORBAX Eclipse XDB C18	250 mm×4.6 mm,5.0 μm,8 nm	60	used
2	traditional	ZORBAX Eclipse Plus C18	250 mm×4.6 mm,5.0 μm,9.5 nm	60	used
3	traditional	ZORBAX Eclipse Plus C8	250 mm×4.6 mm,5.0 μm,9.5 nm	60	new
4	traditional	ZORBAX Eclipse PAH	250 mm×4.6 mm,5.0 μm,9.5 nm	60	new
5	traditional	InfinityLab Poroshell 120 EC C8	150 mm×4.6 mm,2.7 μm,12 nm	60	new
6	biological	ZORBAX 300SB C8	250 mm×4.6 mm,5.0 μm,30 nm	80	new

图2 环孢素A和西罗莫司在不同色谱柱上的色谱图和半峰宽

Fig. 2 Chromatograms and full width at half maxima of cyclosporine A and sirolimus on different chromatography columns

图3 环孢素A和西罗莫司在乙腈-水流动相体系中的色谱峰参数

Fig. 3 Chromatographic peak parameters of cyclosporine A and sirolimus in ACN-water mobile phases

图4 环孢素A和西罗莫司在不同流动相中的保留时间

Fig. 4 Retention of cyclosporine A and sirolimus in different mobile phases

图5 环孢素A和西罗莫司在不同色谱柱上的保留因子与柱温的关系图

Fig. 5 Relationships between retention factor and column temperature of cyclosporine A and sirolimus on different chromatography columns

图6 环孢素A和西罗莫司在不同色谱柱上的柱温与柱效关系图

Fig. 6 Relationships between the column temperature and chromatographic efficiency of cyclosporine A and sirolimus on different chromatography columns

图7 环孢素A和西罗莫司在不同色谱条件下的分离度

Fig. 7 Resolutions of cyclosporine A and sirolimus under different chromatography conditions

图8 空白全血及环孢素A和西罗莫司混合标准储备溶液的色谱图

Fig. 8 Chromatograms of a blank sample of whole blood and a mixed standard solution of cyclosporine A and sirolimus

表2 环孢素A和西罗莫司的线性范围、线性方程、相关系数、检出限和定量限

Table 2 Linear ranges,linear equations,correlation coefficients(r),LODs,and LOQs of cyclosporine A and sirolimus

Immunosuppressant drug	Linear range/(ng/mL)	Linear equation	r	LOD/(ng/mL)	LOQ/(ng/mL)
Cyclosporine A	30-2000	Y=0.053X+0.045	0.9976	10	30
Sirolimus	2-200	Y=0.098X+0.538	0.9989	1	2

表3 空白全血样品中环孢素A和西罗莫司的加标回收率及相对标准偏差(n=5)

Table 3 Spiked recoveries and RSDs of cyclosporine A and sirolimus in a blank whole blood sample(n=5)

Immunosuppressant drug	Added/ (ng/mL)	Found/ (ng/mL)	Recovery/ %	RSD/ %
Cyclosporine A	30	25.06	83.5	9.0
	60	51.26	85.4	6.0
	300	269.01	89.7	3.2
Sirolimus	2	1.94	97.0	6.7
	4	3.83	95.8	4.2
	20	19.56	97.8	3.4

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