开管毛细管电色谱分析3种解热镇痛药物

doi:10.3724/SP.J.1123.2020.01006

摘要/Abstract

摘要：

针对生物体液样品开展药物的绿色高效毛细管电泳分离分析具有重要的研究意义。该研究以3种解热镇痛药（4-氨基安替比林、氨基比林及非那西汀）为研究对象，以嵌段聚合物为涂层，建立了药物的开管毛细管电色谱（OT-CEC）分析新策略。首先，采用活性/可控自由基可逆加成-断裂链转移聚合方法，合成制备得到了两亲性嵌段聚合物-聚（苯乙烯-甲基丙烯酸缩水甘油酯）（P（St-GMA）），并将其涂覆到毛细管内壁；其次，通过考察影响OT-CEC分离效率的关键因素，包括嵌段聚合物的聚合时间、涂覆毛细管嵌段聚合物的浓度、电泳运行缓冲液的种类和pH值、有机溶剂添加剂等，优化了3种解热镇痛药物的OT-CEC分离条件；最终发现，不需添加任何有机溶剂及表面活性剂，仅采用50.0 mmol/L乙酸钠-乙酸（pH 5.7）作为OT-CEC的缓冲溶液，就能实现3种解热镇痛药物的基线分离。在8.0~2.5×10³ μmol/L范围内，分析物峰面积与其对应的浓度呈现良好的线性关系，相关系数（R²）均大于0.995，检出限为1.0~2.5 μmol/L。结果表明：P（St-GMA）在溶液中自组装所形成的类表面活性剂胶团结构增强了两亲性嵌段聚合物与解热镇痛药物之间的相互作用，显著提升了解热镇痛药物的OT-CEC分离效率。该工作不仅为制备新型聚合物及调控嵌段聚合物的自组装行为提供了研究思路，也展示了两亲性嵌段聚合物在药物的绿色OT-CEC分析中的实际应用潜力。

关键词: 开管毛细管电色谱, 两亲性嵌段聚合物, 解热镇痛药物

Abstract:

The advantages of capillary electrophoresis, such as small sample consumption, high separation efficiency, and multiple separation modes, have been known for decades. However, exploring unique capillary electrophoresis techniques for the analysis of fluid drugs in living bio-systems remains an important and urgent task. Owing to the similar structures and mass-to-charge ratios of antipyretic analgesic drugs, efficient baseline separation of these analytes by capillary zone electrophoresis method cannot be easily achieved. Micellar electrokinetic chromatography can improve the baseline separation of these drugs, but the substantial amounts of non-volatile surfactants (such as sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium deoxycholate and cetylammonium bromide) in running buffer solutions would pollute the ion source during mass spectrometric analysis. For this reason, it is difficult to analyze unknown drugs by capillary electrophoresis-electrospray ionization-mass spectrometry. To overcome these drawbacks, much attention has been paid to capillary electrochromatography (CEC) because combines the high separation efficiency of capillary electrophoresis with the high selectivity of high performance liquid chromatography (HPLC). Recent challenges encountered in open-tubular capillary electrochromatography (OT-CEC) expanding the range of suitable functional polymer monomers and improvement of the separation efficiency by tuning the characteristics of the polymer coatings without using any organic solvent additives. In this study, a protocol based on OT-CEC using a block co-polymer coating is proposed for the analysis of three test antipyretic analgesic drugs (4-aminoantipyrine, antipyrine and phenacetin), without adding organic solvents and surfactants in the running buffer solutions. First, an amphiphilic block co-poly(styrene-co-glycidyl methacrylate) (P(St-GMA)), was synthesized by reversible addition-fragmentation chain transfer polymerization under mild conditions. Then, P(St-GMA) was coated onto the capillary surface, and an OT-CEC analysis was performed. Next, the effect of some key factors, including the polymerization time for obtaining P(St-GMA) with different molecular weights, coating concentrations of the block copolymer, the species of the running buffer solutions, pH and concentrations of the running buffer solutions, and organic solvent additives, on the OT-CEC separation efficiency were investigated. Under the optimized conditions with 50.0 mmol/L NaAc-HAc as the running buffer solution at pH 5.7, the three test antipyretic analgesic drugs were base-line separated by the constructed OT-CEC system. Good linear relationships between peak area and concentration of the test analytes in the range of 8.0-2.5×10³ μmol/L were obtained (R² ≥ 0.995). The limits of detection (LODs) were 1.0-2.5 μmol/L. Furthermore, the reason for the OT-CEC separation efficiency was clarified based on the decreased electro-osmotic flow in the coated capillary compared with that in the uncoated capillary. Finally, the proposed OT-CEC assay without using any organic solvents and surfactants as additives was applied for analysis of the three test antipyretic analgesic drugs in rat serum samples. Importantly, it was found that despite peak tailing, the OT-CEC separation efficiency of the drugs was dramatically enhanced because the block co-polymer could self-assemble in the solution and form pseudo-micelles, which further increased the interactions between the P(St-GMA) and these drugs. Our results not only reveal the great potential of block co-polymer coatings in OT-CEC for the analysis of drugs in real biological samples, but also serve asa platform for the preparation of diverse block co-polymers to be used in OT-CEC analysis. We believe that in the near future, the peak tailing problem in OT-CEC analysis can be resolved by using the designed unique block co-polymers, which possess a greater number of functional sites, as coatings and by appropriately tuning the interactions between the analytes and the coatings.

Key words: open-tubular capillary electrochromatography (OT-CEC), amphiphilic block co-polymer, antipyretic analgesic drugs

刘丽丽, 乔娟, 张红医, 齐莉. 开管毛细管电色谱分析3种解热镇痛药物[J]. 色谱, 2020, 38(9): 1107-1114.

LIU Lili, QIAO Juan, ZHANG Hongyi, QI Li. Analysis of three antipyretic analgesic drugs by open-tubular capillary electrochromatography[J]. Chinese Journal of Chromatography, 2020, 38(9): 1107-1114.

图/表 7

图1 (a) P(St-GMA)的FT-IR图谱和(b)裸毛细管及(c、d)涂层毛细管的SEM图

Fig. 1 (a) FT-IR of P(St-GMA) and SEM images of (b) bare capillary and (c, d) coated capillary P(St-GMA): poly(styrene-co-glycidyl methacrylate); SEM: scanning electronic microscopy.

图2 (a) 聚合时间、(b)聚合物含量、(c)缓冲溶液种类、(d)有机溶剂添加剂、(e)缓冲液pH值、(f)NaAc浓度对药物OT-CEC分离度的影响和(g)缓冲液pH和(h)NaAc浓度对涂层管EOF的影响(n=3)

Fig. 2 Effect of (a) polymerization time, (b) contents of P(St-GMA), (c) buffer types, (d) organic solvent additives, (e) pH values of buffer, (f) concentrations of NaAc on the resolutions (Rs) and (g) pH values of buffer and (h) concentrations of NaAc on the electroosmotic flow (EOF) of coated capillary (n=3) Rs1: resolution between antipyrine (AP) and 4-aminoantipyrine (4-AA); Rs2: resolution between 4-AA and phenacetin (PH); Buffer 1: K2HPO4-NaH2PO4; Buffer 2: NH4Ac-HAc; Buffer 3: NaAc-HAc; ACN: acetonitrile; MeOH: methanol; DMK: acetone; EtOH: ethanol.

图3 3种药物的(a)裸管毛细管区带电泳图、(b)裸管胶束电动毛细管色谱图和(c)OT-CEC谱图

Fig. 3 (a) Bare tube capillary zone electrophoresis, (b) bare tube micelle electrokinetic capillary chromatogram and (c) OT-CEC electropherogram of the three drugs 1. AP (100.0 μmol/L); 2.4-AA (100.0 μmol/L); 3. PH (80.0 μmol/L).

图4 基于P(St-GMA)涂层与药物相互作用的OT-CEC分离机理示意图

Fig. 4 Mechanism illustration for OT-CEC analysis based on the interaction between P(St-GMA) coating and the drugs

表1 3种药物的线性方程、相关系数、线性范围和检出限

Table 1 Linear equations, correlation coefficients (R2), linear ranges and LODs of the three drugs

Drug	Linear equation	R²	Linear range/ (μmol/L)	LOD/ (μmol/L)
y: peak area, μV·s; x: concentration, μmol/L.
AP	y=1.5×10²x-5.0×10²	0.999	10.0-2.5×10³	2.5
4-AA	y=2.1×10²x-1.4×10³	0.995	8.0-2.0×10³	1.0
PH	y=5.6×10²x-4.3×10⁴	0.997	8.0-2.0×10³	2.0

图5 (a) 血清空白样品和(b)加标血清样品的OT-CEC谱图

Fig. 5 OT-CEC electropherograms of (a) the blank serum and spiked serum sample 1. AP (100.0 μmol/L); 2.4-AA (100.0 μmol/L); 3. PH (80.0 μmol/L).

表2 血清样品中3种药物的回收率(n=3)

Table 2 Recoveries of the three drugs in serum samples (n=3)

Sample	Drug	Added/ (μmol/L)	Detected/ (μmol/L)	Recovery/ %
Sample-1	AP	90.0	92.8	103.1
		180.0	183.1	101.7
		360.0	361.6	100.4
	4-AA	90.0	93.5	103.1
		180.0	178.9	99.4
		360.0	358.0	99.5
	PH	72.0	71.4	99.2
		144.0	147.0	102.1
		288.0	287.6	99.9
Sample-2	AP	90.0	93.7	104.1
		180.0	179.7	99.8
		360.0	360.1	100.0
	4-AA	90.0	91.7	101.9
		180.0	184.6	102.5
		360.0	364.8	101.3
	PH	72.0	72.6	100.9
		144.0	145.5	101.0
		288.0	283.5	98.4
Sample-3	AP	90.0	91.3	101.4
		180.0	181.0	100.5
		360.0	361.2	100.3
	4-AA	90.0	94.1	104.5
		180.0	174.6	97.0
		360.0	361.2	100.3
	PH	72.0	72.4	100.5
		144.0	146.2	101.5
		288.0	288.3	100.1

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