1. 江苏省食品药品监督检验研究院, 南京 210019;
2. 中国药科大学, 南京 211198
收稿日期:2017-09-17
基金项目:江苏省食品药品监督管理局科研项目(20170202)
Screening of 54 illegally added chemical drugs in anti-hypertensive Chinese traditional patent medicine and health foods by liquid chromatography tandem mass spectrometry
1. Jiangsu Institute for Food and Drug Control, Nanjing 210019, China;
2. China Pharmaceutical University, Nanjing 211198, China
我国高血压患者已突破3.3亿,每3名成人中就有1名患高血压,有超过70%的高血压患者需要2种或更多药物联合用药,这使得高血压的控制更加复杂和困难[1]。中国高血压患者存在3个误区:不愿服药,不难受不服药和不按医嘱服药。一些不法商贩瞅准高血压患者的庞大市场,在中成药和保健食品中非法添加降压化学药物,如果消费者在不知情的情况下,替代西药自行服用,或与西药同服,易造成血压骤然下降或大波动,将会造成肝、肾、心脏的损伤及不良反应,严重者可导致死亡[2-4]。由于中成药和保健食品成分复杂,化学降压药物又种类繁多,如果低剂量加入多种降压药,在检测有效成分或指标性成分时不易发觉;而现行国家药品标准——降压类中成药中非法添加化学药品补充检验方法(批准件编号2009032和2012005),仅对阿替洛尔、可乐定、氢氯噻嗪、卡托普利、哌唑嗪、利血平、氨氯地平、尼莫地平、硝苯地平、尼群地平、尼索地平和非洛地平共12种化学药物进行筛查和验证,而许多文献报道,实际样品中发现了其他降压类药物的非法添加,涉及方法主要包括薄层色谱法[5]、近红外光谱法[6]、液相色谱法[7-8]、液质联用法[9-10]和气质联用法[11]。近年来,中药和保健食品非法添加的研究很多,虽然辅助降血压类中成药和保健食品中非法添加化学药物的检测筛查技术和方法的研究及应用取得了一定的进展,但是,由于降压药物种类繁多,联合用药剂量低,许多降压药物没有紫外吸收,需要直接使用质谱检测和确证(如普利类),故而有必要建立一种更通用、更广泛、更灵敏的筛查方法,建立和完善中成药和保健食品中非法添加的检测标准和检验检测体系[12]。
降压药分类方法较多[10],本文为了更系统、全面地研究,将降压药物大致分为以下6类:1)血管紧张素转化酶抑制剂:普利类;2)血管紧张素Ⅱ受体阻滞剂:沙坦类;3)二氢吡啶类钙离子拮抗剂:地平类;4)β受体阻断药:洛尔类;5)利尿药:噻嗪类、潴钾利尿剂(氨苯蝶啶,阿米洛利)、醛固酮拮抗剂(螺内酯等)和袢利尿剂(呋塞米等);6)其他降压药:α1受体阻滞剂(唑嗪类)、α2受体激动剂(甲基多巴)等。本文以疗效确切和廉价易得为筛选原则,结合文献中实际样品检出情况,选择了54种降压药物作为目标物质,建立了液质联用(LC-MS/MS)测定方法。
1 仪器与试药
1.1 仪器
KQ_300型超声波清洗器(昆山市超声仪器有限公司);电子分析天平(精度为0.000 01 g,Sartorius公司);Milli-Q高纯水发生器(Millipore公司);Thermo uHPLC/TSQ Quantum Access三重四极杆质谱仪(Thermo Finnigan公司)
1.2 试药
对照品均购自中国食品药品检定研究院,54种对照品按照前述6大类分类后按照母离子质核比从小到大编号,编号和对照品信息见表 1。甲醇、乙腈、醋酸铵、甲酸和冰醋酸均为色谱纯。纯水,Milli-Q超纯水仪制备;所有溶液均需过0.22 μm滤膜。本试验所检测的12批(编号Ⅰ~Ⅻ)保健食品和中成药均为市售样品。
表 1
Table 1
表 1 MRM质谱参数
Table 1 LC-MS/MS parameters in MRM mode
类型 |
编号 (No.) |
对照品名称 (name of reference substance) |
批号 (lot No.) |
扫描模式 (scan mode) |
母离子 (parent ion)m/z |
定量离子 (quantitative ion) |
|
定性离子 (qualitative ion) |
m/z |
碰撞能量 (collision energy)/eV |
m/z |
碰撞能量 (collision energy)/eV |
普利类 (prils) |
1-1 |
卡托普利(captopril) |
100318-201103 |
ESI+ |
218.09 |
172.2 |
11 |
|
70.1 |
24 |
1-2 |
培哚普利叔丁胺盐(perindopril erbumine) |
101072-201101 |
ESI+ |
369.24 |
172.2 |
20 |
98.1 |
34 |
1-3 |
马来酸依那普利(enalapril maleate) |
100705-201203 |
ESI+ |
377.21 |
234.2 |
19 |
117.1 |
16 |
普利类 (prils) |
1-4 |
贝那普利拉(benazeprilat) |
100771-201001 |
ESI+ |
397.18 |
351.2 |
19 |
190.2 |
29 |
1-5 |
赖诺普利(lisinopril) |
100814-200701 |
ESI+ |
406.23 |
246.1 |
22 |
309.2 |
19 |
1-6 |
盐酸贝那普利(benazepril hydrochloride) |
100768-201403 |
ESI+ |
425.19 |
190.1 |
32 |
251.2 |
15 |
1-7 |
盐酸喹那普利(quinapril hydrochloride) |
100568-200401 |
ESI+ |
439.22 |
234.2 |
20 |
117.1 |
38 |
1-8 |
福辛普利钠(fosinopril sodium) |
100942-200701 |
ESI+ |
564.31 |
436.2 |
15 |
152.2 |
39 |
地平类 (dipines) |
2-1 |
硝苯地平(nifedipine) |
100338-201103 |
ESI+ |
347.12 |
254.1 |
19 |
315.1 |
15 |
2-2 |
尼群地平(nitrendipine) |
100585-201104 |
ESI+ |
361.14 |
315.1 |
15 |
329.2 |
13 |
2-3 |
非洛地平(felodipine) |
100717-201002 |
ESI+ |
384.08 |
352.1 |
15 |
338.1 |
25 |
2-4 |
尼索地平(nisoldipine) |
100574-200401 |
ESI+ |
389.17 |
315.2 |
10 |
357.2 |
7 |
2-5 |
苯磺酸氨氯地平(amlodipine besylate) |
100374-201204 |
ESI+ |
409.15 |
294.1 |
15 |
238.1 |
18 |
2-6 |
尼莫地平(nimodipine) |
100270-200002 |
ESI+ |
419.18 |
343.1 |
12 |
301.1 |
25 |
2-7 |
拉西地平(lacidipine) |
100741-200802 |
ESI+ |
456.24 |
400.1 |
18 |
354.2 |
20 |
2-8 |
盐酸尼卡地平(nicardipine hydrochloride) |
100586-201102 |
ESI+ |
480.21 |
315.1 |
25 |
359.1 |
23 |
2-9 |
西尼地平(cilnidipine) |
100993-200701 |
ESI+ |
493.20 |
375.1 |
15 |
117.1 |
30 |
沙坦类 (sartans) |
3-1 |
氯沙坦钾(losartan potassium) |
100597-200501 |
ESI+ |
423.17 |
207.1 |
20 |
180.1 |
35 |
3-2 |
厄贝沙坦(irbesartan) |
100607-201202 |
ESI+ |
429.24 |
207.1 |
25 |
180.1 |
40 |
3-3 |
缬沙坦(valsartan) |
100651-201203 |
ESI+ |
436.23 |
291.2 |
18 |
207.1 |
27 |
3-4 |
替米沙坦(telmisartan) |
100629-201202 |
ESI+ |
515.24 |
497.3 |
40 |
276.2 |
46 |
3-5 |
奥美沙坦酯(olmesartan medoxomil) |
100864-200601 |
ESI+ |
559.23 |
207.1 |
30 |
429.2 |
18 |
3-6 |
坎地沙坦酯(candesartan cilexetil) |
100685-200401 |
ESI+ |
611.26 |
423.1 |
15 |
338.1 |
15 |
利尿类 (diuretics) |
4-1 |
盐酸阿米洛利(amiloride hydrochloride) |
100310-200201 |
ESI+ |
230.06 |
171.1 |
17 |
116.1 |
33 |
4-2 |
氨苯蝶啶(triamterene) |
100429-200401 |
ESI+ |
254.12 |
104.1 |
38 |
116.1 |
42 |
4-3 |
吲达帕胺(indapamide) |
100257-201204 |
ESI+ |
366.07 |
132.1 |
14 |
117.1 |
34 |
4-4 |
螺内酯(spironolactone) |
100193-201203 |
ESI+ |
417.21 |
341.1 |
13 |
171.1 |
43 |
4-5 |
盐酸维拉帕米(verapamil hydrochloride) |
100223-200102 |
ESI+ |
455.29 |
165.1 |
28 |
150.1 |
39 |
4-6 |
氢氯噻嗪(hydrochlorothiazide) |
101129-201302 |
ESI- |
293.94 |
214.1 |
32 |
215.1 |
30 |
4-7 |
氢氯噻嗪(hydrochlorothiazide) |
100309-201103 |
ESI- |
295.96 |
269.1 |
21 |
205.1 |
25 |
4-8 |
呋塞米(furosemide) |
100544-201102 |
ESI- |
329.00 |
285.1 |
18 |
206.1 |
24 |
4-9 |
氯噻酮(chlortralidone) |
100195-199701 |
ESI- |
337.01 |
146.1 |
19 |
190.1 |
30 |
4-10 |
苄氟噻嗪(bendroflumethiazide) |
100007-200703 |
ESI- |
420.03 |
289.1 |
24 |
239.1 |
30 |
洛尔类 (lols) |
5-1 |
盐酸普萘洛尔(propranolol hydrochloride) |
100783-201202 |
ESI+ |
260.17 |
183.2 |
18 |
155.1 |
25 |
5-2 |
阿替洛尔(atenolol) |
100117-201105 |
ESI+ |
267.17 |
190.2 |
18 |
145.1 |
26 |
洛尔类 (lols) |
5-3 |
酒石酸美托洛尔(metoprolol tartrate) |
100084-201102 |
ESI+ |
268.19 |
191.2 |
18 |
133.1 |
25 |
5-4 |
盐酸索他洛尔(sotalol hydrochloride) |
100737-200501 |
ESI+ |
273.13 |
255.2 |
12 |
213.1 |
18 |
5-5 |
盐酸卡替洛尔(carteolol hydrochloride) |
100345-201102 |
ESI+ |
293.19 |
237.2 |
15 |
202.2 |
21 |
5-6 |
盐酸艾司洛尔(esmolol hydrochloride) |
100749-200501 |
ESI+ |
296.19 |
145.1 |
27 |
219.2 |
19 |
5-7 |
富马酸比索洛尔(bisoprolol fumarate) |
100711-200401 |
ESI+ |
326.23 |
116.1 |
18 |
107.1 |
40 |
5-8 |
盐酸拉贝洛尔(labetalol hydrochloride) |
100484-201001 |
ESI+ |
329.19 |
311.2 |
13 |
294.2 |
20 |
5-9 |
盐酸贝凡洛尔(bevantolol hydrochloride) |
101033-200901 |
ESI+ |
346.20 |
165.2 |
22 |
150.2 |
32 |
5-10 |
盐酸塞利洛尔(celiprolol hydrochloride) |
100807-200501 |
ESI+ |
380.25 |
307.2 |
18 |
251.2 |
25 |
其他类 (others) |
6-1 |
米诺地尔(minoxidil) |
100238-199701 |
ESI+ |
210.14 |
164.2 |
25 |
110.1 |
27 |
6-2 |
甲基多巴(methyldopa) |
100189-199501 |
ESI+ |
212.09 |
166.2 |
16 |
139.1 |
16 |
6-3 |
盐酸可乐定(clonidine hydrochloride) |
100071-201106 |
ESI+ |
230.03 |
160.1 |
36 |
124.1 |
40 |
6-4 |
甲磺酸酚妥拉明(phentolamine mesylate) |
100110-201103 |
ESI+ |
282.16 |
212.2 |
19 |
91.1 |
40 |
6-5 |
盐酸哌唑嗪(prazosin hydrochloride) |
100164-201003 |
ESI+ |
384.17 |
247.2 |
28 |
138.2 |
31 |
6-6 |
盐酸特拉唑嗪(terazosin hydrochloride) |
100375-201103 |
ESI+ |
388.20 |
290.1 |
26 |
247.1 |
34 |
6-7 |
盐酸氟桂利嗪(flunarizine hydrochloride) |
100844-200802 |
ESI+ |
405.21 |
203.1 |
20 |
183.1 |
45 |
6-8 |
盐酸地尔硫䓬(diltiazem hydrochloride) |
100161-200503 |
ESI+ |
415.17 |
178.1 |
25 |
310.1 |
20 |
6-9 |
甲磺酸多沙唑嗪(doxazosin mesylate) |
100566-200401 |
ESI+ |
452.19 |
344.1 |
30 |
247.1 |
40 |
6-10 |
利血平(reserpine) |
100041-201012 |
ESI+ |
609.28 |
195.1 |
36 |
448.1 |
32 |
6-11 |
曲克芦丁(troxerutin) |
100416-201004 |
ESI+ |
743.24 |
435.2 |
22 |
581.2 |
10 |
|
表 1 MRM质谱参数
Table 1 LC-MS/MS parameters in MRM mode |
2 方法
2.1 混合对照品储备液的制备
取各对照品适量,置同一100 mL棕色量瓶中,加甲醇约75 mL,超声处理30 min,放冷,用甲醇稀释至刻度,摇匀,即得每1 mL中约含卡托普利、螺内酯、氢氯噻嗪和氯噻酮各0.25 mg,曲克芦丁0.10 mg,其他对照品各0.050 mg的混合对照品储备液。
2.2 供试品溶液的制备
取供试品一次服用量(片剂、茶剂、丸剂研细混匀;胶囊剂取内容物混匀),置棕色具塞锥形瓶中,精密加入甲醇50.00 mL,超声(300 W,40 kHz)处理30 min,放冷,以0.22 μm有机系滤膜滤过,精密量取续滤液5.00 mL,置10 mL棕色量瓶中,用50%甲醇溶液稀释至刻度。摇匀,用0.22 μm有机系滤膜滤过,取续滤液,即得。阳性样品定量根据需要可用50%甲醇溶液进一步稀释。
2.3 混合基质溶液的制备
取空白茶剂(Ⅰ)、胶囊(Ⅲ)和软胶囊(Ⅴ)的1次口服量,分别置于棕色具塞锥形瓶中,精密加入甲醇50.00 mL,超声(300 W,40 kHz)处理约30 min,放冷,以0.22 μm有机系滤膜滤过,取续滤液备用。将3种基质提取液1:1:1混合后,再与50%甲醇溶液1:1混合,以0.22 μm有机系滤膜滤过,取续滤液,即得。
2.4 标准曲线溶液的配制
精密量取混合对照品储备液适量,用“2.3”项下的混合基质溶液稀释成系列浓度标准曲线溶液。
2.5 色谱条件
色谱柱:Agilent Poroshell EC-C18(100 mm×2.1 mm,2.7 μm);流动相:0.1%甲酸溶液为A相,乙腈(含0.1%甲酸)为B相,线性梯度洗脱(0~15 min,10%B→95%B;15~16 min,95%B;16~16.5 min,95%B→10%B;16.5~20 min,10%B);流速:0.3 mL·min-1;柱温:40 ℃;进样体积:5 μL。
2.6 质谱条件
离子源:电喷雾离子源(ESI);扫描方式:正离子和负离子扫描;监测方式:多反应监测(MRM);毛细管电压:正离子4 000 V,负离子3 500 V;鞘气压力:241 kPa;离子源温度:350 ℃;其他质谱参数见表 1。
3 结果与讨论
3.1 质谱条件的优化
以甲醇为溶剂配制对照品单标溶液,用50%甲醇溶液稀释至5 μg·mL-1,选用流动注射方式,软件自动优化3组MRM模式离子对,再根据碰撞能量合理性和阴性样品专属性,手动选取2个离子对建立分析方法。
3.2 色谱条件的优化
由于三重四极杆质谱扫描驻留时间和灵敏度存在反比关系,UPLC虽然节省了分析时间,但是同时测定54个化合物、108对离子,质谱扫描时间受限,灵敏度下降;加之中成药和保健食品药品基质复杂,UPLC载样量小,系统容易堵塞,故没有选择极小粒径色谱柱,选择了载样量和分离度兼顾的色谱柱(粒径2.7 μm)。
以获得对称尖锐的峰形为目标,分别考察了以不同挥发性酸和缓冲盐作为水相的分离效果。结果发现由于六大降压类化学药品的酸碱性不同,没有1种流动相能满足所有物质的完全分离和峰形要求。其中,随着pH升高,含有羧基的普利类药物峰形变宽,唑嗪类出现裂分;但是随着pH降低,洛尔类色谱峰逐渐出现裂分,当pH < 3时,阿替洛尔、索他洛尔、阿米洛利等物质出现对称、尖锐的双峰;沙坦类和地平类的峰形对流动相pH变化不如以上几类敏感,但沙坦类更适合酸性强的流动相,尤其是含有羧基的缬沙坦和替米沙坦,而地平类偏中性时峰形更尖锐。同时对有机相进行优化,甲醇具有较好的离子化效率且较廉价,但是乙腈可以获得较低的柱压,且有利于氢氯噻嗪和氯噻嗪的分离。故选择乙腈为有机相。
在流动相优化后,最终选择流动相1[20 mmol·L-1乙酸铵溶液(含0.05%乙酸)-乙腈]和流动相2(0.1%甲酸溶液-0.1%甲酸乙腈溶液)2种流动相做进一步考察。
3.3 提取方法的选择和优化
本实验中54种化合物在甲醇中均具有一定溶解性,故选择甲醇作为提取溶剂;地平类化合物对光敏感,应避光操作;阿替洛尔、可乐定、阿米洛利等色谱保留较弱的化合物,纯甲醇进样有明显的溶剂效应,故以甲醇提取后增加50%甲醇溶液稀释步骤。
3.4 线性范围与检测下限、定量下限
将系列浓度含混合基质的标准曲线溶液在2种流动相、正/负离子模式下分别测定,以峰面积均值Y为纵坐标,对照品溶液浓度X(ng·mL-1)为横坐标,进行线性回归,得各组分的回归方程。用线性范围内最低浓度点的溶液进样20针,计算SD,用SD×3/标准曲线斜率和SD×10/标准曲线斜率分别计算各化合物的检测下限和定量下限。结果见表 2、表 3。
表 2
Table 2
表 2 流动相1条件下各化合物的线性范围、拟合方程、检测下限和定量下限
Table 2 The linear ranges, equations, LODs and LOQs of each compound under mobile phase 1
编号 (No.) |
保留时间 (retention time)/ min |
线性范围 (linear range)/ (ng·mL-1) |
r |
回归方程 (regression equation) |
LOD/ (ng·mL-1) |
LOQ/ (ng·mL-1) |
5-2 |
1.55 |
1.814~90.72 |
0.999 3 |
Y=7.495×106 X-3.109×103 |
0.3 |
1 |
4-1 |
1.67 |
9.622~962.2 |
0.998 6 |
Y=1.028×107 X+1.859×105 |
3 |
10 |
1-5 |
1.68 |
2.059~2 05 9 |
0.999 9 |
Y=9.897×105 X-5.965×102 |
0.7 |
2 |
5-4 |
1.72 |
2.449~122.5 |
0.998 5 |
Y=1.311×107 X+2.443×104 |
0.5 |
2 |
1-1 |
1.75 |
487.0~9 740 |
0.998 1 |
Y=5.496×105 X-1.854×105 |
94 |
313 |
4-6 |
2.31 |
10.21~102.1 |
0.999 9 |
Y=6.262×106 X-1.330×104 |
3 |
10 |
6-3 |
2.54 |
1.970~492.5 |
0.999 2 |
Y=1.993×106 X+4.283×103 |
0.6 |
2 |
5-5 |
2.85 |
2.267~113.4 |
0.999 8 |
Y=1.734×107 X+5.505×103 |
0.4 |
2 |
4-7 |
2.88 |
19.63~981.5 |
0.999 8 |
Y=2.154×105 X+1.324×103 |
7 |
23 |
1-4 |
3.47 |
1.969~984.5 |
0.999 6 |
Y=9.529×106 X-8.486×103 |
0.3 |
1 |
4-2 |
3.56 |
2.069~103.5 |
0.998 4 |
Y=1.079×107 X+3.673×104 |
0.3 |
1 |
6-2 |
3.77 |
2.142~107.1 |
0.998 1 |
Y=4.221×107 X+1.453×105 |
0.2 |
1 |
6-1 |
3.79 |
2.148~107.4 |
0.999 5 |
Y=3.854×107 X+9.952×104 |
0.3 |
1 |
6-11 |
4.03 |
4.821~1 928 |
0.999 3 |
Y=7.293×105 X-9.819×103 |
1 |
3 |
6-6 |
4.11 |
1.907~953.5 |
0.999 7 |
Y=5.800×106 X+5.652×104 |
0.7 |
2 |
5-3 |
4.35 |
2.132~106.6 |
0.998 6 |
Y=4.633×105 X+1.179×104 |
0.6 |
2 |
4-8 |
4.73 |
46.88~937.5 |
0.999 3 |
Y=5.446×104 X-2.590×103 |
14 |
47 |
6-5 |
4.78 |
1.893~473.2 |
0.999 8 |
Y=3.338×106 X-1.745×103 |
0.4 |
1 |
4-9 |
4.79 |
49.97~1 999 |
0.999 5 |
Y=4.453×105 X-1.042×104 |
7 |
23 |
5-6 |
4.82 |
2.083~104.2 |
0.999 2 |
Y=1.294×107 X+1.219×104 |
0.3 |
1 |
5-10 |
4.91 |
2.085~104.3 |
0.999 4 |
Y=3.903×107 X+8.559×104 |
0.2 |
1 |
1-3 |
5.30 |
1.925~192.5 |
0.997 6 |
Y=1.376×107 X+6.780×104 |
0.3 |
1 |
5-8 |
5.37 |
1.944~97.21 |
0.998 9 |
Y=4.590×106 X+1.048×104 |
0.5 |
2 |
6-4 |
5.41 |
2.039~101.9 |
0.999 2 |
Y=1.097×108 X+2.774×105 |
0.3 |
1 |
5-7 |
5.54 |
2.071~103.6 |
0.999 2 |
Y=2.371×107 X+4.038×104 |
0.2 |
1 |
1-2 |
5.79 |
2.011~100.6 |
0.999 8 |
Y=6.298×107 X+3.334×104 |
0.1 |
0.3 |
5-1 |
5.97 |
1.960~196.0 |
0.999 0 |
Y=4.569×106 X+2.536×104 |
0.5 |
2 |
3-3 |
6.33 |
2.057~514.3 |
0.999 5 |
Y=3.082×106 X+1.503×104 |
0.7 |
2 |
5-9 |
6.40 |
2.040~102.0 |
0.999 0 |
Y=7.060×107 X+2.026×105 |
0.4 |
1 |
3-1 |
6.45 |
2.149~107.5 |
0.997 8 |
Y=5.466×106 X+1.127×104 |
0.5 |
2 |
1-6 |
6.69 |
1.996~199.6 |
0.998 9 |
Y=2.024×107 X+4.156×104 |
0.7 |
2 |
6-9 |
6.84 |
2.947~147.4 |
0.999 8 |
Y=1.290×107 X+1.794×104 |
0.6 |
2 |
6-8 |
7.03 |
2.270~113.5 |
0.999 4 |
Y=6.318×107 X+2.032×105 |
0.6 |
2 |
1-6 |
7.15 |
2.088~104.4 |
0.996 0 |
Y=5.880×107 X+3.256×105 |
0.3 |
1 |
4-3 |
7.21 |
18.31~457.8 |
0.999 6 |
Y=1.967×106 X-1.706×103 |
4 |
13 |
3-2 |
7.31 |
1.967~19.67 |
0.999 3 |
Y=7.900×107 X+3.156×104 |
0.1 |
0.3 |
3-5 |
7.32 |
1.904~47.60 |
0.997 3 |
Y=7.668×106 X+2.095×103 |
0.7 |
2 |
2-5 |
7.39 |
2.096~104 8 |
0.999 9 |
Y=5.929×106 X-1.022×103 |
0.5 |
2 |
4-5 |
7.77 |
2.024~101.2 |
0.999 9 |
Y=9.774×107 X+7.655×104 |
0.2 |
1 |
4-10 |
7.98 |
4.796~95.91 |
0.999 7 |
Y=2.649×106 X+3.495×103 |
1 |
4 |
2-1 |
8.72 |
1.976~98.80 |
0.999 7 |
Y=7.644×106 X+7.319×102 |
0.4 |
2 |
3-4 |
8.88 |
1.963~98.15 |
0.999 3 |
Y=8.178×107 X+1.697×105 |
0.1 |
0.3 |
4-4 |
9.51 |
1 998~19 975 |
0.999 3 |
Y=3.939×103 X-2.808×103 |
244 |
813 |
6-10 |
9.62 |
1.975~987.5 |
0.999 6 |
Y=6.004×106 X+3.984×104 |
0.3 |
1 |
1-8 |
10.11 |
10.09~1 009 |
0.997 2 |
Y=2.244×106 X+8.056×104 |
3 |
10 |
2-2 |
10.20 |
49.35~1 974 |
0.999 8 |
Y=9.533×104 X-3.014×103 |
14 |
47 |
2-6 |
10.58 |
2.001~201.1 |
0.999 1 |
Y=1.083×107 X+1.003×104 |
0.5 |
2 |
2-8 |
10.60 |
2.030~101.5 |
0.999 6 |
Y=9.356×107 X+1.681×104 |
0.2 |
1 |
3-6 |
10.75 |
2.121~106.1 |
0.999 5 |
Y=6.911×107 X+1.398×105 |
0.6 |
2 |
2-4 |
11.11 |
2.162~108.1 |
0.999 9 |
Y=8.586×106 X-6.392×103 |
0.3 |
1 |
2-3 |
11.50 |
10.30~1 030 |
0.998 9 |
Y=7.406×105 X+5.434×103 |
3 |
10 |
2-9 |
12.31 |
19.93~996.5 |
0.999 3 |
Y=1.026×106 X+9.242×103 |
2 |
7 |
6-7 |
13.08 |
1.993~99.65 |
0.999 5 |
Y=2.211×108 X+5.213×105 |
0.4 |
1 |
2-7 |
13.17 |
4.985~99.70 |
0.999 1 |
Y=2.916×106 X-2.575×102 |
0.6 |
2 |
|
表 2 流动相1条件下各化合物的线性范围、拟合方程、检测下限和定量下限
Table 2 The linear ranges, equations, LODs and LOQs of each compound under mobile phase 1 |
表 3
Table 3
表 3 流动相2条件下各化合物的线性范围、拟合方程、检测下限和定量下限
Table 3 The linear range, equation, LOD and LOQ of each compound under mobile phase 2
编号 (No.) |
保留时间 (retention time)/min |
线性范围 (linear range)/ (ng·mL-1) |
r |
回归方程 (regression equation) |
LOD/ (ng·mL-1) |
LOQ/ (ng·mL-1) |
6-2 |
0.80,1.13 |
2.142~107.1 |
0.999 6 |
Y=6.792×107 X+1.714×105 |
0.2 |
0.7 |
5-2 |
0.81,1.36 |
1.814~453.6 |
0.998 8 |
Y=4.692×106 X+2.742×104 |
1 |
3 |
5-4 |
0.82,1.49 |
2.449~122.5 |
0.998 7 |
Y=1.804×107 X+6.827×104 |
0.4 |
1 |
4-1 |
1.45 |
1.924~481.1 |
0.998 2 |
Y=1.065×107 X+1.010×105 |
1 |
2 |
6-3 |
2.18 |
1.970~985.0 |
0.999 8 |
Y=1.654×106 X+2.248×103 |
0.5 |
2 |
4-6 |
2.28 |
5.105~102 1 |
0.998 4 |
Y=1.161×106 X+1.170×104 |
2 |
5 |
1-5 |
2.47 |
2.059~205 9 |
0.998 7 |
Y=4.023×106 X+4.721×104 |
0.8 |
3 |
5-5 |
2.55 |
2.267~113 4 |
0.999 9 |
Y=1.624×107 X+3.202×103 |
0.5 |
2 |
4-7 |
2.57 |
49.08~196 3 |
0.999 6 |
Y=2.433×105 X-4.701×103 |
12 |
40 |
4-2 |
3.06 |
2.069~103.5 |
0.999 5 |
Y=1.500×107 X+6.413×104 |
0.2 |
1 |
6-1 |
3.27 |
2.148~107.4 |
0.999 6 |
Y=6.185×107 X+1.728×105 |
0.2 |
1 |
6-6 |
3.76 |
1.907~476.7 |
0.998 1 |
Y=2.532×107 X+2.739×105 |
0.5 |
2 |
1-1 |
3.77 |
48.70~9 740 |
0.999 4 |
Y=2.359×106 X-9.574×105 |
14 |
47 |
6-11 |
3.98 |
3.857~964.1 |
0.999 8 |
Y=6.538×106 X+1.382×103 |
1 |
3 |
5-3 |
4.03 |
2.132~106.6 |
0.999 8 |
Y=6.769×106 X+1.077×104 |
0.4 |
1 |
6-5 |
4.31 |
1.893~946.4 |
0.999 1 |
Y=1.100×107 X+1.734×105 |
0.4 |
1 |
5-6 |
4.50 |
2.083~104.2 |
0.999 9 |
Y=1.761×107 X+1.445×104 |
0.3 |
1 |
1-4 |
4.62 |
1.969~984.5 |
0.999 9 |
Y=3.925×107 X+2.498×105 |
0.5 |
2 |
5-10 |
4.64 |
2.085~104.3 |
0.999 7 |
Y=5.141×107 X+1.017×105 |
0.2 |
1 |
4-8 |
4.70 |
46.88~1 875 |
0.998 9 |
Y=1.028×105 X+2.959×103 |
16 |
53 |
5-8 |
5.05 |
1.944~972.1 |
0.999 9 |
Y=1.287×107 X+3.272×104 |
0.6 |
2 |
6-4 |
5.06 |
2.039~101.9 |
0.999 8 |
Y=1.416×108 X+3.203×105 |
0.1 |
0.3 |
5-7 |
5.19 |
2.071~103.6 |
0.998 9 |
Y=3.288×107 X+5.382×104 |
0.3 |
1 |
1-3 |
5.52 |
1.925~96.26 |
0.999 5 |
Y=2.894×107 X+4.268×104 |
0.3 |
1 |
5-1 |
5.57 |
1.960~980.0 |
0.998 1 |
Y=5.130×106 X+1.177×105 |
0.5 |
2 |
1-2 |
5.75 |
2.011~100.6 |
0.999 6 |
Y=9.226×107 X+5.769×104 |
0.1 |
0.3 |
5-9 |
5.80 |
2.040~510.0 |
0.998 3 |
Y=6.886×107 X+9.336×105 |
0.4 |
1 |
6-9 |
5.85 |
2.947~147.4 |
0.999 6 |
Y=1.850×107 X+1.202×105 |
0.5 |
2 |
6-8 |
6.38 |
2.270~567.5 |
0.999 0 |
Y=1.231×108 X+9.692×105 |
0.2 |
1 |
4-9 |
6.44 |
4.997~999.5 |
0.999 5 |
Y=2.484×106 X-1.809×103 |
1 |
3 |
1-6 |
6.54 |
1.996~998.0 |
0.999 4 |
Y =7.187×107 X+1.237×105 |
0.3 |
1 |
2-5 |
7.02 |
2.096~104 8 |
0.999 7 |
Y=1.766×107 X-2.684×104 |
0.5 |
2 |
2-8 |
7.07 |
2.030~507.5 |
0.997 5 |
Y=1.040×108 X+1.340×106 |
0.1 |
0.3 |
4-3 |
7.15 |
1.831~915.6 |
0.999 8 |
Y=1.040×107 X+3.050×104 |
0.9 |
3 |
3-5 |
7.20 |
4.760~190.4 |
0.998 2 |
Y=1.342×107 X+4.520×104 |
1 |
3 |
4-5 |
7.22 |
2.024~506.0 |
0.998 4 |
Y=8.564×107 X+9.066×105 |
0.3 |
1 |
1-6 |
7.31 |
2.088~104.4 |
0.997 2 |
Y=8.721×107 X+3.720×105 |
0.3 |
1 |
3-4 |
7.35 |
1.963~98.2 |
0.997 4 |
Y=5.933×107 X+2.301×105 |
0.5 |
2 |
3-2 |
7.40 |
1.967~98.4 |
0.997 5 |
Y=1.246×108 X+ 5.048×105 |
0.1 |
0.3 |
3-1 |
7.55 |
2.149~107 5 |
0.999 7 |
Y=2.077×107 X+1.798×105 |
0.4 |
1 |
6-10 |
7.67 |
1.975~987.5 |
0.999 8 |
Y=1.318×107 X+3.610×104 |
0.3 |
1 |
4-10 |
7.98 |
4.796~1 918 |
0.999 7 |
Y=1.485×106 X+2.163×103 |
1 |
3 |
2-1 |
8.65 |
1.976~494.0 |
0.999 6 |
Y=1.784×107 X+8.433×104 |
0.4 |
1 |
3-3 |
9.15 |
5.143~1 029 |
0.999 9 |
Y=1.715×107 X+5.136×104 |
0.7 |
2 |
6-7 |
9.25 |
1.993~498.3 |
0.998 7 |
Y=1.603×108 X+2.126×106 |
0.3 |
1 |
4-4 |
9.45 |
19.98~998.8 |
0.999 1 |
Y=1.924×106 X-2.379×104 |
3 |
10 |
2-2 |
10.15 |
4.935~987.0 |
0.998 6 |
Y=3.157×106 X-3.633×104 |
2 |
7 |
2-6 |
10.33 |
2.001~1 006 |
0.999 2 |
Y=2.343×107 X +2.280×104 |
0.5 |
2 |
2-4 |
11.02 |
2.162~540.5 |
0.998 6 |
Y=1.467×107 X+1.374×105 |
0.5 |
2 |
2-3 |
11.44 |
2.059~1 030 |
0.999 9 |
Y=6.169×106 X-5.614×103 |
0.8 |
3 |
2-9 |
12.25 |
4.983~996.5 |
0.999 8 |
Y=5.656×106 X+2.378×103 |
1 |
3 |
1-8 |
12.79 |
5.047~1 009 |
0.999 9 |
Y=3.491×106 X+1.912×104 |
2 |
7 |
3-6 |
12.90 |
5.303~1 061 |
0.999 2 |
Y=6.100×107 X+1.304×106 |
0.6 |
2 |
2-7 |
13.08 |
4.985~997.0 |
0.999 0 |
Y=4.484×106 X+6.555×104 |
0.7 |
2 |
|
表 3 流动相2条件下各化合物的线性范围、拟合方程、检测下限和定量下限
Table 3 The linear range, equation, LOD and LOQ of each compound under mobile phase 2 |
结果显示:虽然流动相2中各化合物出峰接近,分离度差,且阿替洛尔、卡替洛尔、阿米洛利均裂分为双峰,但是线性考察结果发现:不管是正离子监测还是负离子监测,流动相2的标准曲线斜率更大,线性范围更宽,灵敏度显著提升,尤其是螺内酯、卡托普利和尼群地平。故选择流动相2作为最终流动相,其典型色谱图如图 1所示。
3.5 专属性、精密度和方法回收率考察
回收率加标溶液的配制:精密量取混合对照品储备液1.00、5.00和25.00 mL,分别置50 mL棕色容量瓶中,以甲醇定容至刻度,混匀,即得低、中和高3个浓度加标溶液。
选取5种剂型降压类供试品:茶剂(Ⅰ)、丸剂(Ⅱ)、胶囊(Ⅲ)、片剂(Ⅳ,中西药复方制剂,标识含有氢氯噻嗪和可乐定)和软胶囊(Ⅴ)。分别取15次服用量混匀(片剂、茶剂、丸剂研细;软胶囊挤出内容物、硬胶囊取内容物研细),分别称取一次口服剂量(分别为3.0、6.0、1.2、0.25和1.6 g)各10份,置棕色具塞锥形瓶中,1份加入甲醇1.00 mL作为专属性实验,3份加入1.00 mL低浓度加标溶液,3份加入1.00 mL中浓度加标溶液,3份加入1.00 mL高浓度加标溶液,再加入提取溶剂甲醇50.00 mL,超声处理30 min,放冷,以0.22 μm有机系滤膜滤过,取续滤液备用;同时平行处理低、中、高浓度对照品溶液各一份。分别精取上述续滤液各5.00 mL,置10 mL棕色量瓶中,用50%甲醇溶液稀释至刻度,摇匀,用0.22 μm有机系滤膜滤过,取续滤液测定,以加标后样品中各化合物峰面积均值(n=3)与对照品溶液中峰面积均值(n=6)之比计算方法回收率(%),对照品溶液重复进样6针,计算精密度RSD(%)。
专属性实验结果:均未检出标识以外的非法添加的降压化学药品;方法回收率实验结果见表 4。
表 4
Table 4
表 4 回收率测定结果
Table 4 Results of recovery tests
编号 (No.) |
回收率(recoveries)/% |
|
RSD/% |
L-Ⅰ |
L-Ⅱ |
L-Ⅲ |
L-Ⅳ |
L-Ⅴ |
均值(mean) |
M-1 |
M-Ⅱ |
M-Ⅲ |
M-Ⅳ |
M-Ⅴ |
均值(mean) |
H-Ⅰ |
H-Ⅱ |
H-Ⅲ |
H-Ⅳ |
H-Ⅴ |
均值(mean) |
L |
M |
H |
6-2 |
139.6 |
138.2 |
100.5 |
112.4 |
124.0 |
122.9 |
110.1 |
109.8 |
99.1 |
95.7 |
112.2 |
105.4 |
82.8 |
83.7 |
95.7 |
81.9 |
110.3 |
90.9 |
|
9.5 |
3.4 |
6.3 |
5-2 |
105.7 |
135.9 |
115.8 |
99.5 |
114.7 |
114.3 |
98.9 |
117.2 |
105.5 |
99.2 |
107.5 |
105.7 |
93.5 |
109.7 |
93.1 |
99.1 |
101.2 |
99.3 |
6.1 |
4.3 |
9.9 |
5-4 |
50.6 |
127.4 |
110.4 |
139.0 |
168.9 |
119.3 |
55.4 |
120.3 |
110.2 |
102.1 |
123.8 |
102.4 |
51.2 |
120.9 |
103.6 |
115.3 |
124.2 |
103.0 |
5.1 |
5.5 |
3.0 |
4-1 |
120.7 |
148.9 |
110.2 |
109.9 |
118.3 |
121.6 |
104.8 |
121.4 |
102.3 |
104.0 |
111.7 |
108.8 |
102.1 |
130.2 |
93.1 |
106.7 |
106.3 |
107.7 |
5.1 |
5.9 |
4.2 |
6-3 |
118.5 |
145.3 |
101.1 |
/* |
104.1 |
117.3 |
109.9 |
120.6 |
97.7 |
/* |
104.5 |
108.2 |
103.2 |
115.9 |
93.6 |
/* |
108.3 |
105.3 |
16.3 |
6.2 |
1.9 |
4-6 |
148.8 |
129.7 |
76.1 |
127.0 |
103.1 |
116.9 |
131.1 |
118.7 |
92.4 |
116.6 |
103.3 |
112.4 |
121.3 |
119.2 |
89.5 |
106.2 |
106.1 |
108.5 |
1.1 |
4.3 |
1.1 |
1-5 |
52.9 |
49.2 |
43.7 |
47.1 |
58.7 |
50.3 |
55.6 |
57.1 |
44.1 |
66.9 |
70.2 |
58.8 |
52.3 |
49.6 |
28.2 |
76.3 |
83.1 |
57.9 |
2.2 |
3.8 |
3.6 |
5-5 |
123.1 |
149.6 |
112.4 |
91.7 |
102.2 |
115.8 |
113.9 |
145.2 |
102.5 |
96.9 |
101.8 |
112.1 |
105.5 |
164.9 |
96.5 |
97.0 |
110.4 |
114.9 |
3.2 |
2.3 |
2.6 |
4-7 |
98.8 |
69.5 |
79.1 |
/* |
119.4 |
91.7 |
102.4 |
86.7 |
90.2 |
/* |
104.2 |
95.9 |
105.1 |
92.5 |
95.1 |
/* |
102.7 |
98.9 |
5.1 |
6.0 |
5.4 |
4-2 |
84.0 |
97.7 |
91.3 |
83.6 |
89.9 |
89.3 |
95.1 |
103.2 |
96.2 |
95.5 |
98.9 |
97.8 |
101.8 |
108.3 |
99.9 |
105.3 |
98.8 |
102.8 |
7.0 |
2.5 |
2.8 |
6-1 |
87.5 |
118.8 |
98.8 |
96.6 |
96.0 |
99.5 |
89.7 |
102.9 |
99.3 |
101.6 |
96.8 |
98.1 |
86.5 |
92.9 |
98.2 |
103.3 |
101.7 |
96.5 |
5.7 |
3.5 |
3.8 |
6-6 |
97.4 |
133.1 |
99.5 |
106.4 |
97.2 |
106.7 |
98.3 |
113.8 |
97.2 |
111.4 |
98.6 |
103.9 |
98.5 |
126.2 |
90.8 |
126.9 |
93.6 |
107.2 |
5.1 |
4.1 |
3.6 |
1-1 |
34.8 |
40.8 |
75.1 |
58.2 |
41.6 |
50.1 |
34.7 |
27.3 |
89.4 |
69.0 |
58.5 |
55.8 |
32.5 |
6.1 |
93.5 |
76.8 |
74.1 |
56.6 |
2.2 |
1.8 |
8.5 |
6-11 |
99.1 |
70.4 |
98.5 |
95.6 |
109.4 |
94.6 |
95.2 |
65.4 |
99.3 |
96.3 |
103.2 |
91.9 |
86.5 |
61.9 |
96.3 |
96.2 |
105.3 |
89.2 |
2.1 |
0.5 |
0.03 |
5-3 |
97.6 |
116.3 |
101.5 |
108.5 |
95.7 |
103.9 |
98.4 |
103.4 |
98.9 |
104.8 |
100.3 |
101.2 |
98.1 |
91.7 |
94.7 |
107.8 |
105.1 |
99.5 |
5.3 |
2.5 |
0.9 |
6-5 |
68.4 |
69.9 |
90.2 |
78.7 |
83.8 |
78.2 |
73.1 |
66.9 |
94.8 |
88.2 |
93.5 |
83.3 |
70.4 |
66.7 |
95.1 |
86.1 |
93.2 |
82.3 |
7.2 |
3.1 |
6.0 |
5-6 |
102.4 |
94.7 |
100.1 |
101.6 |
71.5 |
94.1 |
99.9 |
95.3 |
99.6 |
103.1 |
88.7 |
97.3 |
95.4 |
83.3 |
95.8 |
98.0 |
97.2 |
93.9 |
7.9 |
1.3 |
4.9 |
1-4 |
110.7 |
93.6 |
38.1 |
109.2 |
116.9 |
93.7 |
92.7 |
88.9 |
66.2 |
103.4 |
109.2 |
92.1 |
76.5 |
72.3 |
66.4 |
96.3 |
111.2 |
84.5 |
4.1 |
3.2 |
2.3 |
5-10 |
112.3 |
112.8 |
105.6 |
106.6 |
89.7 |
105.4 |
102.8 |
99.4 |
102.1 |
101.9 |
94.2 |
100.1 |
90.7 |
81.0 |
93.1 |
96.0 |
96.3 |
91.4 |
6.9 |
2.5 |
3.5 |
4-8 |
96.4 |
86.6 |
77.5 |
100.2 |
120.3 |
96.2 |
93.6 |
94.1 |
96.5 |
101.9 |
109.2 |
99.1 |
97.8 |
96.5 |
95.3 |
105.1 |
110.3 |
101.0 |
8.9 |
3.9 |
7.9 |
5-8 |
148.6 |
121.8 |
111.7 |
121.9 |
118.4 |
124.5 |
110.7 |
111.1 |
105.3 |
115.8 |
112.1 |
111.0 |
105.9 |
101.3 |
98.3 |
113.8 |
118.1 |
107.5 |
6.3 |
1.9 |
0.9 |
6-4 |
108.3 |
104.1 |
113.4 |
112.0 |
93.8 |
106.3 |
98.3 |
99.1 |
101.7 |
101.2 |
95.6 |
99.2 |
87.4 |
77.0 |
92.4 |
97.6 |
93.8 |
89.6 |
2.6 |
2.7 |
0.6 |
5-7 |
119.7 |
110.4 |
108.7 |
112.5 |
100.2 |
110.3 |
104.4 |
100.8 |
101.7 |
103.0 |
106.2 |
103.2 |
94.8 |
90.0 |
96.0 |
100.1 |
101.5 |
96.5 |
5.9 |
1.7 |
0.6 |
1-3 |
117.5 |
121.3 |
96.5 |
114.4 |
100.9 |
110.1 |
101.9 |
102.3 |
98.1 |
99.6 |
102.3 |
100.8 |
94.2 |
92.0 |
90.3 |
99.9 |
103.9 |
96.1 |
4.4 |
3.9 |
2.3 |
5-1 |
129.7 |
117.2 |
119.4 |
114.1 |
120.4 |
120.2 |
102.6 |
106.4 |
105.9 |
100.8 |
104.4 |
104.0 |
97.1 |
97.4 |
95.7 |
99.8 |
110.8 |
100.2 |
2.2 |
5.1 |
3.0 |
1-2 |
113.0 |
130.2 |
93.6 |
108.3 |
103.8 |
109.8 |
102.5 |
101.1 |
95.3 |
104.1 |
102.3 |
101.1 |
89.2 |
88.4 |
92.3 |
96.8 |
103.6 |
94.1 |
2.0 |
3.2 |
4.8 |
5-9 |
122.6 |
128.0 |
111.3 |
122.2 |
111.5 |
119.1 |
107.2 |
108.8 |
104.1 |
107.1 |
107.4 |
106.9 |
98.1 |
92.9 |
95.7 |
104.0 |
109.0 |
99.9 |
2.0 |
2.9 |
2.2 |
6-9 |
98.5 |
77.2 |
95.7 |
82.3 |
68.7 |
84.5 |
95.5 |
77.3 |
95.3 |
89.0 |
79.1 |
87.2 |
85.9 |
73.7 |
91.5 |
90.7 |
82.8 |
84.9 |
5.0 |
3.6 |
1.0 |
6-8 |
99.4 |
101.2 |
92.5 |
96.1 |
102.5 |
98.3 |
97.0 |
99.8 |
94.9 |
98.2 |
99.8 |
97.9 |
96.5 |
98.2 |
94.7 |
100.3 |
105.1 |
99.0 |
3.1 |
2.7 |
7.3 |
4-9 |
65.6 |
89.0 |
104.0 |
94.1 |
97.3 |
90.0 |
88.1 |
95.6 |
102.3 |
101.2 |
103.1 |
98.1 |
90.1 |
99.5 |
110.1 |
105.8 |
101.2 |
101.3 |
2.2 |
2.4 |
4.3 |
1-6 |
111.7 |
108.3 |
102.3 |
100.2 |
104.1 |
105.3 |
97.1 |
91.6 |
99.8 |
95.5 |
94.9 |
95.8 |
93.1 |
89.2 |
95.7 |
101.6 |
88.4 |
93.6 |
4.4 |
2.9 |
3.8 |
2-5 |
139.4 |
133.0 |
112.1 |
106.8 |
119.3 |
122.1 |
112.1 |
114.2 |
102.1 |
105.9 |
109.1 |
108.7 |
90.0 |
101.1 |
94.6 |
103.2 |
113.6 |
100.5 |
3.9 |
3.8 |
4.5 |
2-8 |
88.7 |
96.6 |
103.3 |
93.5 |
95.8 |
95.6 |
88.9 |
90.6 |
100.2 |
97.3 |
99.1 |
95.2 |
85.0 |
87.5 |
93.6 |
94.5 |
98.1 |
91.7 |
8.8 |
6.6 |
2.3 |
4-3 |
97.2 |
105.0 |
105.0 |
99.6 |
96.8 |
100.7 |
92.3 |
93.8 |
102.8 |
97.9 |
101.2 |
97.6 |
84.3 |
86.7 |
92.7 |
97.7 |
100.8 |
92.4 |
3.5 |
1.6 |
0.2 |
3-5 |
635.4 |
727 |
120.5 |
119.1 |
145.1 |
349.4 |
134.0 |
155.3 |
98.1 |
104.2 |
112.2 |
120.8 |
122.9 |
150.4 |
83.4 |
86.5 |
89.8 |
106.6 |
18.6 |
3.4 |
2.6 |
4-5 |
102.4 |
107.4 |
103.6 |
104.6 |
104.8 |
104.6 |
93.8 |
100.2 |
102.8 |
103.2 |
101.9 |
100.4 |
89.4 |
89.8 |
92.9 |
98.5 |
102.2 |
94.6 |
4.9 |
3.9 |
3.7 |
1-6 |
134.1 |
128.5 |
100.6 |
106.6 |
101.4 |
114.2 |
113.1 |
107.7 |
96.5 |
102.3 |
104.6 |
104.8 |
90.6 |
87.6 |
90.5 |
97.2 |
103.5 |
93.9 |
2.7 |
3.8 |
0.9 |
3-4 |
99.5 |
95.1 |
104.8 |
97.2 |
100.2 |
99.4 |
99.0 |
99.8 |
98.7 |
97.2 |
101.8 |
99.3 |
97.2 |
92.6 |
94.4 |
102.1 |
104.2 |
98.1 |
6.5 |
2.5 |
3.9 |
3-2 |
95.7 |
99.0 |
109.2 |
95.8 |
97.8 |
99.5 |
90.0 |
93.3 |
101.2 |
97.5 |
100.9 |
96.6 |
87.3 |
86.5 |
95.1 |
96.6 |
104.8 |
94.1 |
6.2 |
3.8 |
2.9 |
3-1 |
55.4 |
78.8 |
112.5 |
101.1 |
99.7 |
89.5 |
66.8 |
87.3 |
102.6 |
100.7 |
100.6 |
91.6 |
68.2 |
86.7 |
94.3 |
99.3 |
108.3 |
91.4 |
4.4 |
3.4 |
1.4 |
6-10 |
95.8 |
105.3 |
99.4 |
99.9 |
99.3 |
99.9 |
87.6 |
98.1 |
99.8 |
97.8 |
96.9 |
96.0 |
75.3 |
86.9 |
96.8 |
101.0 |
102.0 |
92.4 |
4.4 |
3.5 |
8.9 |
4-10 |
159.2 |
98.8 |
84.2 |
96.3 |
98.9 |
107.5 |
120.9 |
98.9 |
92.5 |
96.8 |
100.4 |
101.9 |
119.2 |
97.2 |
94.3 |
102.3 |
100.3 |
102.7 |
2.0 |
3.4 |
5.1 |
2-1 |
102.8 |
115.8 |
105.4 |
107.0 |
96.1 |
105.4 |
98.2 |
102.4 |
100.8 |
100.5 |
100.2 |
100.4 |
90.5 |
90.9 |
95.7 |
98.9 |
102.4 |
95.7 |
2.3 |
2.6 |
5.5 |
3-3 |
119 |
120.3 |
11.5 |
60.5 |
30.8 |
68.4 |
102.1 |
102.3 |
45.3 |
88.1 |
77.2 |
83.0 |
94.6 |
96.4 |
47.1 |
100.7 |
22.9 |
72.3 |
9.2 |
3.5 |
1.4 |
6-7 |
107.8 |
101.1 |
103.6 |
99.3 |
103.1 |
103.0 |
100.6 |
100.2 |
99.2 |
98.2 |
103.7 |
100.4 |
94.7 |
89.9 |
95.5 |
99.9 |
105.6 |
97.1 |
5.4 |
3.5 |
2.4 |
4-4 |
111.6 |
112.2 |
110.0 |
103.3 |
98.0 |
107.0 |
100.3 |
99.2 |
102.7 |
1 021 |
102.3 |
101.1 |
89.5 |
84.5 |
93.0 |
98.4 |
104.4 |
94.0 |
3.9 |
3.4 |
3.7 |
2-2 |
124.5 |
137.6 |
115.1 |
110.9 |
103.1 |
118.2 |
112.6 |
115.2 |
106.2 |
102.8 |
103.4 |
108.0 |
96.0 |
100.4 |
95.1 |
104.0 |
103.5 |
99.8 |
15.1 |
2.6 |
1.8 |
2-6 |
109.8 |
105.4 |
102.9 |
103.7 |
102.4 |
104.8 |
99.8 |
102.6 |
104.7 |
102.1 |
100.1 |
101.9 |
99.2 |
97.6 |
99.5 |
96.3 |
98.2 |
98.2 |
7.2 |
4.3 |
2.6 |
2-4 |
110.5 |
120.5 |
104.9 |
90.8 |
101.2 |
105.6 |
100.6 |
102.5 |
102.4 |
97.6 |
103.1 |
101.2 |
91.6 |
92.9 |
93.6 |
100.9 |
102.3 |
96.3 |
6.2 |
2.8 |
3.6 |
2-3 |
103.7 |
109.5 |
102.1 |
96.7 |
96.3 |
101.7 |
96.4 |
98.4 |
99.3 |
98.8 |
99.3 |
98.4 |
87.0 |
85.4 |
94.5 |
95.5 |
104.8 |
93.4 |
2.1 |
3.5 |
1.8 |
2-9 |
112.6 |
114.8 |
112.9 |
104.2 |
104.3 |
109.8 |
103.6 |
100.4 |
104.8 |
105.1 |
104.3 |
103.6 |
84.6 |
82.5 |
90.2 |
100.4 |
99.3 |
91.4 |
3.7 |
3.1 |
5.5 |
1-8 |
100.5 |
88.7 |
10.1 |
76.7 |
59.0 |
67.0 |
92.3 |
88.1 |
33.2 |
87.3 |
91.9 |
78.6 |
82.2 |
63.1 |
41.5 |
88.5 |
103.4 |
75.7 |
6.8 |
2.8 |
2.2 |
3-6 |
112.9 |
117.9 |
121.7 |
113.7 |
102.0 |
113.6 |
97.2 |
99.8 |
105.4 |
105.6 |
103.6 |
102.3 |
94.5 |
92.0 |
96.5 |
103.0 |
106.0 |
98.4 |
9.0 |
2.7 |
3.2 |
2-7 |
111 |
120.6 |
108.9 |
109.6 |
98.6 |
109.7 |
98.3 |
95.6 |
100.5 |
104.7 |
99.4 |
99.7 |
94.6 |
95.2 |
98.0 |
99.8 |
99.4 |
97.4 |
5.7 |
3.9 |
4.3 |
注(note):*标签标识含有的降压药物(drugs on the label);L、M、H:低、中、高浓度水平(low,medium,high concentration levels) |
|
表 4 回收率测定结果
Table 4 Results of recovery tests |
由表中可以看出,赖诺普利(1-5)、卡托普利(1-1)、哌唑嗪(6-5)、多沙唑嗪(6-9)、氯沙坦(3-1)缬沙坦(3-3)和福辛普利(1-8)的方法回收率较低,可能与其结构相关;不同剂型回收率也有较大差异,其中片剂的回收率最接近均值,可能与其辅料相对简单、一次口服剂量最小(辅料少)有关;低浓度的回收率普遍比高浓度高,可能与基质效应有关;奥美沙坦酯不同浓度回收率计算结果不一致,可能存在较大基质效应,有待进一步优化方法。实际样品中的非法添加物含量常超出本法线性范围(见表 5),测定时样品需要进一步稀释,稀释后基质效应会显著降低,方法回收率将有所增加。
表 5
Table 5
表 5 12批样品测定结果
Table 5 Quantitative results of 12 samples
样品 (sample) |
剂型 (dosage form) |
检出的非法添加物质,峰号 (illegally added substances detected,peak number) |
含量(content)/ (mg·kg-1) |
Ⅰ |
茶剂(tea) |
— |
— |
Ⅱ |
丸剂(pills) |
— |
— |
Ⅲ |
胶囊(capsules) |
— |
— |
Ⅳ |
片剂(tablets) |
— |
— |
Ⅴ |
软胶囊(soft capsules) |
— |
— |
Ⅵ |
茶剂(tea) |
— |
— |
Ⅶ |
茶剂(tea) |
— |
— |
Ⅷ |
胶囊(capsules) |
— |
— |
Ⅸ |
胶囊(capsules) |
氨苯蝶啶(triamterene,A) |
6 219.5 |
氨氯地平(amlodipine,B) |
5 365.9 |
氢氯噻嗪(hydrochlorothiazide,C) |
35 975.6 |
Ⅹ |
胶囊(capsules) |
阿替洛尔(atenolol,D) |
8 032.8 |
卡托普利(captopril,E) |
1 311.5 |
硝苯地平(nifedipine,F) |
8 524.6 |
氢氯噻嗪(hydrochlorothiazide,G) |
17 049.2 |
Ⅺ |
软胶囊(soft capsules) |
普萘洛尔(propranolol,H) |
2 307.7 |
氨氯地平(amlodipine,I) |
2 769.2 |
尼群地平(nitrendipine,J) |
374.4 |
氢氯噻嗪(hydrochlorothiazide,K) |
5 384.6 |
Ⅻ |
软胶囊(soft capsules) |
氨氯地平(amlodipine,L) |
1 682.5 |
比索洛尔(bisoprolol,M) |
516.6 |
|
表 5 12批样品测定结果
Table 5 Quantitative results of 12 samples |
3.6 实际样品的测定
利用本方法分别对市售12批(10个品种)降压类保健食品和中成药进行检测,其中3批茶剂(2个品种)、1批丸剂、4批硬胶囊、1批片剂、3批软胶囊(2个品种)。结果见表 5,阳性样品典型图谱见图 2。其中Ⅺ和Ⅻ为同一品种2个批次的软胶囊,2批次内容物性状即可见不同,Ⅺ测出氢氯噻嗪、普萘洛尔、氨氯地平和尼群地平,Ⅻ(批号新)测出比索洛尔和氨氯地平,可见不法商贩非法添加的随意性。
4 结论与展望
降压类保健食品和中成药添加化学药物无疑会对本身需要长期服药的高血压患者带来严重风险,本文建立的LC-MS/MS方法可以筛查常见的54种降压类化学药品,经过优化流动相,提高了方法灵敏度和专属性。随着时代的发展,虽然非法添加物质种类会不断增加,但随着色谱-质谱联用仪的推广使用,建立并更新化学药品的标准质谱库,完善检验检测的标准体系,并在监管上加大抽样范围和处罚力度,将令非法添加无所遁形,不给不法商贩以可乘之机。