方法:选用通用性较好的新型紧压式三相混合填料的固相萃取柱(SPEC Multi-Modal),采用酸性和碱性两步洗脱,建立同时提取血浆中几十种具不同理化性质的酸性、中性和碱性药物的系统化固相萃取方法。GC-MS系统分析方法将色谱系统的保留性质和质谱系统的特征离子、谱库检索相结合实行多指标定性。利用所选的每类药物及其代谢物代表碎片离子的质量色谱,对几类药物及其代谢物进行筛选。结果:测定了21种常见药物毒物在血浆中的绝对回收率,回收率均在78%以上,相对标准偏差小于7%(n=5)。所建系统分析方法对急性中毒病人进行了定性定量分析,取得满意的结果,使方法在实际应用中得到了验证。结论:此方法系统性强、选择性好、灵敏度高,结果可靠。
No. | Compound | RT/min | RRT | MW | Characteristic ion |
1 |
Amphetamine |
3.03±0.008 |
0.286±0.0008 |
135 | 91,65,63,120,77 |
2 | Methamphetamine | 3.48±0.012 | 0.328±0.0011 | 149 | 58,65,91,77,117 |
3 | Nicotine | 4.96±0.012 | 0.468±0.0011 | 162 | 84,133,162 |
4 | MDA | 6.12±0.030 | 0.578±0.0028 | 179 | 136,135,77,105,179 |
5 | Barbital | 6.38±0.012 | 0.602±0.0012 | 184 | 141,156,98,112,83 |
6 | MDMA | 6.61±0.028 | 0.624±0.0026 | 193 | 58,135,193,177 |
7 | Aprobarbital | 7.42±0.018 | 0.700±0.0017 | 210 | 167,124,97,168,195 |
8 | Meprylcaine | 7.72±0.000 | 0.728±0.0000 | 235 | 100,105,58,70,84 |
9 | Amobarbital | 8.15±0.008 | 0.769±0.0008 | 226 | 156,141,55,167,142 |
10 | Pentobarbital | 8.34±0.011 | 0.787±0.0010 | 226 | 141,156,98,157,112 |
11 | Pethidine | 8.40±0.000 | 0.793±0.0000 | 247 | 71,103,172,247,218 |
12 | Isobutyl aminobenzoate | 8.57±0.005 | 0.809±0.0005 | 193 | 120,137,92,65,193 |
13 | Secobarbital | 8.73±0.005 | 0.823±0.0004 | 238 | 168,167,141,97,156 |
14 | Caffeine | 9.03±0.008 | 0.852±0.0008 | 194,109,67,82,55 | |
15 | Prilocaine | 9.08±0.000 | 0.857±0.0000 | 220 | 86,107,106,134,220 |
16 | Lidocaine | 9.40±0.000 | 0.887±0.0000 | 234 | 86,58,87,72,120 |
17 | Phenobarbital | 10.14±0.004 | 0.956±0.0028 | 232 | 204,232,117,156,141 |
18 | Procaine | 10.46±0.013 | 0.987±0.0013 | 236 | 86,99,120,58,164 |
19 | n-Heneicosane (IS) | 10.60±0.000 | 1.000 | ||
20 | Methadone | 11.25±0.008 | 1.061±0.0008 | 309 | 72,91,165,178,294 |
21 | Methaqualone | 11.38±0.000 | 1.074±0.0000 | 250 | 91,235,233,250,132 |
22 | Amitriptyline | 11.59±0.011 | 1.093±0.0011 | 277 | 58,59,202,91,203 |
23 | Cocaine | 11.65±0.000 | 1.094±0.0000 | 303 | 82,83,182,94,105 |
24 | Imipramine | 11.77±0.000 | 1.110±0.0000 | 280 | 58,234,85,235,193 |
25 | Doxepine | 11.81±0.011 | 1.114±0.0011 | 279 | 58,165,178,152,189 |
26 | Pramocaine | 12.03±0.000 | 1.135±0.0000 | 293 | 100,128,70,101,129 |
27 | Bupivacaine | 12.15±0.000 | 1.146±0.0000 | 288 | 140,84,141,96,56 |
28 | Promethazine | 12.15±0.000 | 1.146±0.0000 | 284 | 72,199,180,167,213 |
29 | Carbamazepine | 12.47±0.019 | 1.176±0.0015 | 236 | 193,236,192,165,139 |
30 | Codeine | 12.83±0.000 | 1.210±0.0000 | 299 | 299,162,124,115,70 |
31 | Butacaine | 13.13±0.000 | 1.239±0.0000 | 306 | 120,142,100,178,263 |
32 | Diazepam | 13.17±0.000 | 1.242±0.0000 | 284 | 256,283,284,257,255 |
33 | Morphine | 13.22±0.006 | 1.247±0.0005 | 285 | 285,162,115,109,215 |
34 | Chlorprothixene | 13.53±0.008 | 1.276±0.0008 | 315 | 58,221,255,315 |
35 | Chlorpromazine | 13.53±0.008 | 1.276±0.0008 | 318 | 58,86,221,318,272 |
36 | Codeine acetyl | 13.67±0.008 | 1.290±0.0008 | 341 | 341,282,229,115,124 |
37 | Chlordiazepoxide | 13.70±0.008 | 1.292±0.0004 | 299 | 283,282,247,124,163 |
38 | Thebaine | 13.73±0.006 | 1.295±0.0005 | 311 | 311,296,140,152,115 |
39 | 6-Acetylmorphine | 13.78±0.004 | 1.300±0.0004 | 327 | 268,327,165,211,242 |
40 | Heroin | 14.69±0.011 | 1.386±0.0011 | 369 | 327,268,369,310,204 |
41 | Trifluoperazine | 14.94±0.011 | 1.409±0.0011 | 407 | 70,113,141,127,248 |
42 | Dibucaine | 15.16±0.010 | 1.430±0.0010 | 343 | 86,58,116,144,228 |
43 | Nitrazepam | 16.26±0.042 | 1.534±0.0010 | 281 | 253,280,206,234,222 |
44 | Papaverine | 16.83±0.006 | 1.588±0.0041 | 339 | 338,324,339,154,308 |
45 | Clozapine | 17.41±0.010 | 1.642±0.0005 | 326 | 243,256,192,227,326 |
46 | Estazolam | 18.15±0.026 | 1.712±0.0026 | 294 | 205,259,239,293,294 |
47 | Alprazolam | 18.90±0.026 | 1.783±0.0026 | 308 | 77,204,279,273,308 |
48 | Dihydroetorphine | 20.91±0.020 |
1.973±0.0020 |
413 | 380,395,176,115,296 |
有相似结构的同类药物,往往有相同的特征离子。另外,某类药物的代谢物,往往有与药物原型相同的特征离子。参考有关文献[3],并通过对几类药物及其代谢物的特征离子进行综合分析比较,选出了此类药物及其代谢物的代表特征离子,见表2。
Tab 2 The representative ions of six category drugs and their metabolites for reconstructed chromatograms
Drug category | Representative characteristic ion |
Barbiturates, methaqualone and their metabolites | 141,156,167,204,235,251,219 |
Benzodiazepines and their metabolites? | 283,243,268,308,253,299,251,259 |
Tricyclic antidepressants, phenothiazines and their metabolites | 58,72,193,113,179,180,215,234 |
Local anaesthetics and their metabolites | 82,86,100,120,140,182,58,123 |
Analgesics and their metabolites | 57,71,72,162,327,324,311,277 |
Amphetamine stimulants and their metabolites | 56,58,91,135,136,137 |
In view of the fact that clozapine and its metabolites have similar ions with the benzodiazepines, it is attributed to benzodiazepines
用所选代表特征离子的质量色谱图来筛选可能存在的药物及其代谢物,可以简化色谱图,排除一些杂质的干扰,同时节省时间,满足了毒物分析快速、准确的需要,这一点在实际中毒样品测定中得到证明。
2 血浆中21种毒物药物绝对回收率的测定
按前述方法处理样品后,用GC-FID对加入到血浆中的21种常见毒物药物进行了回收率测定,结果见表3。
Tab 3 Absolute recoveries of 21 drugs in plasma at concentration of 20 μg.mL-1 and 5 μg.mL-1 (n=5)
Peak No.? |
Drug | 20 μg.mL-1 | 5 μg.mL-1 | ||
Recoveries/% | RSD/% | Recoveries/% | RSD/% | ||
1 | Amobarbital |
96.3 |
2.90 |
95.0 |
2.61 |
2 | Pentobarbital | 96.3 | 2.43 | 93.5 | 2.80 |
3 | Pethidine | 94.0 | 5.51 | 95.9 | 2.00 |
4 | Secobarbital | 96.1 | 2.01 | 94.5 | 2.03 |
5 | Caffeine | 93.1 | 6.75 | 93.9 | 4.18 |
6 | Lidocaine | 91.5 | 5.72 | 85.7 | 3.95 |
7 | Phenobarbital | 102.5 | 3.43 | 101.5 | 2.93 |
8 | Methaqualone | 95.2 | 1.40 | 90.2 | 2.33 |
9 | Amitriptyline | 93.2 | 1.54 | 89.3 | 4.31 |
10 | Diazepam | 96.5 | 3.15 | 87.9 | 2.70 |
11 | Chlorpromazine | 89.8 | 4.08 | 79.1 | 4.46 |
12 | Methadone | 92.1 | 2.12 | 91.0 | 2.25 |
13 | Cocaine | 93.5 | 1.68 | 86.9 | 4.47 |
14 | Imipramine | 93.7 | 2.62 | 89.6 | 3.23 |
15 | Promethazine | 91.4 | 3.93 | 96.9 | 3.39 |
16 | Codeine | 99.4 | 4.18 | 102.8 | 2.58 |
17 | Codeine acetate | 89.8 | 4.64 | 83.1 | 2.88 |
18 | 6-Acetyl morphine | 98.8 | 6.98 | 101.8 | 2.63 |
19 | Heroin | 87.5 | 4.18 | 83.7 | 2.16 |
20 | Clozapine | 90.5 | 6.36 | 92.6 | 3.54 |
21 | Dihydroetorphine | 86.2 | 6.63 | 85.8 | 5.35 |
Fig 2 a. The total ion chromatogram of the patient plasma extract, RT 10.60 min (IS); b. Reconstructed mass chromatogram of selected ions for benzodiazepines (m/z 243+251+253+268+283+299+308), RT 13.15 min (diazepam), RT 13.68 min(chlordiazepoxide); c. Reconstructed mass chromatogram of selected ions for tricyclic antidepressants and phenothiazines (m/z 58+72+113+179+180+193+215+234), RT 12.42 min (carbamazepine), RT 9.98 min (carbamazepine metabolite)
结果表明,在血浆药物浓度为20 μg.mL-1和5 μg.mL-1情况下,药物绝对回收率为78%~103%,相对标准偏差小于7%。
由以上可见,所建系统化固相萃取方法通用性较好,对所选具有不同理化性质的药物均有较高的回收率。具有简单、快速,半自动化操作易于规范化、重现性好、提取干净和有机溶剂用量少等优点,能满足系统分析简单、快速、准确的要求。
3 应用
取中毒病人血浆0.5 mL,按上述所建方法处理后,加内标正二十一烷2 μg,乙酸乙酯100μL定容,用于GC-MS分析。利用质量色谱法结合谱库检索确认为安定、利眠宁和卡马西平及其代谢物,如图2所示。表4列出了GC-MS定性定量分析结果。
Tab 4 The quantitative results of toxicants in plasma
Toxicant | Concentration/μg.mL-1 |
Diazepam |
12.2 |
Chlordiazepoxide | 14.9 |
Carbamazepine | 115.1 |
Carbamazepine metabolite |
致谢:本文得到杨进生教授、刘荫棠副研究员以及胡绪英老师的指导和帮助。
REFERENCES:
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[2]Chen XH, Wijsbeek J, Veen JV, et al. Solid-phase extraction for the screening of acidic, neutral and basic drugs in plasma using a single column procedure on Bond Elut Certify [J]。 J Chromatogr, 1990,529(1):161-166.
[3]Pfleger K, Maurer HH, Weber A. Tables of measured compounds. Dyll