全血中二氮卓类药物检测方案(组织研磨仪)

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UCT， LLC·2731 Bartram Road·Bristol， PA 19007·800.385.3153·215.781.9255·www.unitedchem.com·Email： methods@unitedchem.comOUCT， LLC 2014· All rights reserved Quantitative Analysis of Benzodiazepines in Whole Bloodby QuEChERS and LC-MS/MSUCT Part Numbers：Enviro-Clean ECQUUS15CT (15 mL centrifuge tube with 400 mgMgSO4 and 100 mg NaOAc)Enviro-Clean CUMPSC18CT (2 mL dSPE tube with 150 mg MgSO4， 50mg PSA and 50 mg C18)SLDA100ID21-3UM (Selectra DA LC column， 100x2.1 mm，3 um)SLDAGDC21-3UM (Selectra° DA guard column， 10x2.0 mm， 3um)SLDGRDHLDR (Guard cartridge holder) November 2014 Summary： Benzodiazepines (Benzos) are psychoactive drugs widely prescribed for treatinganxiety， insomnia， agitation， seizures， muscle spasms， and alcohol withdrawal. Benzosare deemed safe and effective for short term use. However， frequent use of these drugsmay lead to dependence and abuse. Because of this attribute clinical， forensic andtoxicological laboratories are interested in monitoring these compounds in biologicalsamples. Common sample preparation methods for biological samples include a proteinprecipitation step followed by liquid-liquid extraction (LLE) or solid phase extraction(SPE). This application describes an easy， fast， and effective method using QuEChERSfor the quantitative analysis of benzodiazepines in whole blood. 1 mL of negative whole blood sample is extracted using 2 mL of acetonitrile (MeCN)with 0.4 % formic acid (FA). 400 mg magnesium sulfate (MgSO4) and 100 mg sodiumacetate (NaOAc) (pre-packed in 15-mL centrifuge tube) are employed to enhance thephase separation and the partition of benzodiazepines into the organic phase. Aftershaking and centrifugation， 1 mL of the supernatant is purified by a 2-mL dSPE tubecontaining 150 mg MgSO4， 50 mg PSA， and 50 mg C18. MgSO4 absorbs residual waterin the extract， while PSA and C18 remove organic acids and fatty matrix co-extractives，resulting in a clean extract for LC-MS/MS analysis. Matrix matched calibration curves were constructed for the benzodiazepinesquantification. The responses for 10 representative compounds were linear with Rranging from 0.9963 to 1.0000 over the concentration range of 10 -500 ng/mL. Matrixeffects were evaluated by comparing the slopes of the matrix matched calibrationcurves to those of the calibration curves of solvent standards. The matrix effects werefound to be minor， ranging from -22 to 18%. This indicated that the QuEChERS methodwith dSPE cleanup sufficiently removed matrix interferences that may cause significantion suppression or enhancement. Excellent recoveries (85.5-105%) and relativestandard deviations (RSD%≤10.7%) were obtained. This method was also applied to 8real whole blood samples， no benzodiazepines were detected above the limit ofquantitation of 10 ng/mL. Procedure： QuEChERS extraction a) Add 2 mL of MeCN with 0.4% FA to 15-mL centrifuge tube containing 400 mgMgSO4 and 100 mg NaOAc (ECQUUS15CT). b) Add internal standards (IS)， and appropriate amounts of benzos spikingsolution to fortified samples. c) Add 1 mL of the negative whole blood into the 15-mL tubes d) Cap and shake for 1 min at 1000 strokes/min using a Spex 2010 Geno-Grinder. e) Centrifuge at 3000 g for 5 min. dSPE cleanup a) Transfer 1 mL of the supernatant to a 2-mL dSPE tube (CUMPSC18CT). b) Shake 1 min at 1000 strokes/min using the Spex 2010 Geno-Grinder. c) Centrifuge at 3000 g for 5 min. d) Transfer 0.4 mL of the cleaned extract into a 2-mL auto-sampler vial， add 0.4mL of reagent water， and vortex for 30 sec. e) The samples are ready for LC-MS/MS analysis. LC-MS/MS method： System： AB Sciex API 4000 QTrap MS/MS with Agilent 1200 Binary Pump SL Column： UCT Selectra DA LC column， 100x2.1 mm， 3 um Guard Column： UCT Selectra[DA guard column， 10x2.0 mm， 3 pm Column Temperature： 50°℃ Column Flow Rate： 0.3 mL/min Injection Volume： 10 pL Gradient Program： Time (min) % Mobile Phase A(0.1% FA in water) % Mobile Phase B(0.1% FA in methanol) 0 70 30 0.5 70 30 2 25 75 6.5 25 75 7 0 100 9 0 100 10.1 70 30 14 70 30 MRM transitions (ESl positive， dwell time： 50 ms) Compound Rt (min) Q1 ion Q3 ion 1 Q3 ion 2 7-aminoclonazepam 7.58 286.1 222.3 250.2 Alpha-Hydroxy-Alprazolam 9.26 325.2 297.1 216.3 Alprazolam 9.72 309.2 205.3 281.2 Clonazepam 9.03 316.1 270.2 241.2 Diazepam 9.87 285.1 193.2 154.1 Lorazepam 8.94 321.1 303.3 275.0 Midazolam 8.53 326.0 291.0 222.0 Nordiazepam 9.30 271.1 140.1 165.2 Oxazepam 9.00 287.1 241.3 104.2 Temazepam 9.45 301.1 255.2 177.2 Alprazolam D5 9.69 314.2 286.3 Oxazepam D5 8.98 292.1 246.2 Results： Linearity and Matrix Effect Compound Solvent standard Matrix-matched standard Matrix effect Slope Linearity (R2) Slope Linearity (R2) 7-aminoclonazepam 0.00823 0.9993 0.00646 0.9998 -22 Alpha-Hydroxy-Alprazolam 0.00646 0.9990 0.00764 0.9996 18 Alprazolam 0.000413 0.9990 0.000486 0.9989 18 Clonazepam 0.00443 0.9995 0.00497 0.9999 12 Diazepam 0.0133 0.9997 0.0146 0.9996 10 Lorazepam 0.00306 0.9999 0.0034 0.9997 11 Midazolam 0.00656 0.9989 0.00675 0.9963 3 Nordiazepam 0.00703 0.9999 0.00754 0.9998 7 Oxazepam 0.00987 1.0000 0.0107 1.0000 8 lemazepam 0.00641 0.9998 0.00709 0.9999 11 Recovery and RSD% from Whole Blood Spiked at 3 Levels Compound Spiked at 10 ng/mL Spiked at 50 ng/mL Spiked at 200 ng/mL Recovery% RSD% (n=6) Recovery% RSD% (n=6) Recovery% RSD% (n=6) 7-aminoclonazepam 88.6 7.5 96.9 2.1 99.7 3.8 Alpha-Hydroxy-Alprazolam 101.2 3.4 91.0 2.0 90.3 2.7 Alprazolam 92.3 10.7 90.2 4.0 86.5 3.5 Clonazepam 96.4 3.6 105.0 3.2 103.0 2.0 Diazepam 85.5 3.3 103.0 2.7 100.4 1.9 Lorazepam 96.9 5.1 93.7 4.1 91.6 2.7 Midazolam 96.7 2.7 101.6 2.7 100.6 1.9 Nordiazepam 88.4 3.9 99.7 2.5 97.8 2.3 Oxazepam 86.5 1.9 93.8 2.4 92.6 1.7 iemazepam 96.7 2.7 101.6 2.7 100.6 1.9 Chromatogram of a whole blood sample spiked with 200 ng/mL benzos Peak list： 1.7-aminoclonazepam； 2. Midazolam； 3. Lorazepam；4. Oxazepam；5. Clonazepam；6. Alpha-Hydroxy-Alprazolam； 7.Nordiazepam；8. Temazepam； 9. Alprazolam； 10. Diazepam Matrix Matched Calibration Curve of 7-aminoclonazepam(R=0.9998) 3.43.2- 3.0- 2.8- 2.6- 2.4- 2.2- 2.0- 1.8- 1.6- 1.4- 1.2- 1.0- 0.8- 0.6- 0.4- 0.2- 0.0- c 4111-01-01 苯二氮卓类(BZ)是精神活性药物，广泛用于治疗焦虑、失眠、烦躁、癫痫、肌肉痉挛和酒精戒断。BZ短期使用被认为是安全有效的。然而，频繁使用这些药物可能导致依赖和滥用。由于这一特性，临床、法医和毒理学实验室对监测生物样品中的这些化合物很感兴趣。生物样品常用的样品制备方法包括：先将蛋白质沉淀，然后是液-液萃取(LLE)或固相萃取(SPE)。本应用介绍了一种简便、快速、有效的全血苯二氮卓类药物定量分析方法。用含0.4%甲酸(FA)的2ml乙腈(MeCN)提取1mL阴性全血。采用400mg硫酸镁(MgSO4)和100mg醋酸钠(NaOAc)(预装在15 ml离心管中)使相分离加强，并将苯二氮杂卓分离至有机相中。摇匀离心后，用含150mg MgSO4，50mg PSA， 50mg C18的2ml dSPE管纯化，取1 mL上清液。MgSO4可除去萃取物中残余的水，PSA和C18去除有机酸和脂肪基质共萃取物。得到一个纯净的提取物，用于LC-MS/MS分析。建立苯二氮卓类化合物的基质匹配的校准曲线。10个基质匹配溶液浓度在10 ~ 500 ng/mL范围内均呈线性关系，R范围为0.9963 ~ 1.0000。通过比较同基质校准曲线与标准溶液的校准曲线的斜率来评价基质效应。基质效应很小，从-22%到18%不等。这表明带有dSPE清除的QuEChERS方法可以充分去除基质干扰，这些干扰可能导致离子的显著抑制或增强。加样回收率为85.5 ~ 105%，相对标准偏差(RSD%)为10.7%。该方法也应用于8份真实全血样本，未检出苯二氮卓类药物超过10 ng/mL的定量限。