药物分析文献

Journal of Pharmaceutical and Biomedical Analysis 108(2015)97–101

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Journal of Pharmaceutical and Biomedical

Analysis

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Development of the MAE/UHPLC-MS-TOF method for determination of benzodiazepines in human bio-fluids for toxicological analysis

Aneta Wo ´zniakiewicz,Renata Wietecha-Posłuszny ∗,MichałWo ´zniakiewicz,

Julia Nowak,PawełKo´s cielniak

Laboratory for Forensic Chemistry,Department of Analytical Chemistry,Jagiellonian University,3Ingardena Street,30-060Kraków,Poland

a r t i c l e

i n f o

Article history:

Received 24October 2014

Received in revised form 3February 2015Accepted 5February 2015

Available online 16February 2015

Keywords:

Benzodiazepines

Human serum and blood analysis Microwave-assisted extraction Liquid chromatography Mass spectrometry

a b s t r a c t

A rapid method of microwave-assisted extraction (MAE)followed by ultrahigh performance liquid chro-matography with mass spectrometry with time of flight detection (UHPLC-MS-TOF)was optimized and validated for the purpose of determination of five benzodiazepines in human serum and blood sam-ples.Extraction parameters and conditions of the UHPLC-MS-TOF method were defined.Validation of the developed method was performed at three concentration levels:10,100and 250ng/mL of each drug for both serum and blood samples.For serum and blood the limit of detection was found in the ranges 0.46–2.58ng/mL and 0.43–1.87ng/mL,precision (RSD):0.3–6.7%and 0.9–8.4%,accuracy of the assay (RE):−5.3to +2.4%and −5.7to +7.6%,recovery:80.5–104.3%and 79.9–106.9%,matrix effects:95.9–110.5%and 97.5–114.2%,respectively.Moreover,the optimized and validated MAE/UHPLC-MS-TOF method was applied to analysis of blood samples.

©2015Elsevier B.V.All rights reserved.

1.Introduction

Benzodiazepines (BZDs)are a large group of psychoactive drugs which demonstrate unique properties,depending on the substi-tuted groups attached to the core [1–3].Long-term use of BZDs can induce physical and mental addiction.However,despite these threats,they are the most frequently prescribed medications due to their positive properties and high treatment effectiveness [4].Therefore,it is important to develop new analytical methods for the determination of BZDs in the human body.In recent years,much attention has been directed to the application of microwave radiation to the sample preparation step [5–7].Moreover,liquid chromatography with mass spectrometry has been widely used to screen,identify and quantify BZDs or their metabolites in various materials [4,8,9].

The aim of this research was to exploit microwave-assisted extraction (MAE)in the preparation step of the serum and blood samples in order to determine five BZDs:alprazolam,estazolam,lorazepam,diazepam and tetrazepam.Extracts of the studied compounds were analyzed by means of ultrahigh performance liquid chromatography (UHPLC)coupled to mass spectrometry with a time of flight mass analyzer (MS-TOF).The validated

∗Corresponding author.Tel.:+48126632084;fax:+48126632084.E-mail address:wietecha@.pl (R.Wietecha-Posłuszny).

MAE/UHPLC-MS-TOF method was then applied to analysis of serum reference materials and forensic blood samples.

2.Material and methods

2.1.Standards,reagents and materials

Drug standards:alprazolam (Alp),estazolam (Est),lorazepam (Lor),diazepam (Dia),tetrazepam (Tetr)were purchased from LGC Standards (Teddington,UK).The deutered analogues of BZDs:estazolam-d5(Est-d5),lorazepam-d4(Lor-d4)and diazepam-d5(Dia-d5)were obtained from Lipomed AG (Arlesheim,Switzerland).Drug stock solutions (10mg/mL)were prepared in methanol and stored in −20◦C.Spiking solutions were prepared daily by appro-priately diluting stock solutions with water.Standard drug solu-tions were prepared by diluting stock solutions with a mobile phase to concentrations of 75,150ng/mL and 1.88␮g/mL –(correspond-ing to 10,20and 250ng/mL,respectively,within 1mL of biofluid sample).LC–MS grade acetonitrile and methanol,ammonium for-mate,isopropyl alcohol,electrospray calibrant solution and sodium tetraborate decahydrate were purchased from Sigma–Aldrich (St.Louis,MO,USA).Analytical grade formic acid was supplied by Merck (Darmstadt,Germany).Ethyl acetate and 30%NaOH water solution,both of analytical grade,were purchased from POCH (Gli-wice,Poland).Water (18.2M cm,TOC <5ppm)was ultrapurified and filtered through a Milli-Q Plus system (Millipore,Bedford,MA,

/10.1016/j.jpba.2015.02.009

0731-7085/©2015Elsevier B.V.All rights reserved.