The Application of Ultra-Fast Triple Quadrupole LC-MS/MS to Forensic Analysis. Abstract

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1 SR14_008E S h i m a d z u R e v i e w The Application of Ultra-Fast Triple Quadrupole LC-MS/MS to Forensic Analysis by Toshikazu MINOHATA 1, Keiko KUDO 2, Kiyotaka USUI 3, Noriaki SHIMA 4, Munehiro KATAGI 4, Noriaki IKEDA 2, Hitoshi TSUCHIHASHI 5, and Koichi SUZUKI 5 Abstract In recent years, drug abuse and the use of illegal drugs have become a social problem as there are an increasing number of incidents of crime and addiction involving the use of illicit drugs such as psychotropics and hypnotics. The search for and identification of illegal substances has therefore become an issue in the forensic, toxicological, and clinical sectors, and there is a need for fast and highly sensitive simultaneous analysis methods. Using fast polarity switching and MRM (Multiple Reaction Monitoring) triggered automatic MS/MS, a new method has been developed for the simultaneous screening and quantification of the most widely abused drugs that are available in Japan and that are relevant in clinical and forensic cases. In product ion scan measurement (automatic MSMS, or Synchronized Survey Scan ) using MRM as the trigger, mass spectra of a precursor ion can be obtained during an MRM transition. Compound structures can be simultaneously confirmed by the resulting product ion spectra due to the high selectivity achieved during MRM with no interference from co-eluting substances. Keyword:Forensic, Toxicology, LC-MS/MS, MRM (Multiple Reaction Monitoring), Synchronized Survey Scan 1. Introduction Incidences of drug addiction and crimes using medical products such as psychotropics and hypnotics have increased in recent years and become a problem for society. Accordingly, the investigation, identification, and quantification of compounds presumed to have been taken by a subject, from a complex matrix such as blood or urine has become an important topic for analytical scientists in a clinical setting such during critical care, as well as researchers of forensic medicine and forensic toxicology tasked with performing judicial autopsy and post-mortem work. These topics call for methods of simultaneous analysis that are both fast and highly sensitive. At present, gas chromatography/mass spectrometry (GC/MS) is used widely in the field of forensic medicine. GC/MS is an extremely useful technique since using full-scan mode with electron ionization (EI) allows for scanning over an extensive range of compounds, and a sizable spectral library exists that is useful for the identification of non-target compounds. However, polar compounds such as metabolites of benzodiazepine hypnotics and compounds that are unstable when exposed to heat are difficult to analyze using GC/MS without derivatization. Also, some compounds simply cannot be derivatized for the purpose of improving chromatograph behavior. The presence of phospholipids and other impurities can also prevent acquisition of a good chromatogram and require thorough pretreatment by solid-phase extraction or other methods. Liquid chromatography/triple quadrupole mass spectrometry (LC-MS/MS) is an effective analysis platform capable of both excellent selectivity and quantitative performance at low substance (Received December 12, 2013) 1 MS Business Unit, Life Science Business Department, Analytical & Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan 2 Department of Forensic Pathology and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 3 Department of Public Health and Forensic Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan 4 Forensic Science Laboratory, Osaka Prefectural Police, Osaka, Japan 5 Department of Legal Medicine, Osaka Medical Collage, Takatsuki, Japan concentrations. The use of LC-MS/MS for toxicological drug screening and quantitative applications is finding its way into university forensic medicine laboratories, prefectural foundation hospitals, and companies that perform clinical laboratory tests. LC-MS/MS does not require derivatization of the target compound and therefore brings associated time and cost savings. We have used a high-speed liquid chromatograph/mass spectrometer LCMS-8040 designed for use in forensic medicine to examine a pretreatment method and a method of product ion scan measurement that is triggered by multiple reaction monitoring (MRM) (automatic MS/MS, Synchronized Survey Scan ). The methods we developed allowed us to use an MRM mode to provide improved specificity in the detection of compounds from within a complex matrix such as blood, the acquisition of an MS/MS spectrum in product ion scan mode, and compound identification by the library search of a spectral database. We also used a pretreatment method that consists of the QuEChERS method normally employed for residual pesticide analysis of food and agricultural materials, modified here for toxicological drug screening, which allowed us to recover medicinal toxicants with a wide range of properties, from acidic to basic compounds. 2. Experimental Method 2.1 Reagents We used acetonitrile and methanol for LCMS from Wako Pure Chemical Industries, Ltd., and analytical grade ammonium formate and trifluoroacetic acid (TFA) also from Wako. Water was acquired from a Merck ultra-pure water (Milli-Q water) production system. QuEChERS reagents used for sample pretreatment were the Q-sep QuEChERS extraction salt packet (conforming to AOAC method) from Restek Corporation, and the diazepam-d5 used as an internal standard reagent was from Hayashi Pure Chemical Ind., Ltd. The 4 mm stainless steel beads were from TAITEC Corporation. 2.2 Instruments and Analytical Conditions We used a Shimadzu Nexera ultra high performance liquid chromatograph (two-liquid high-pressure gradient system) and a Shimadzu LCMS-8040 liquid chromatograph/mass spectrometer. Analytical conditions were as follows: Column: Shim-pack FC-ODS (2.0 mmi.d. 150 mml, 3 µm); Mobile phase A: 10 mm ammonium

2 formate in water; B: methanol; Gradient conditions: 5% B (0 min) 95% B (15 20 min) 5% B ( min); Ionization method: electrospray ionization (ESI); Detection polarity: positive and negative ions; Nebulizer gas flowrate: 1.5 ml/min; Drying gas flowrate: 10 ml/min; DL temperature: 250 C; HB temperature: 400 C. Other settings were used at values obtained by automatic adjustment. 2.3 Pretreatment Method First, 0.5 ml of blood was placed in a 15 ml centrifuge tube, to which 1 ml of distilled water was added and a vortex mixer used to agitate the mixture. Two 4 mm stainless steel beads, 1.5 ml of acetonitrile, and 10 µl of acetonitrile solution containing 10 ng/µl of diazepam-d5 were then added to the tube and mixed with a vortex mixer. Next, 0.5 g of the packing from the Q-sep QuEChERS extraction salt packet (0.4 g MgSO g NaOAc) was added and after shaking vigorously several times by hand, mixed thoroughly with a vortex mixer then centrifuged for 10 minutes at 3,000 rpm. The supernatant liquid was transferred to a new tube, to which 100 ml of 0.1% TFA acetonitrile solution was added, the resulting material mixed with a vortex mixer then dried in a concentration/drying unit using blown nitrogen gas. To the dry sample was added 200 µl of methanol, and using a vortex mixer to assist with dissolution, the mixture was transferred to a micro tube and centrifuged for 5 minutes at 10,000 rpm. From this, 150 µl of supernatant liquid was placed in a 1.5 ml HPLC vial containing a micro-volume insert, and 5 µl of this sample analyzed by LC-MS/MS. 3. Results and Discussion 3.1 Investigation of QuEChERS Method for Forensic Toxicological Drug Screening Analysis of biological samples such as blood or urine requires pretreatment of the sample by protein extraction, solid-phase extraction or another method. However, there is currently no definitive method of pretreatment with current methods entailing problems that include the inability to remove impurities effectively, complex handling procedures, and only being able to recover compounds that are either acidic or basic. We investigated developing a pretreatment method based on the QuEChERS method for the purpose of recovering all compounds, whether acidic or basic, with ease. The QuEChERS method as a concept derives its name from its characteristics of being "Quick," "Easy," "Cheap," "Effective," "Rugged" and "Safe," and was developed to effectively process large quantities of samples. It is mainly used as pretreatment for the analysis of residual pesticides in food, and there are few reports of its use for samples analyzed in forensic medicine such as blood, urine and organ fluids. We added the 24 medicinal toxicants shown in Table 1 to a whole blood control and investigated the analysis of this mixture 2) 4). The results showed percentage recovery from a 0.5 ml sample (blood) was improved by diluting with 1 ml of distilled water. To this was added an equal amount (1.5 ml) of acetonitrile and the mixture agitated. After the protein had clumped together, 0.5 g of QuEChERS extraction salt was added to the mixture then mixed vigorously and centrifuged. Doing so moved the drugs to the acetonitrile layer with high percentage recovery. Then, 0.1% TFA acetonitrile solution was added to prevent volatilization of the drugs, such as stimulants and nicotine, during solvent removal. The QuEChERS method normally uses materials such as C18 or PSA (primary-secondary amine) to remove lipids and fatty acids and purify the sample, but due to the improved percentage recovery we omitted this purification step. After the sample solvent was dried, 200 µl of methanol was added for re-dissolution and the resulting solution used as the sample. A 24-component standard mixture solution [A], blood pretreated after the addition of these 24 components [B], and blood pretreated before the addition of these 24 components [C] were analyzed. The peak area was calculated for each compound and the percentage recovery and matrix effect were confirmed. The percentage recovery (%) of each compound was calculated according to "peak area [B]/peak area [C] 100," and the percentage matrix effect (%) was calculated according to "peak area [C]/peak area [A] 100." The results are shown in Table 1. Percentage recovery was 80% or more with 16 of the 24 components, and there was a mean percentage recovery of 82.6%. The percentage matrix effect was 90 to 110% for 18 of the 24 components and the mean percentage matrix effect was 94.6%. Our investigation based on the QuEChERS method demonstrated it provides a consistent percentage recovery regardless of acidic or basic component, and can be applied for use on biological samples such as blood. Table 1 Recovery and matrix effect for 24 compounds Analytes Recovery (%) Matrix effect (%) IS Abused Drugs Hypnotic Drugs Psychotropic Drugs Medical Drugs Diazepam-d5 Dihydrocodeine Methamphetamine Methylephedrine 7-Aminoflunitrazepam 7-Aminonitrazepam Bromovalerylurea Flunitrazepam Phenobarbital (neg) Quazepam Zolpidem Amoxapine Chlorpromazine Clomipramine Fluvoxamine Levomepromazine Olanzapine Paroxetine Quetiapine Sertraline Zotepine Acetaminophen Chlorpheniramine Diclofenac Mirtazapine Average

3 3.2 Development of an MRM-Triggered Product Ion Scan Method The LCMS-8040 has a Synchronized Survey Scan function that automatically performs MS/MS when a threshold is exceeded, thereby allowing for a combined MRM and MRM-dependent product ion scan in a single analysis. With MRM it is possible to detect compounds at high sensitivity simultaneously with performing a product ion scan, and obtain an MS/MS spectra. Furthermore, since the Collision Energy (CE) can be configured for each product scan, it is possible to choose an optimum CE for each compound. The MRM-product ion scan setup windows used in the LabSolutions software are shown in Fig. 1. The MRM chromatograms and library search results based on MS/MS spectra data are shown in Fig. 2-1, Fig. 2-2 for urine with four added compounds (allylisopropylacetylurea, diclofenac, amobarbital (neg), thiamylal (neg): each at 1 ng/µl). When fast positive/negative ionization switching was used to perform simultaneous measurements, we confirmed that all four compounds were identified from the MS/MS spectra by matching against the highest scoring library spectra in terms of degree of similarity. Positive Negative MRM parameter Product Ion Scan parameter Fig. 1 The user interfaces for setting MRM-product ion scan parameters in LabSolutions software 4.0 ( 100,000) >55.05(+) Allylisopropylacetylurea ( 100,000) >215.05(+) >214.00(+) Diclofenac Fig. 2-1 MRM chromatograms and library search results for 4 compounds spiked into urine

4 6.0 ( 1,000) >42.00(-) Amobarbital (neg) ( 10,000) >58.10(-) >101.00(-) Thiamylal (neg) Fig. 2-2 MRM chromatograms and library search results for 4 compounds spiked into urine 3.3 Development of a Simple Quantification Method Quantitative analysis of medicinal toxicants in a biological sample requires the preparation of a calibration curve using a standard reference of the medicinal substance, and sample preparation for quantitative analysis entails considerable use of time and labor. Quantitative analysis cannot be performed when there is no standard reference material. By using diazepam-d5 as an internal standard reference and registering calibration curve information (slope and intercept) calculated from this material to a method, we were able to develop a simple quantification method that allows for identification and an approximate quantitative analysis of medicinal substances that are not readily obtainable. We evaluated this method by adding 103 compounds to blood, pretreating the blood with a QuEChERS method, and using that as the sample. The 103 compounds were divided into three groups where group A compounds (16 components) were added to a concentration of 0.1 ng/µl, group B compounds (78 components) to 1 ng/µl, and group C compounds (9 components) to 10 ng/µl. The results are shown in Table 2. The simple quantitative values obtained immediately after analysis were calculated based on calibration curve information taken from the ratio between the peak area of the internal standard reference material (diazepam-d5) and the data obtained from the component in the sample. The simple quantitative results obtained for each compound group were a mean concentration of 0.08 ng/µl for group A compounds, 0.79 ng/µl for group B compounds, and 13.4 ng/µl for group C compounds. The simple quantitative value is likely to vary substantially based on percentage recovery of the medicinal substance and matrix effects during pretreatment, as well as conditions such as the mobile phase, column and mass spectrometer used. The simple quantitative value must be regarded as merely an estimate, and expert testimony would require a method utilizing the correct standard reference material.

5 Table 2 Semi-quantitated results for 103 compounds spiked into blood group Analytes Alprazolam Aripiprazole Atropine Brotizolam Colchicine Estazolam Ethyl loflazepate Etizolam Flunitrazepam Haloperidol Lidocaine Nimetazepam Risperidone THC THC-COOH Triazolam Average of group A 7-Aminoflunitrazepam 7-Aminonimetazepam 7-Aminonitrazepam 8-Hydroxyetizolam (M-III) Acetaminophen Aconitine Allylisopropylacetylurea Alpha-Hydroxybrotizolam Alpha-Hydroxytriazolam Amitriptyline Amoxapine Amphetamine Benzoyl ecgonine Biperiden Bromazepam Bupivacaine Carbamazepine Chlorpheniramine Conc. (ng/µl) group Analytes Chlorpromazine Clomipramine Acetylpheneturide Clonazepam Cocaine Codeine Desmethyldiazepam Dextromethorphan Diazepam Dihydrocodeine Diltiazem Diphenhydramine Diprophyline Dosulepin Ecgonine methyl ester Ephedrine Ethenzamide Fenitrothion(MEP) Flurazepam Fluvoxamine Glibenclamide Hydroxyzine Imipramine Ketamine Levomepromazine Lorazepam Lormetazepam Malathion Maprotiline MDA MDMA Mepivacaine Methamphetamine Methomyl Methylephedrine Methylphenidate Conc. (ng/µl) group Analytes Mexiletine Mianserin Midazolam Mirtazapine Nitrazepam Nortriptyline Olanzapine Oxazepam Pancuronium Paroxetine Pentazocine Promethazine Quazepam Quetiapine Sertraline Sildenafil Sulpiride Temazepam Trazodone Trihexyphenidyl Warfarin Zolpidem Zopiclone Zotepine Average of group B Amobarbital (neg) Barbital (neg) Bromovalerylurea Diclofenac Loxoprofen (neg) Pentobarbital (neg) Phenobarbital (neg) Salicylic_acid (neg) Thiamylal (neg) Average of group C Conc. (ng/µl) Conclusion We investigated the development of a screening system intended for forensic medicine using the LCMS We examined using a QuEChERS method, which is mainly used for residual pesticide analysis in food, as a method of sample pretreatment and showed it gave consistent percentage recovery results irrespective of the compound being acidic or basic, and confirmed the method can be used for biological samples such as blood. We developed the Synchronized Survey Scan analysis method capable of simultaneously detecting compounds at high sensitivity and acquiring the MS/MS spectra and demonstrated this method is capable of quantitative and qualitative analysis in a single measurement. We also developed a simple quantification method able to provide a quantification result immediately after measurement by registering calibration curve information. Since the quantification results provided by this method are only a rough estimate of the concentration in a sample, they must always be used while aware that the results acquired vary greatly depending on errors in percentage recovery and concentration factor of the medicinal substance during analysis pretreatment, matrix effects and conditions in the mass spectrometer. However, the method is useful for quickly getting a rough idea for the concentration of a medicinal substance when this kind of estimated quantitative result is desired simultaneously with the performance of a qualitative analysis, particularly when a standard reference material is not immediately available. References 1) Katagi M, Tsutsumi H, Miki A, Nakajima K, Tsuchihashi H: Jpn. J. Forensic. Toxicol. 20: 303 (2002) 2) Usui K, Hayashizaki Y, Hashiyada M, Funayama M: Leg Med (Tokyo). 14 (6), (2012) 3) Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ: J AOAC Int, 86 (2), (2003) 4) Matuszewski BK, Constanzer ML, Chavez-Eng CM: Anal Chem. 75 (13), (2003) 5) Ishida T, Kudo K, Inoue H, Tsuji A, Kojima T, Ikeda N: J Anal Toxicol. 31 (1), 66-8 (2007) 6) Ishida T, Kudo K, Naka S, Toubou K, Noguchi T, Ikeda N: Rapid Commun Mass Spectrom. 21(18), (2007)

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