Method Development for the Detection of Basic/Weak Basic Drugs in Hair by LCMSMS: Comparison between Methanolic and Alkaline Extraction on Real Samples ()
1. Introduction
Drug analysis in hair has grabbed the attention of toxicology analysts and researchers in recent years. This is mainly because it has provided some ability of proving drug ingestion when conventional samples could not. Hair differs from other traditional biological samples used for human toxicological analysis such as urine, blood, liver or saliva with its significantly longer detection window (months) allowing retrospective investigation and measurement of drug consumption. Hair analysis is becoming accepted in many developed countries for substance consumption related issues in a wide range of sectors; the medico-legal sector, workplace testing, treatment monitoring, schools, forensics, research, insurance companies, environmental biomonitoring and driving licensing [1-4].
Extraction of drugs from hair is considered one of the most important steps in hair analysis. Apart from external drug deposition on hair, drugs are mainly enclosed tightly in the hair shaft and to a certain extent maybe bound to proteins, melanin or lipids of the cell membrane complex. Therefore, hair matrix type, structure of the drug, method and duration of extraction, and solvent used are all important factors affecting the final extraction yield [5].
There are numerous reports of screening strategies published for analysing different forensic basic drugs groups. Hypnotic drugs such as benzodiazepines are at the top of the list of drug facilitated crimes. These are weak basic drugs and have been reported to be extracted from hair by different methods, with phosphate buffers, methanol or digestive enzymes [6-8]. Other basic drug groups like antipsychotics, antidepressants and amfetamines have frequently been reported to be extracted with alkaline digestion (NaOH) [9-12]. However, recently there has been some focus on using methanolic solutions as an extraction medium consisting of varying ratios of methanol, acetonitrile and formate buffer for extraction of several groups of basic drugs simultaneously [13-15]. However, the extent of their efficiency was not examined most of the time. Therefore, in the present study the aim was to develop a method to enable the detection and quantification of basic and weak basic drugs in hair simultaneously based on the methanolic solution extraction technique and to compare its efficiency with the alkaline digestion technique which was followed by liquid-liquid extraction (LLE).
2. Materials
2.1. Chemicals
All chemicals were of HPLC or analytical grade. Methanol, acetonitrile, acetone, propanol, methyl-tert-butylether (MTBE), citalopram, clobazam, clonazepam, cocaine, codeine, desmethyldiazepam, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-Methylenedioxyme-thamphetamine (MDMA), midazolam, sulpiride, zaleplon and zolpidem were purchased from Sigma-Aldrich Co, Dorset UK. 7-aminoclonazepam, amfetamine, amfetamineD11, benzoylecgonine-D8, benzoylecognine, codeine-D3, diazepam, diazepam-D5, ecognine methyl ester, medazepam, oxazepam and temazepam were purchased from LGC standards, Middlesex UK. Other prescription drug standards were supplied from their manufacturer. Deionised water was prepared on site (ELGA Limited). Dichloromethane, 7.5 M ammonium acetate solution and all consumables (tubes, etc) are from VWR International Ltd, Lutterworth UK. The rest of the chemicals used in the solutions below were from Sigma-Aldrich Co, Dorset UK.
2.2. Solutions
2.2.1. Standard Solutions
1000 mg/L or 100 mg/L working standards made with MS grade methanol of each analyte and internal standard. All stored at –20˚C and diluted to the required concentration prior to the experiment.
2.2.2. 0.1 M Phosphate Solution
One litre of solution was prepared by dissolving 1.7 g of anhydrous disodium orthophosphate and 12.14 g of sodium dihydrogen orthophosphate monohydrate in 800 mL of de-ionized water. The pH was adjusted to 6 with 1 M potassium hydroxide and the solution made up to volume with de-ionized water.
2.2.3. 0.1% Formic Acid (v/v)
100 µL of concentrated formic acid (98/100) was mixed with 99.9 mL of de-ionized water, mixed well and stored at room temperature.
2.2.4. Methanolic Extraction Solution
60 mL of methanol, 10 mL of acetonitrile and 30 mL of 0.1% formic acid were measured into measuring cylinder, mixed well and stored at room temperature.
2.2.5. 1 M NaOH Solution
40 g of NaOH (MW 40.0) pellets were measured into a 1000 mL volumetric flask and made up to the 1000 mL mark with de-ionized water. This was mixed thoroughly and stored at room temperature.
2.2.6. 2 M Ammonium Acetate
266.6 mL of 7.5 M ammonium acetate solution were measured into a 1000 mL volumetric flask and made up to the 1000 mL mark with de-ionized water.
3. Methods and Results
3.1. Hair Preparation
The decontamination process was by using three washing steps with two solvents and one aqueous solution: 0.01 M phosphate solution (pH 6), dichloromethane and propanol/acetone (1:1). The uncut hair was put in a 6 ml glass tube, then 2 mL of the first solution added and vortex mixed for 1 minute. After that the solvent was decanted and then the next solvent was added. After the last washing step (2 mL propanol/acetone) the hair was left to dry at room temperature or the process was speeded up with the speed-vac. This decontamination method was found to be quick and very efficient in cleaning postmortem hair samples.
After the hair had dried segments of ~0.6 cm each were cut into small pieces (1 - 3 mm) with scissors and ideally 25 mg were weighed directly. If this was not possible any amount between 10 and 80 mg was considered and placed into a 2 mL glass tube with a screw cap.
3.2. Methanolic Extraction
Five hundred micro-litres of the methanolic extraction solution (described earlier) was added to the hair in each tube and left in a sonicator for 16 hours at 40˚C. In the case of calibrators, the required concentrations were made based on adding 25 μL of drugs standard (e.g. 1 mg/L in methanol) to the calibrators’ tubes containing 25 mg blank hair each and those 25 μL were deducted from the extraction solution (475 μL). Also 25 μL of internal standards (bromperidol and the dueterated standards amfetamine-D11, benzoylecgonine-D8, codeine-D3 and diazepam-D5) were added as well at a concentration similar to the middle calibrator. After centrifugation for 10 minutes the extracted solutions were transferred to autosampler vials and evaporated to dryness followed by reconstitution with 125 μL of 25% methanol and filtered with the syringeless mini-uniprep filters (Whatman GE). Finally 20 μL of this filtrate was injected into the LCMSMS system.
3.3. Alkaline Digestion
Twenty five milligrams of washed blank/patient hair was incubated with 0.5 mL of 1 M NaOH for 3 hours in a water bath (50˚C) to allow for digestion of the hair. Samples were then extracted after digestion with 2 mL MTBE by 30 minutes rotary mixing and 10 minutes centrifugation. Solvent layers were transferred into clean tubes containing 150 µL 1% formic acid, then mixed on a rotary mixer for 20 minutes and removed after 10 minutes of centrifugation by aspiration. From the remaining aqueous layer 20 µL was injected into the LCMSMS system.
3.4. Method Development with Some Validation for Selected Analytes
3.4.1. Instrumentation and Ion Identification
The LCMSMS analysis was carried out with an HPLC system consisting of a Perkin Elmer PE200 series autosampler, pump and column oven. The MSMS part was a SCIEX API 2000 Triple Quadrupole MS-MS instrument (Applied Biosystems) equipped with an electrospray interface (Turbo Ion Spray) set on the positive mode. Ion spray voltage was set to 5500 V. Nitrogen was used as the nebulizer gas-GS1 (30 psi), auxiliary gas-GS2 (20 psi heated to 300˚C), curtain gas (20 psi) and as the CAD gas (set on 3).
Analytes were infused at 0.25 mL/minute to the LCMSMS system at a concentration of 1 mg/L in 50% methanol/2mM ammonium acetate solution. The precursor and product ion identification and parameter tuning were completed by automatic and manual tuning. Optimum tuning parameters, precursor and product quantitation ions are shown in Table 1. Mobile phase was 50% methanol, 0.1% formic acid and 2 mM ammonium acetate (isocratic). Separation with Altech Alltima C18 column (15 cm × 2.1 mm, 5 μm) maintained at 50˚C.
3.4.2. Comparison of Mini-Uniprep Filter Media
Three filter materials were compared by using spiked blank hair samples at concentrations of 0.5 ng/mg hair. 25 mg of blank hair were weighted out in glass tubes. Then the whole extraction/incubation method was applied to all the samples. The three materials were 0.45 µm pore size Glass Microfiber (GMF), 0.2 µm pore size Polytetrafluroethylene (PTFE) and 0.2 µm pore size Polyvinylidenefluoride (PVDF) used in manufacturing Whatman Mini-UniPrep Syringeless Filters. The filters efficiency was calculated by comparing the percentage of extraction recovery through their peak area ratio of product ion for each drug after filtration with unfiltered calibrators.
Table 2 shows clearly that the PTFE filter medium has the greatest recovery (mean = 100%) of all the analytes while the GMF and PVDF filters (mean = 89% and 72%, respectively) reduced significantly the response of amisulpride and citalopram. Also the responses of medazepam, midazolam and zolpidem were considerably reduced by the PVDF filter.